黃耀輝臺灣大學：職業醫學與工業衛生研究所許家晴Hsu, Chia-ChinChia-ChinHsu2007-11-282018-06-292007-11-282018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/59872在評估職場的低濃度無機砷暴露時，作為生物指標的尿中砷物種如三價砷、五價砷及其代謝物單甲基砷酸、雙甲基砷酸等，易受到食入含有有機砷之海產後所產生的部份相同代謝物的干擾，如雙甲基砷酸等。且近年來發現有機砷代謝時的中間產物，包括三價的單甲基砷酸及雙甲基砷酸，可能具有細胞毒性。因此需進一步了解攝食含有有機砷食物後尿中砷物種的分佈情形，協助釐清其對無機砷暴露評估的干擾。 本次研究徵求未受無機砷暴露之21名受試者進行飲食控制實驗，並以問卷收集基本資料。實驗期共七天半，受試者在實驗期間規律作息，並統一供餐以避免攝食含有有機砷的食物。在第四天晚餐提供定量牡蠣以作為有機砷之暴露。除了第四天外，其他時間均避免食入海產，並以問卷詳細記錄全天飲食。實驗期第一至第三天收集早晨初尿以監測是否尿中砷物種濃度已降至背景值，之後四天半收集早晨初尿和傍晚各一次尿液，來觀察尿中砷代謝的趨勢。以感應耦合電漿質譜儀量測尿中總砷量，尿中砷物種則使用高效能液相層析儀（HPLC）串聯感應耦合電漿質譜儀(ICP-MS)進行分析。在HPLC中，使用陰離子交換層析管柱分析AsIII、AsV、MMA、DMA四種砷物種，以及使用陽離子交換層析管柱分析arsenobetaine（AsBe）、arsenocholine（AsCho）、trimethylarsine oxide（TMAO）、tetramethylarsonium ion（TetMA）等另外四種砷物種。 結果顯示，攝入有機砷後，尿中代謝物以DMA和AsBe為主，還有AsIII、MMA、TMAO和TetMA，以及一個未知的砷物種，推測可能是AsSug群或dimethylarsinoylethanol、dimethylarsinoylacetate其中的一種。另外，AsV和AsCho則較少在尿中被偵測到。 在受試者具有相同的生活作息下，受試者尿中DMA濃度在攝食牡蠣後的12~36小時達到高峰期，顯示大部分的受試者能快速地代謝排除DMA；但尿中DMA濃度回降的速率較緩，約在高峰值出現後48小時才降回相對低點。除了牡蠣中原本的DMA外，同時偵測到的五種未知砷物種中也應包括部分的AsSug，代謝後也會使尿中DMA濃度增加。 大部分受試者尿中的AsBe濃度在36小時後出現高峰值，尿中AsBe濃度的上昇及下降速率相當，但有3名受試者在食入牡蠣後，AsBe濃度並無明顯的變化趨勢。在考慮受試者性別、單位體重攝食牡蠣量、年齡等基本資料對攝食牡蠣後尿中砷物種濃度的影響，發現受試者的單位體重攝食牡蠣量是影響DMA、AsBe和總砷濃度重要的因子。 利用mixed model對單位體重攝食量（牡蠣重/受試者體重，g/kg）影響尿中總砷、DMA和AsBe的在尿中的變化趨勢進行模式預測後，發現此模式隨著單位體重攝食量增加1g/kg，會使DMA上昇17.1μg/g creatinine；若受試者為男性的情況下，此模式隨著單位體重攝食量增加1g/kg，會使總砷上昇73.3μg/g creatinine，或使AsBe上昇29.9μg/g creatinine。 雖然AsIII和MMA在尿中的濃度並不高，但在食入牡蠣後的實驗期間，AsIII和MMA之間具有顯著的相關。由於此次實驗所食用的牡蠣中含有2.6%的AsIII，因此尿中AsIII的來源可能和食入牡蠣中含有的AsIII有關，MMA則為AsIII的代謝物。 整體而言，攝食49.9±7.4g的牡蠣可能會造成尿中無機砷相關代謝物種（AsIII、MMA、DMA）在攝食後第一天濃度增加35.5±9.4μg/g creatinine，干擾對無機砷暴露情形的評估。同時，由研究結果可知，牡蠣中DMA和AsBe被攝食進入人體後的吸收與代謝速率不同，有個體性差異存在。When assessing low inorganic arsenic exposure, the urinary biomarkers, including arsenite(AsIII), arsenate(AsV), methylarsonic acid(MMA) and dimethylarsinic acid(DMA), might be interfered by the metabolites of organic arsenic contained in the ingested seafood. Besides, it was reported in recent years that the intermediate metabolites of organic arsenic, i.e., MMAIII and DMAIII, were concerned of their cytotoxicity. So it is important to explore the metabolism of organic arsenic by characterizing urinary arsenic species. Total 21 volunteers with no inorganic arsenic exposure were recruited in this study. During the experimental period of 7.5 days, participants were provided with daily meals by the study team to prevent eating any foods containing organic arsenic. From the 1st to the 3rd day, participants provided with their first morning voided urine samples for arsenic species determination in order to make sure that their urinary arsenic levels have been lowered down to the background levels. At dinner on the 4th day, participants had an aliquot of cooked oyster around 50 g. In the following 4.5 days, participants were asked to provid with both their first morning voided and the evening voided urine samples. Urinary arsenic species were determined with the high performance liquid chromatography-inductively coupled plasma-mass spectrometer (HPLC-ICP-MS), using anion-exchange column for AsIII, AsV, MMA and DMA speciation, and cation-exchange column for arsenobetaine(AsBe), arsenocholine(AsCho), trimethylarsine oxide(TMAO) and tetramethylarsonium ion(TetMA) speciation, respectively. Detected urinary arsenic metabolites after oyster ingestion included DMA, AsBe, AsIII, MMA, TMAO, TetMA and