臺灣博碩士論文加值系統

台灣目前估計約有350萬吸菸人口，每年推估有超過18,800人死於吸菸相關疾病，死因統計資料亦顯示與吸菸相關的死因約佔16%；另外台灣嚼食檳榔的人口數約為180萬，研究指出，嚼食檳榔者有87%有吸菸的習慣。香菸中的尼古丁經由nitrosation會產生強致癌物質4-(methylnitrosamino)-1-(3 pyridyl)-1- butanone (NNK)，而NNK在人體中，會經由CYPA2As酵素作用形成代謝物4-(Methylnitrosamino)-1-(3-Pyridyl)-1- Butanol (NNAL)，再經phase II酵素加成作用，最後由尿液中排出。檳榔中含有許多物質，其中的黃樟素，經動物實驗證實，對於CYP2As酵素具有誘發的作用;實驗室以往數據指出，倉鼠在投予黃樟素後第九天後給予NNK，尿中的之代謝物NNAL的量會降低。然而，此現象是否在人體出現，仍未見諸報導。
此實驗是比較純吸菸者以及吸菸同時嚼食檳榔者尿中NNAL含量的差異，而尿中NNAL的濃度是利用液相層析串聯質譜儀搭配同位素稀釋法來進行定量。由於NNAL在尿液中的含量非常的低，為ppt (pg/ml)等級，因此必須使用固相萃取（Solid Phase Extraction, SPE）的方式，來進行濃縮。本實驗的研究樣本為台灣吸菸者與吸菸又同時嚼食檳榔者的尿液，在尿液中加入β-glucuronidase來檢測尿中的total NNAL含量，並且用尿液中的creatinine (Cre)的含量來進行濃度的校正。
實驗的結果，單純抽香菸者的16個樣本，尿液中Total NNAL的平均濃度為149.46ng/mg Cre，而抽菸且嚼食檳榔者的33個樣本，尿液中NNAL的平均濃度為85.97 ng/mg Cre，且此差異達到統計上的顯著意義(p=0.007); free NNAL的部分為60.04及23.97 (pg/mg Cre)，且此差異達到統計上的顯著意義(p=0.004)。而從此結果可以合理的懷疑，由於CYP2As酵素活化，使NNK的α-hydroxylation上升，導致NNAL的排出量下降。從NNK的解毒效果的指標NNAL-Gluc/free NNAL ratio來看，純吸菸者為4.73 (1.8~7.66)，抽菸且嚼食檳榔者為3.44 (2.45~4.43)，明顯的較純吸菸者來的低，因此可以合理的懷疑，檳榔中有物質會影響到NNK在體內的解毒途徑，可能會增加NNK的毒性。
影響人體代謝的物質很多，且不同個體的代謝速率差異很大，因此要從單一物質來探討對人體代謝的影響是很困難的。另外此實驗並沒有將受測者的吸菸量及吸菸習慣(如菸品的濃淡、吸菸的深度)加入探討及校正，因為這些條件很直接的會影響到人體中的尼古丁的含量，因此也會很直接的影響到尿液中NNAL的含量，若能將這些條件也加入進行校正，實驗數據可能會更加精準。

According to recent estimates, there are about 3.5 million smokers in Taiwan. Statistical data have shown that more than 18,800 deaths are attributed to smoking-related diseases in Taiwan annually and 16% of all deaths are smoking-related. In addition, there are about 2.5 million betel quid chewers in Taiwan, and 94% of betel quid chewers are also cigarette smoker. 4-(Methylnitro samino)-1-(3 pyridyl)-1-butanone (NNK), derived from nicotine nitrosation, is a strong cigarette related carcinogen. NNKis metabolized either though CYP2As to reactive metabolites and bind to DNA or reduced to 4-(Methylnitrosamino)-1- (3-Pyridyl)-1- Butanol (NNAL) and conjugated by phase II enzyme and is finally excreted in urine. Betel quid contain many substances, including safrole, which have been reported to induce CYP2As enzyme in animal experiments. The data also indicated that the urinary NNAL levels were decreased following safrole treatment in hamster for 9 days. Nevertheless, whether this interaction happens in human has yet to be identified.
