以質譜法分析 (一)人類尿液中五個DNA加成產物 (二)乙二醛誘導形成的DNA交聯產物 (三)從採血紙上萃取血紅蛋白上乙二醛與甲基乙二醛修飾程度的穩定性

Analysis of (1) 5 DNA adducts in human urine, (2) glyoxal-induced DNA cross-links, and (3) stability of glyoxal- and methylglyoxal- induced modifications in hemoglobin from DBS by LC-MS/MS

鄧儀君

指導教授 ： 陳皓君

國立中正大學/理學院/化學暨生物化學系/碩士(2016年)

摘要

人類生活環境中充滿了許多對人體具有危害性的反應物質。這些化學物質進入人體中會與DNA反應造成DNA的損傷，進而產生DNA加成產物，甚至演變癌症。我們發展出穩定同位素稀釋法搭配奈升流速液相層析奈電噴灑游離串聯質譜法分析這五種DNA加成產物。目前已測量了9個抽菸的食道癌患者尿液樣品以及7個抽菸的正常人尿液樣品，人類尿液樣品中每個所測得的DNA加成產物含量，經由Mann Whitney U-test統計計算後，發現在抽菸者中cotinine以及7-EtG的含量相較於食道癌病患都有明顯的增加。希望藉此分析方法比較出εCyt、εAde、8-OH-dG、 3-EtA及7-EtG與癌症的相關性，以此做為癌症風險評估的指標。
文獻指出乙二醛和甲基乙二醛在糖尿病患者體內容度高於正常人，且他們又是誘導性突變物質，會與生物分子反應。其中，乙二醛會與DNA反應造成DNA交聯產物，如dG-gx-dA和dG-gx-dC。在此研究，我們利用奈升流速效能液相層析奈電噴灑串聯式質譜法 (nanoLC-NSI/MS/MS)，分析15個糖尿病患者與15個正常人血液中交聯產物含量，發現糖尿病患者體內dG-gx-dA或dG-gx-dC的含量相較於正常人都有明顯的增加。
乙二醛與甲基乙二醛是具有反應性的α-雙羧基醛，會與蛋白質發生反應，造成蛋白質胺基酸殘基的修飾。DBS收集血液的方式與傳統的靜脈注射相比，多了許多的優點，為了確保使用DBS保存糖尿病患者與正常人血液時，血液中乙二醛與甲基乙二醛轉譯後修飾的穩定度，我們將DBS保存在室溫與冰箱(4℃)中，且每隔一周分析樣品中修飾的含量，進而得到DBS保存的最佳條件。結果顯示，在室溫下以及4度C下時，這些轉譯後修飾分別在14天以及21天內是穩定的。

關鍵字

人類尿液 ； DNA加成產物 ； DNA交聯產物 ； 乙二醛 ； 甲基乙二醛 ； 採血紙 ； 血紅蛋白

並列摘要

Exposure to environmental exogenous chemicals and endogenous reactive species in human can lead to the formation of structurally modified DNA bases (DNA adducts), which play a role key on multi-stage carcinogenesis process, and are the initial step of carcinogenesis. If DNA adducts haven’t been repaired by our body, these modified DNA bases could mispair by base to base, and lead to mutation during DNA replication, and develop into cancer eventually. A highly specific and sensitive assay based on stable isotope dilution nanoLC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was used to measureεCyt, εAde, 8-OH-dG, cotinine, 3-EtA and 7-EtG in human urine. We can analyze five adducts and cotinine at the same time more easily by using stable isotope dilution nanoLC-NSI/MS/MS. This highly specific and sensitive analysis should be clinically useful in assessing the possibility of measuring adducts in human urine as biomarkers in cancer risk assessment.
Glyoxal (gx) is an α-dicarbonyl species widely distributed in foods and the environment. It is also derived endogenously from oxidation of lipids and nucleic acids and the metabolism of carbohydrates. It has been shown that the levels of glyoxal in diabetes mellitus patients are higher than in non-diabetics. Glyoxal reacts with biomolecules, causing cross-links of proteins and DNA. The cross-linked 2′-deoxyribonucleoside products of glyoxal with have been characterized as dG-gx-dA and dG-gx-dC. In this study, we have developed a highly sensitive and quantitative assay for simultaneous detection and quantification of dG-gx-dA and dG-gx-dC cross-links by stable isotope dilution nanoflow performance liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selective reaction monitoring (H-SRM) mode. Levels of dG-gxdC and dG-gx-dA in 15 diabetes mellitus patients’ leukocyte DNA are higher than those in 15 non-diabetics.
Advantages of using dried blood spot (DBS) samples—compared with venipuncture—include the relative low amount of blood, low cost of sample collection, transport, and storage. The reactive α-dicarbonyl aldehydes glyoxal (gx) and methylglyoxal (Mgx) react with proteins, lipids and nucleic acids, resulting in the formation of advanced glycation end products (AGEs). Particularly, the levels of glyoxal and methylglyoxal in diabetes mellitus patients are higher than in non-diabetics. Briefly, hemoglobin was extracted from the DBS cards stored at 4℃ (in the refrigerator) and at room temperature (in a dry box). After 1, 7, 14, 28, and 42 days, the extents of these modifications were quantified by nanoflow LC nanospray ionization tandem mass spectrometry (nanoLCNSI/MS/MS) after trypsin digestion. The results showed that these PTMs are stable for 14 days at room temperature (except glyoxal-α-Lys-11 and methylglyoxal-β-Lys-66) and 21 days at 4℃.

並列關鍵字

human urine ； DNA adduct ； DNA cross-link ； glyoxal ； methylglyoxal ； DBS ； hemoglobin

參考文獻

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