2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38046Humic acids are naturally occurring biopolymers that are frequently found in the soil, drinking water and a variety of plants. It has been suggested that humic acid is a potential toxin, can generate reactive oxygen species (ROS) and to induce oxidative damage in many animal/human cell cultures. Humic acid is also a possible etiological factor for blackfoot disease. However, humic acid is a complex substance and its composition varies from region to region. Most previous studies conducted their experiments using a commercial product of HA or synthetic compound, little work was ever done with a fractionated product of HA. Therefore, it would be useful to identify what compound of HAs is the major cause of oxidative stress by using the fractionated HA. 8-hydroxy-2’-deoxyguanosine (8-OHdG) is one of the major oxidative DNA damage formed by radical-induced damage at C-8 position of guanine and has been regard as the biomarker of oxidative stress. Several methods have been applied for analysis of 8-OHdG, such as high performance liquid chromatography with electrochemical detection (HPLC-ECD), gas chromatography with mass spectrometry (GC-MS), liquid chromatography with tandem mass (LC-MS/MS), and enzyme-linked immunosorbent assay (ELISA). Among these methods, LC-MS/MS has a high sensitivity and selectivity, and can be applied with the isotope-dilution method, such analytical technique is the most accurate and reliable analytical method. However, it was noted that many previous efforts on the determining of 8-OHdG induced by HA was made by ELISA rather than LC-MS/MS. In the present study, an isotope dilution LC-MS/MS method will be developed for the quantification of 8-OHdG. By using this method, we are going to investigate: 1. the oxidative DNA damage in cell/animals caused by a variety sources of HA, 2. the interaction between HA and metals (i.e. As), 3. which compounds of HA are responsible for the oxidative DNA damage by cooperating with a suitable fractionation method. When a representative metabolite of HA can be identified, we will try to demonstrate a suitable and specific DNA adduct for assessing the HA exposure.腐植酸(humic acid)是來自於植物體或動物體的殘留物，經由化學作用和生 物作用之分解及合成所產生的一種複雜的天然化合物，普遍存在於土壤， 植物，井水等。過去的研究發現腐質酸含有多酚類之物質可能會產生自由， 進而攻擊核酸、蛋白質和造成脂質過氧化，同時也被懷疑與烏腳病的致病 機轉有關。先前大部分的研究係運用腐植酸的粗萃物進行實驗，然而腐植 酸之成分結構複雜並且隨氣候及地理位置不同其成分變異也大，因此各學 者的研究成果難以互相驗證。因此，若能藉由適當的分離技術(fractionation method)找出容易會導致基因氧化傷害的腐植酸成份將更能幫助我們了解其 作用機制。 液相層析串聯質譜儀(LC-MS/MS)配合同位素稀釋法(isotope dilution) 是公 認最精準的定量方法，對於8-OHdG 定量具高特異性及敏感性，同時樣品 前處理簡單。本研究的主旨乃是運用液相層析串聯質譜儀(LC-MS/MS)配合 同位素稀釋法，建立不同生物樣本8-OHdG 分析技術。接著將此分析法運 用於各種生物樣本分析，藉以探討以下問題：1.各種不同環境來源的腐植酸 粗萃物所造成細胞或動物器官的基因氧化傷害。2.探討腐植酸與金屬間的交 互作用是否提升基因的氧化傷害。3.搭配分離方法(fractionation method)的建 立，試圖探知腐植酸之化學特性與基因氧化傷害之關連性。未來若能找出 腐植酸的特定代謝物，則針對腐植酸合成特定DNA 鍵結物(specific DNA adduct)，以作為腐植酸暴露之有效生物指標。Project