The aim of this study was to compare the NNAL levels in urine from smokers and smokers with betel quid chewing habits using isotope-dilution LC-MS/MS method. Because the content of NNAL in human urine can only be measured in parts per trillion (ppt) level, this experiment applied Solid Phase Extraction (SPE) to enrich the NNAL before analyses. The total NNAL in urine samples were analyzed following β-glucuronidase digestion and the free and total NNAL level were calibrated by the content of ceatinine (Cre) in urine.
In the 16 smoking only urine samples, the average concentration of total NNAL was 149.46 ng/mg Cre; however, in the 33 samples from betel quid chewing smokers, the average concentration of total NNAL was 85.97 ng/mg Cre. The difference of total NNAL contents between smokers and smokers also chewed betel quid is statistically significant. The average concentration of free NNAL in the smokers and smokers with betel quit chewing is 60.04 and 23.97 (pg/mg Cre). The difference of free NNAL contents between smokers and smokers also chewed betel quid is also statistically significant. The results could be reasonable doubt that the CYP2As enzyme was activated, so that the α-hydroxylation of NNK rose and leading to decreased the NNAL excretion. In addition, the ratio NNAL-Gluc to free NNAL is a useful biomarker for NNK detoxification in smokers. In this study, this NNAL-Gluc to free NNAL ratio in smokers was higher than smokers with betel quit chewing ( 4.73 v.s. 3.44). Therefore, it is reasonable to assume that betel quid components might affect NNK metabolism in vivo.
Many substances have potential to affect the metabolism in human body. As a result, it is difficult to discuss the metabolic rate of a single chemical across different subjects. Furthermore, this study did not consider specific smoking habits. For instance, what brand of tobacco, or how often they smoked. These conditions would directly affect the nicotine and NNAL levels in the human body. Only when these factors can be corrected, the experiment would be more accurate.

目錄
中文摘要…………………………………………………………………………VI
Abstract…………………………………………………………………………VIII
英文縮寫對照表……………………………………………………………………X
壹、緒論
檳榔………………………………………………………………………………1
香菸………………………………………………………………………………6
液相層析質譜儀…………………………………………………………………9
貳、實驗動機與目的
研究背景與動機………………………………………………………………13
研究目的………………………………………………………………………14
參、實驗材料與方法
實驗材料………………………………………………………………………15
實驗方法………………………………………………………………………18
肆、實驗結果
一、利用LC/MS/MS偵測尿中的NNK代謝產物NNAL…………………21
二、分析方法之驗證…………………………………………………………22
三、統計結果…………………………………………………………………22
伍、討論
一、利用LC/MS/MS偵測尿液中的NNAL…………………………………24
二、固項萃取管之比較………..………………………………………………25
三、香菸與檳榔間的交互作用探討…………………………………………26
四、抽菸與嚼食檳榔對於尿液中Total NNAL及Free NNAL的影響……27
五、有無嚼食檳榔對於尿液中Creatinine的影響……………………………30
六、基質效應 (Matrix effect)…………………………………………………31
七、吸菸與嚼食檳榔對於尿液中NNAL與DNA損傷之探討………………31
八、研究限制…………………………………………………………………34
陸、結論……………………………………………………………………………35
柒、參考文獻………………………………………………………………………36
實驗圖表……………………………………………………………………………43
附錄…………………………………………………………………………………56

圖目錄
Fig 1. NNAL的母離子在TIC掃描下的層析圖譜…………………………………43
Fig 2. 使用positive ion mode 進行NNAL 的full scan的層析圖………44
Fig 3. 水中的NNAL與內標之層析圖譜…………………………………………45
Fig 4. 尿中的NNAL與內標之層析圖譜 ………………………………………46
Fig 5. 水中NNAL之檢量線箱圖…………………………………………………47
Fig 6. 吸菸者與吸菸同時嚼食檳榔者Total NNAL之箱型圖……………………48
Fig 7. 吸菸者與吸菸同時嚼食檳榔者Free NNAL之箱型………………………49
Fig 8. 經MIP萃取後之層析圖譜…………………………………………………50

表目錄
Table 1. NNAL的精確度及準確度偵測結果………………………………………51
Table 2. 尿中Total及Free NNAL含量……………………………………………52
Table 3. Glucuronide /Free NNAL ratio及尿中Creatinine含量……………………53
Table 4. 吸菸者與有嚼食檳榔習慣之吸菸者尿中內標的peak area比較………54
Table 5. NNAL與N7-MethG及8-OhdG相關性比較………………………………55

1. 葛應欽, 台灣嚼時檳榔與健康, 1994: 屏東醫療網通訊.
