| 摘要: | 為確保土壤及地下水資源的永續利用，世界各國大都訂有土壤重金屬的濃度標準。近年來，我國與歐美先進國家相繼提出污染場址健康風險評估，但在估算具包封度受污土壤健康風險時，其生物有效性(bioavailability)大都使用保守情境 (100 %被人體胃腸吸收)，導致復育成本增加。美國環保署訂定超級基金場址 (Superfund site)復育標準時，允許考量生物有效性，所謂絕對生物有效係數 (absolute bioavailability factor, ABF)是指污染物進入血液的劑量與暴露劑量的百分比，相對生物有效係數 (relative bioavailability factor, RBF)為待測樣品與標準樣品之ABF比值。若使用動物實驗評估，程序複雜且昂貴費時，因此美國於1992年開始發展一套模擬人體胃腸環境之體外試驗方法。本研究嘗試研擬我國之標準操作方法，建立系統品管基準，特別著重試驗設備與方法之簡化。
本研究參考美國環保署第VIII區生物有效性physiologically based extraction test (PBET)與前期研究體外試驗方法，第一階段模擬胃消化環境(pH 1.8)，第二階段模擬小腸環境 (pH 7.0)。文獻中對於體外試驗操作參數與程序，如樣品前處理、溫度、酵素添加量、胃腸相pH值與反應時間等參數已漸趨一致，本研究針對尚未深入探討之重要參數進行試驗，以砷酸鈉 (Na2HAsO4．7H2O)與標準土讓 (NIST 2710 soil)，探討蠕動強度、液固比、小腸相萃取時間以及萃取液前處理方式對生物有效性之影響。
本研究建議標準操作程序：首先將樣品經風乾且過篩取粒徑< 250 μm，取樣品量0.5 gm，使用密閉血清瓶，有效容積500 mL，且採用較易控制轉速之磁力攪拌，速度梯度 (G值)為470 sec-1，同時採用氣控式溫度37±0.5 oC；第一階段模擬胃消化環境，液固比1000:1 mL/gm，僅添加0.15 M NaCl及1 % porcine pepsin製備胃液，以HCl溶液調整至pH 1.8±0.1，反應1小時後，取萃取液經離心去除粒狀物後，以ICP-MS分析總砷濃度；第二階段為模擬小腸環境，僅添加pancreatin與bile，以飽合NaHCO3溶液將pH調整至7.0±0.1做為小腸液，反應1小時後，取樣經離心去除粒狀物後，以ICP-MS分析總砷濃度。
研究結果顯示，當液固比1000:1 mL/gm時，在95%信賴水準 (95% CI)時，三種速度梯度 (G=0, 470, 1000 sec-1)對生物有效性無顯著性差異，胃相與腸相之砷酸鈉ABF為82-96 %，標準土壤RBF分別為26-35 %與27-29 %。在三種液固比 (200:1, 1000:1, 5000:1 mL/gm)時，除NIST 2710 soil之腸相外，其餘均無顯著性差異，胃相與腸相砷酸鈉ABF為85-101 %，標準土壤RBF分別為29-33 %與25-29 %。在砷酸鈉與NIST 2710 soil之腸相萃取方面，以第1個小時為基準，第2-11小時平均濃度之相對誤差皆小於10 %，與前期研究相符，表示以1小時做為小腸相萃取時間是合宜的。萃取液前處理試驗中，砷酸鈉與NIST 2710 soil之胃與小腸相總砷濃度，95 % CI皆無顯著性差異。綜合上述結果，擬定四種品管基準：(1) 試劑空白砷濃度低於方法偵測極限；(2) 重複樣品管制，相對差異百分比為±10 %；(3) 砷酸鈉ABFG與ABFI平均值標準差分別為90±8 %與90±5 %；(4) 標準土壤RBFG與RBFI分別為35±2 %與30±3 %。
爲進一步簡化試驗方法，建議將開放式反應槽改為密閉式；使用可調速磁力攪拌及具載重環磁石，取代昂貴之氬氣攪拌；將水浴溫控改為氣控式，以利試驗觀察與採樣。此外，小腸相萃取不使用吸附劑，萃取液前處理利用高速離心取代壓阻較高之過濾方式。本研究二階段試驗之生物有效性皆低估動物試驗RBF，建議可將二階段胃腸體外試驗方法改為單相一階段生物有效性萃取(simplified bioaccessibility extraction, SBET)試驗方法。
In order to preserve underground water resource for sustainable use, most countries in the world have promulgated heavy metal standards for soil. Recently Taiwan and many other countries have proposed a variety of risk assessment meth-ods for site remediation. But most of the methods normally assume 100% bioavail-ability for contaminated soils. The US Environmental Protection Agency (USEPA) allows for a modification of bioavailability in the Superfund Program. The absolute bioavailability factor (ABF) is defined as the dose percentage of contaminant in soil entering the blood circulation system, while relative bioavailability factor (RBF) is defined as the ABF ratio in percentage between soil samples to the standard sample. Various in-vitro bioavailability test methods based on gastrointestinal physiology have been proposed since 1992. The aim of this study is to propose a standard test procedure for bioavailability focusing on establishing quality control (QC) criteria and simplifying test procedure and apparatus.