an unknown species. This unknown species was thought dimethylarsinoylethanol, dimethylarsinoylacetate or one of arsenosugars. AsV and AsCho were rarely detected in the urine samples. With the similar circadian daily activities, the participants’ DMA concentrations reached their maxima about 12~36 hours after oyster ingestion, and they took another 48 hours to decline and return to the respective backgrounds. Urinary DMA contents might be attributed to the original DMA in oyster and that metabolized from the AsSug contained in oyster. Most study participants’ maximum urinary AsBe levels occurred about 36 hours after oyster ingestion. The rates of AsBe increasing and decreasing between the participants’ respective backgrounds and maximum levels were equivalent. However, urinary AsBe levels of 3 study participants did not show any significant elevation during the study period. Urinary DMA, AsBe and the total arsenic levels were demonstrated significantly associated with the amount of ingested oyster per unit bodyweight (g/kg bodyweight). The trends of urinary DMA, AsBe and the total arsenic levels after oyster ingestion could be well predicted by using mixed model under given conditions. An increase of 17.7μg/g creatinine for DMA is expected after eating oyster at the dose of 1 g/kg bodyweight. If the participant is male, the total arsenic and AsBe may increase by 73.3 and 29.9μg/g creatinine, respectively, with oyster ingestion of 1 g/kg bodyweight. Urinary AsIII and MMA levels were found very low but highly correlated with each other after oyster ingestion. Because AsIII consisted of 2.6% of arsenic species in the study oyster, the urinary AsIII might originally come from this part, and the urinary MMA might also resulted from this part of AsIII as metabolite. In conclusions, 1st day after ingesting 49.9±7.4g of oyster, the urinary inorganic arsenic metabolites, including AsIII, AsV, MMA and DMA, might be significantly interfered by an increase of 35.5±9.4μg/g creatinine. The excretion rates of DMA and AsBe in urine were observed varying in the present study. So were the different individual susceptibility of the study participants for the organic arsenic metabolism.目 錄 第一章 前 言 7 1.1 研究背景 7 1.2 研究目的 8 第二章 文獻回顧 9 2.1 有機砷在生物體分佈的情形 9 2.2 生物體代謝有機砷的相關研究 16 2.2.1 動物實驗 16 2.2.2 人體試驗 16 2.2.3 其他影響有機砷物種的條件 22 2.3 毒性介紹 22 2.3.1 AsBe、AsCho、TMAO、TetMA、AsSug 22 2.3.2 DMAV 23 2.3.3 MMAIII、DMAIII 24 2.4 分析方法 25 2.5 HPLC-ICP-MS原理介紹 26 2.5.1 HPLC 26 2.5.2 ICP-MS 27 第三章 材料與方法 31 3.1 研究設計 31 3.2 研究對象 31 3.3 儀器參數 32 3.4 食物樣本收集及處理 36 3.4.1 樣本來源 36 3.4.2 食物樣本前處理 36 3.4.3 砷物種之測定 37 3.4.4 總砷之測定 37 3.5 尿液樣本收集及處理 37 3.5.1 尿液收集 37 3.5.2 尿液樣本前處理 37 3.5.3 尿中總砷分析 38 3.5.4 尿中砷物種分析 38 3.6 儀器設備 39 3.7 試藥與試劑 40 3.8 試藥配製 41 3.8.1 標準品 41 3.8.2 動相溶液 41 3.9 分析方法品質控制與保證 42 3.9.1 檢量線與偵測極限 42 3.9.2 食物及尿中砷物種檢量線及偵測極限 43 3.9.3 回收率 46 第四章 結 果 49 4.1 受試者基礎資料 49 4.2 食物中砷物種分布情形 49 4.3 受試者尿中砷代謝物種分佈之情形 50 4.3.1 DMA與AsBe 51 4.3.2 AsIII、MMA與As_x4 54 4.3.3 TMAO與TetMA 54 4.4 砷代謝物種分布之性別差異 55 4.5 早晨初尿與傍晚尿液樣本之砷物種分布差異 56 4.6 食入牡蠣後尿中砷物種濃度變化情形之模式預測 58 第五章 討 論 62 5.1 個體吸收AsBe之能力差異 62 5.2 尿中DMA來源及個體代謝DMA之能力差異 62 5.3 牡蠣砷物種分布與尿液中AsIII、MMA之關係 64 5.4 TMAO與TetMA尿中分布情形探討 64 5.5 尿中未知物種As_x4之推測 65 5.4 本次研究限制 66 第六章 結 論 67 第七章 參考文獻 68931962 bytesapplication/pdfen-US牡蠣代謝液相層析串聯感應耦合電漿質譜儀有機砷易感受性HPLC-ICP-MSSusceptibilityOyster metabolismOrganic arsenicthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59872/1/ntu-93-R91841020-1.pdf