2. Awang, M.N., Betel quid and oral carcinogenesis. Singapore Med J, 1988. 29(6): p. 589-93.
3. Wang, C.K. and M.J. Wu, The separation of phenolics from Piper betle leaf and the effect on the mutagenicity of arecoline. The Chinese Agricultural Chemical Society., 1996. 34(5): p. 638-647.
4. Branshaw, S.a., UPPER ALIMENTARY TRACT CANCER IN NATAL INDIANS WITH SPECIAL REFERENCE TO THE BETEL-CHEWING HABIT. 1969.
5. Nair, U.J., et al., Effect of lime composition on the formation of reactive oxygen species from areca nut extract in vitro. Carcinogenesis, 1990. 11(12): p. 2145-8.
6. Shirname, L.P., M.M. Menon, and S.V. Bhide, Mutagenicity of betel quid and its ingredients using mammalian test systems. Carcinogenesis, 1984. 5(4): p. 501-3.
7. Jeng, J.H., et al., Genotoxic and non-genotoxic effects of betel quid ingredients on oral mucosal fibroblasts in vitro. J Dent Res, 1994. 73(5): p. 1043-9.
8. J.Nair, H.O., M.Friesen, A.Croisy, S.V.Bhide and H.Bartsch, Tobacco-specific and betel nut-specific N-nitroso compounds: occurrence in saliva and urine of betel quid chewers and formation in vitro by nitrosation of betel quid. 1985.
9. Wenke, G. and D. Hoffmann, A study of betel quid carcinogenesis. 1. On the in vitro N-nitrosation of arecoline. Carcinogenesis, 1983. 4(2): p. 169-72.
10. Rosin, M.P., The influence of pH on the convertogenic activity of plant phenolics. Mutat Res, 1984. 135(2): p. 109-13.
11. Nair, U.J., et al., Formation of reactive oxygen species and of 8-hydroxydeoxyguanosine in DNA in vitro with betel quid ingredients. Chem Biol Interact, 1987. 63(2): p. 157-69.
12. Wang, C. and L. Hwang, phenolic compounds of betel quid chewing juice. Food Sci, 1993. 20: p. 458-471.
13. Bolton, J.L., N.M. Acay, and V. Vukomanovic, Evidence that 4-allyl-o-quinones spontaneously rearrange to their more electrophilic quinone methides: potential bioactivation mechanism for the hepatocarcinogen safrole. Chem Res Toxicol, 1994. 7(3): p. 443-50.
14. Miller, E.C., et al., Sulfuric acid esters as ultimate electrophilic and carcinogenic metabolites of some alkenylbenzenes and aromatic amines in mouse liver. Carcinog Compr Surv, 1985. 10: p. 93-107.
15. Jeng, J.H., et al., Reactive oxygen species are crucial for hydroxychavicol toxicity toward KB epithelial cells. Cell Mol Life Sci, 2004. 61(1): p. 83-96.
16. Nakagawa, Y., et al., Biotransformation and cytotoxic effects of hydroxychavicol, an intermediate of safrole metabolism, in isolated rat hepatocytes. Chem Biol Interact, 2009. 180(1): p. 89-97.