This study refers to the physiologically based extraction test (PBET) developed by USEPA Region VIII and the method developed in the early study by our group. The first phase of this method simulates stomach condition (pH 1.8) and the second phase simulates intestinal condition (pH 7.0). Many test parameters proposed in these methods have widely accepted in the literature for sample pretreatment, tem-perature, enzyme dose, pH, and reaction time. This study used sodium arsenate (Na2HAsO4‧7H2O) and NIST 2710 soil as the standards to investigate the effect of mixing intensity, liquid to solid (L/S) ratio, extraction time, and extract pretreatment methods for the determination of bioavailability.
Testing at a constant L/S ratio of 1000:1 mL/gm, all the ABFs (n = 5) obtained for the 2 phases from the 3 velocity gradients (G) of 0, 470, and 1000 1/sec had no significant difference (95 % CI) for both standards. The ABFs (n = 5) from both phases were 82-96 % for sodium arsenate and were 26-35% for the NIST soil. When the test was conducted at a constant G of 470 1/sec with 3 L/S ratios of 200:1, 1000:1, and 5000:1 mL/gm, only the ABFs obtained from the intestinal phase for the standard soil resulted in significant difference (p =0.02). The ABFs (n= 5) ob-tained from both phases were 85-101 % for sodium arsenate and were 25-33 % for the standard soil. The As concentrations extracted for both standards from the intes-tinal phase were within 10% of difference for 2-11 hours of extraction time based on the first hour of extraction. Thus 1 hour of extraction appears to be appropriate. Sample pretreatment using centrifugation with or without a follow-up filtration yielded no significant difference. In conclusion, we propose 3 types of QC criteria: (1) a concentration less than the method detection limit (MDL) for the reagent blank; (2) a ABFG and ABFI of 90±8 % and 90±5 % for sodium arsenate, respectively, (3) a RBFG and RBFI of 35±2 % and 30±3 % for NIST 7210 soil, respectively.
Based on the test results, this study proposes a standard procedure for the two-phase bioavailability test. The soil sample is first dried by air and sieved for the particle size of 250 μm or less. A 0.5 gram of sample is fed into a serum bottle with a liquid volume of 500 mL at a liquid to solid (L/S) ratio of 1000:1 mL/gm in the stomach phase. A velocity gradient (G) of 500 1/sec is provided using magnetic stir-ring and a temperature of 37±0.5 oC is maintained in a air-circulated incubator. The extraction liquid contains 0.15 M of NaCl and 1% of porcine pepsin and the pH is adjusted to 1.8±0.1 by HCl stock solution. After 1 hour of extraction, a sample of extract is taken and centrifuged to remove particulate matters. The supernatant is determined for total arsenic. In the intestinal phase, pancreatin and bile are added to the extract liquid and is adjusted to pH 7.0±0.1 by NaHCO3 stock solution. After I hour of extraction, a sample extract is taken and centrifuged for the measurement of arsenic.
In order to simply the method, we further propose using magnetic stirring with weighted ring in stead of argon sparging; using air-circulated incubator in stead of water bath for better observation and sample collection; using centrifuging in stead of filtration for extract pretreatment; using one phase of simplified bioaccessibility extraction (SBET) method instead of the two-phase method for a better estimate to the in-vivo method. |