17. Borchert, P., et al., The metabolism of the naturally occurring hepatocarcinogen safrole to 1'-hydroxysafrole and the electrophilic reactivity of 1'-acetoxysafrole. Cancer Res, 1973. 33(3): p. 575-89.
18. Jeurissen, S.M., et al., Human cytochrome p450 enzyme specificity for bioactivation of safrole to the proximate carcinogen 1'-hydroxysafrole. Chem Res Toxicol, 2004. 17(9): p. 1245-50.
19. Ueng, Y.F., et al., Identification of the main human cytochrome P450 enzymes involved in safrole 1'-hydroxylation. Chem Res Toxicol, 2004. 17(8): p. 1151-6.
20. Ioannides, C., M. Delaforge, and D.V. Parke, Interactions of safrole and isosafrole and their metabolites with cytochromes P-450. Chem Biol Interact, 1985. 53(3): p. 303-11.
21. 施惠瀅, 檳榔嚼塊組成物影響香菸中NNK代謝物肢之體內研究, 2006, 國立陽明大學: 醫學院藥理學研究所.
22. Yamazaki, H., et al., Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes. Carcinogenesis, 1992. 13(10): p. 1789-94.
23. Schuller, H.M., Mechanisms of smoking-related lung and pancreatic adenocarcinoma development. Nat Rev Cancer, 2002. 2(6): p. 455-63.
24. Hecht, S.S., Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst, 1999. 91(14): p. 1194-210.
25. Hoffmann, D., I. Hoffmann, and K. El-Bayoumy, The less harmful cigarette: a controversial issue. a tribute to Ernst L. Wynder. Chem Res Toxicol, 2001. 14(7): p. 767-90.
26. Hecht, S.S., Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer, 2003. 3(10): p. 733-44.
27. Hecht, S.S., Human urinary carcinogen metabolites: biomarkers for investigating tobacco and cancer. Carcinogenesis, 2002. 23(6): p. 907-22.
28. Hecht, S.S., DNA adduct formation from tobacco-specific N-nitrosamines. Mutat Res, 1999. 424(1-2): p. 127-42.
29. van Zeeland, A.A., et al., 8-Hydroxydeoxyguanosine in DNA from leukocytes of healthy adults: relationship with cigarette smoking, environmental tobacco smoke, alcohol and coffee consumption. Mutat Res, 1999. 439(2): p. 249-57.
30. Hecht, S.S., Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem Res Toxicol, 1998. 11(6): p. 559-603.
31. von Weymarn, L.B., J.A. Chun, and P.F. Hollenberg, Effects of benzyl and phenethyl isothiocyanate on P450s 2A6 and 2A13: potential for chemoprevention in smokers. Carcinogenesis, 2006. 27(4): p. 782-90.
32. Messina, E.S., R.F. Tyndale, and E.M. Sellers, A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes. J Pharmacol Exp Ther, 1997. 282(3): p. 1608-14.
33. Yamazaki, H., et al., Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes. Arch Toxicol, 1999. 73(2): p. 65-70.
34. Goniewicz, M.L., et al., Comparison of urine cotinine and the tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and their ratio to discriminate active from passive smoking. Nicotine Tob Res, 2011. 13(3): p. 202-8.
35. Thomson, J.J., Rays of positive electricity, and their application to chemical analyses. 1913: London, Longmans.
36. Kloepfer, A., J.B. Quintana, and T. Reemtsma, Operational options to reduce matrix effects in liquid chromatography-electrospray ionization-mass spectrometry analysis of aqueous environmental samples. J Chromatogr A, 2005. 1067(1-2): p. 153-60.
37. Niessen, W.M.A., In Liquid Chromatography-Mass Spectrometry. Atmospheric pressure ionization., 2006: p. 141.
38. Kitamura, R., et al., Improvement in precision of the liquid chromatographic-electrospray ionization tandem mass spectrometric analysis of 3'-C-ethynylcytidine in rat plasma. J Chromatogr B Biomed Sci Appl, 2001. 754(1): p. 113-9.
39. Lindberg, R., et al., Determination of antibiotic substances in hospital sewage water using solid phase extraction and liquid chromatography/mass spectrometry and group analogue internal standards. Chemosphere, 2004. 57(10): p. 1479-88.
40. Wen, C.P., et al., Uncovering the relation between betel quid chewing and cigarette smoking in Taiwan. Tob Control, 2005. 14 Suppl 1: p. i16-22.
41. Shah, K.A., M.S. Halquist, and H.T. Karnes, A modified method for the determination of tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human urine by solid phase extraction using a molecularly imprinted polymer and liquid chromatography tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 2009. 877(14-15): p. 1575-82.
42. Chiang, H.C., et al., 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone is correlated with 8-hydroxy-2'-deoxyguanosine in humans after exposure to environmental tobacco smoke. Sci Total Environ, 2012. 414: p. 134-9.
43. Castonguay, A., et al., Metabolism and tissue distribution of tobacco-specific N-nitrosamines in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 1985. 6(11): p. 1543-50.
44. Morse, M.A., et al., Characterization of a glucuronide metabolite of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its dose-dependent excretion in the urine of mice and rats. Carcinogenesis, 1990. 11(10): p. 1819-23.
45. Hecht, S.S., et al., A tobacco-specific lung carcinogen in the urine of men exposed to cigarette smoke. N Engl J Med, 1993. 329(21): p. 1543-6.
46. Carmella, S.G., et al., Analysis of N- and O-glucuronides of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human urine. Chem Res Toxicol, 2002. 15(4): p. 545-50.
47. Byrd, G.D. and M.W. Ogden, Liquid chromatographic/tandem mass spectrometric method for the determination of the tobacco-specific nitrosamine metabolite NNAL in smokers' urine. J Mass Spectrom, 2003. 38(1): p. 98-107.
48. Benowitz, N., et al., Urine cotinine underestimates exposure to the tobacco-derived lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in passive compared with active smokers. Cancer Epidemiol Biomarkers Prev, 2010. 19(11): p. 2795-800.
49. Ko, Y.C., et al., Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med, 1995. 24(10): p. 450-3.
50. 黃惠慈, 香菸中致癌物NNK與黃樟素之倉鼠體內基因毒性探討, 2009: 國立陽明大學醫學院 環境與職業衛生研究所.
51. Muscat, J.E., et al., Racial differences in exposure and glucuronidation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Cancer, 2005. 103(7): p. 1420-6.
52. Tricker, A.R., et al., Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mouse lung and effect of cigarette smoke exposure on in vivo metabolism to biological reactive intermediates. Adv Exp Med Biol, 2001. 500: p. 451-4.
53. Zhang, X., et al., CYP2A13: variable expression and role in human lung microsomal metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. J Pharmacol Exp Ther, 2007. 323(2): p. 570-8.
54. Chiang, H.C., et al., Metabolic effects of CYP2A6 and CYP2A13 on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced gene mutation--a mammalian cell-based mutagenesis approach. Toxicol Appl Pharmacol, 2011. 253(2): p. 145-52.
55. Liu, J., et al., Global gene expression associated with hepatocarcinogenesis in adult male mice induced by in utero arsenic exposure. Environ Health Perspect, 2006. 114(3): p. 404-11.
56. Waalkes, M.P., J.M. Ward, and B.A. Diwan, Induction of tumors of the liver, lung, ovary and adrenal in adult mice after brief maternal gestational exposure to inorganic arsenic: promotional effects of postnatal phorbol ester exposure on hepatic and pulmonary, but not dermal cancers. Carcinogenesis, 2004. 25(1): p. 133-41.
57. Jalas, J.R., S.S. Hecht, and S.E. Murphy, Cytochrome P450 enzymes as catalysts of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco specific carcinogen. Chem Res Toxicol, 2005. 18(2): p. 95-110
58. Chiu, H.F., et al., Does arsenic exposure increase the risk for liver cancer? J Toxicol Environ Health A, 2004. 67(19): p. 1491-500.
59. Lee, H.L., et al., Enhancements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism and carcinogenic risk via NNK/arsenic interaction. Toxicol Appl Pharmacol, 2008. 227(1): p. 108-14.
60. Ono, S., et al., Specificity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica, 1996. 26(7): p. 681-93.
61. Draper, A.J., A. Madan, and A. Parkinson, Inhibition of coumarin 7-hydroxylase activity in human liver microsomes. Arch Biochem Biophys, 1997. 341(1): p. 47-61.
62. Takeuchi, H., et al., Pretreatment with 8-methoxypsoralen, a potent human CYP2A6 inhibitor, strongly inhibits lung tumorigenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in female A/J mice. Cancer Res, 2003. 63(22): p. 7581-3.
63. Miyazaki, M., et al., Mechanisms of chemopreventive effects of 8-methoxypsoralen against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung adenomas. Carcinogenesis, 2005. 26(11): p. 1947-55.
64. Takeuchi, H., et al., 8-Methoxypsoralen, a potent human CYP2A6 inhibitor, inhibits lung adenocarcinoma development induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in female A/J mice. Mol Med Rep, 2009. 2(4): p. 585-8.
65. Kresty, L.A., et al., Metabolites of a tobacco-specific nitrosamine, 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK), in the urine of smokeless tobacco users: relationship between urinary biomarkers and oral leukoplakia. Cancer Epidemiol Biomarkers Prev, 1996. 5(7): p. 521-5.
66. Chung, C.J., et al., Low ratio of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol-glucuronides (NNAL-Gluc)/free NNAL increases urothelial carcinoma risk. Sci Total Environ, 2011. 409(9): p. 1638-42.
67. Rostron, B., NNAL exposure by race and menthol cigarette use among U.S. smokers. Nicotine Tob Res, 2013. 15(5): p. 950-6.
68. Lee, H.L., et al., Correlation between the urine profile of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolites and N7-methylguanine in urothelial carcinoma patients. Cancer Epidemiol Biomarkers Prev, 2008. 17(12): p. 3390-5.
69. Wu, K.D., et al., The milk-alkali syndrome caused by betelnuts in oyster shell paste. J Toxicol Clin Toxicol, 1996. 34(6): p. 741-5.
70. Yiang, G.-T., et al., The milk-alkali syndrome caused by chewing betel nuts. J Med Sci, 2000. 20: p. 429-434.
71. Lin, S.H., et al., Hypercalcaemia and metabolic alkalosis with betel nut chewing: emphasis on its integrative pathophysiology. Nephrol Dial Transplant, 2002. 17(5): p. 708-14.
72. Pilger, A., et al., Longitudinal study of urinary 8-hydroxy-2'-deoxyguanosine excretion in healthy adults. Free Radic Res, 2001. 35(3): p. 273-80.
73. Chuang, C.Y., et al., Oxidative DNA damage estimated by urinary 8-hydroxydeoxyguanosine: influence of taxi driving, smoking and areca chewing. Chemosphere, 2003. 52(7): p. 1163-71.
74. Chao, M.R., et al., Rapid and sensitive quantification of urinary N7-methylguanine by isotope-dilution liquid chromatography/electrospray ionization tandem mass spectrometry with on-line solid-phase extraction. Rapid Commun Mass Spectrom, 2005. 19(17): p. 2427-32.
75. 廖芸儹, 嚼食檳榔與吸菸造成氧化性傷害之研究, 2008, 國立陽明大學 環境與職業衛生研究所.
76. Dietz, B.M. and J.L. Bolton, Biological reactive intermediates (BRIs) formed from botanical dietary supplements. Chem Biol Interact, 2011. 192(1-2): p. 72-80.
77. Maser, E., Significance of reductases in the detoxification of the tobacco-specific carcinogen NNK. Trends Pharmacol Sci, 2004. 25(5): p. 235-7.