| 摘要: | 生物有效性 (bioavailability)是毒學的重要觀，用以評估污染物經由同途徑，於一段時間後，目標污染物進入血液循環系統的劑與總劑的比，稱為絕對生物有效性係(absolute bioavailability factor, ABF)，將該物質與某一標準物質者之 ABF相除，即為該物質之相對生物有效性係(relative bioavailability factor, RBF)。本研究乃考USEPA Region VIII，研擬一種模擬腸胃環境的階段生物有效性體外試驗(in-vitro test)程序。第一階段模擬胃消化環境，加入 0.15 M NaCl 及 1% porcine，於 pH 1.8萃取 1hr；第二階段模擬腸吸收環境，加入 3.5 gm╱L bile 及 0.35 gm╱L pancreatin，但使用吸收劑，於pH 5.5 萃取 1hr。本研究使用砷酸鈉(Na2HAsO47H2O)及美國 NIST 2710 土壤為標準物質，以建系統品管基準，並探討操作：氧化還原電位(ORP)、蠕動強、液固比，期能將評估程序應用於玩具、牙材、中藥、食品等之健康風險評估。本研究首先於0.5 L 密閉容器下以磁石進攪拌，餵入樣品時控制厭氧，進胃腸相ORP 監測，結果顯示種標準物質胃相 ORP 分別為 490 及 448 mv，腸相分別為 256 及 225mv，明未進厭氧控制，將高估人體腸胃相 ORP，唯推應會導致 RBF 之誤差。進一步探討蠕動對生物有效性的影響，種標準物質均加入0.5 gm (1.0 gm╱L)進階段萃取，當液固比為1000 mL╱gm 時，於三種速梯(G=0, 470, 1006 sec-1)下，砷酸鈉胃及腸相ABF 為 94 ~ 99 %，標準土壤 RBFG 為 31 ~ 42 %，RBFI 為 28 ~37 %，於信賴水準 95%下，僅NIST 2710 胃相之 ABF 有顯著差(p=0.048) ，但一般而言，當 pH 較低時， G 值對 ABF 影響應會較小，但本研究之胃相有顯著性差之可能結果為實驗誤差所造成，因此G 似乎是一個重要的實驗條件。本研究仍建議 G 值暫訂為 470 sec-1。進一步探討於G 為 470 sec-1 時，三種液固比(200, 1000, 5000 mL╱gm)對生物有效性的影響，結果顯示胃及腸相砷酸鈉ABF 為 80 ~ 119 %，標準土壤 RBFG 為 27 ~ 34 %，RBFI 為22 ~ 33 %。標準土壤於腸相和胃相時均無顯著性差，可能原因為樣品 NIST 2710 soil 本身包封較高，因此在pH 較低之胃相或 pH 較高之腸相，對溶解之影響大，故液固比對砷ABF 之影響程較小，所以液固比在胃相中似乎是重要的試驗。本研究仍建議液固比為1000 mL╱gm。綜合本研究砷之生物有效性研究實證結果，初擬三種系統品管基準：空白管制樣品濃相對於實測質之貢獻小於10%；標準砷酸鈉胃腸相之 ABF 平均值與標準偏差為 100 ±10%，NIST 標準土壤之 RBFG 為 35 ± 6%，RBFI 為 30 ± 4 %。
Bioavailability is an important concept in toxicology. The absolute bioavailability factor (ABF) is defined as the portion of a target toxicant entering the blood circulation system via a specific pathway in a certain period of time after exposure. To avoid inaccurate measurement, the relative bioavailability factor (RBF) is normally used in practice and is defined as the ratio of the ABF of the test material to the ABFof a surrogate reference material (SRM). In reference of the USEPA Region VIII’s method, this study proposed a two-step in-vitro bioavailability test for soil materials and the like. In the first step, 0.15 M NaCl and 1% porcine were used as the stomach liquid at a pH of 1.8 for 1 hour of extraction, while in the second step, 3.5gm╱L bile and 0.35gm╱L pancreatin were used as the intestinal liquid at a pH of 5.5 for 1 hr. Sodium arsenate (Na2HAsO47H2O) and NIST 2710 Montana soil were used as the SRM and matrix standard, respectively, for developing test criteria of oxidation reduction potential (ORP), peristalsis intensity, liquid to solid (L╱S) ratio. The in-vitro test was first continuously monitored in a sealed vessel of 0.5 L after feeding for ORP during the extraction. ORPs were obtained from the tests on the two standard materials were 490 and 448 mv for the stomach phase and 256 and 225 mv for the intestinal phase, respectively. Comparing with the ORP data (150 and -50 mv) reported for the human gastrointestinal system, the ORP values appeared to be overestimated because it was not intended to remove oxygen from air in this study. The two-step procedure was evaluated at three levels of mixing intensity (G = 0, 470, 1006 sec-1) using a dosage of 0.5 gm (1.0 mg╱L) and a L╱S ratio of 1000 mL╱gm for the two standard materials. The ABF obtained from both phases of the test ranged 94 ~ 99 % for the SRM; and the RBFG and RBFI ranged 31 ~ 42 % and 28 ~ 37 %, respectively, for the standard soil materials. At a confidence level of 95 %, only the ABF obtained from the stomach phase for the NIST 2710 was significantly different (p=0.048) at the three G values under study. However, in general, it should be easier to break the encapsulation at the lower pH value and reduce the mixing effect on the ABF value in the stomach phase. We therefore suggested that mixing intensity was not a key parameter under the test conditions in this study. We proposed a G value of 470 sec-1 to be used for the mixing intensity. Three L╱S ratios (200, 1000, 5000 mL╱gm) were also evaluated at a constant G value of 470 sec-1. The ABF obtained from both test phases ranged 80~ 119 % for the SRM; and the RBFG and RBFI ranged 27 ~ 34 % and 22 ~ 33 %, respectively, for the standard soil material. The three RBFI values were no significantly different with respect to the L╱S values for both the intestinal and stomach phase. It was speculated that the degree of encapsulation was too high to make the difference in ABF at the three different L╱S ratios for both standard material. We proposed a L╱S ratio of 1000 mL╱gm to be used. Based on the results of this study, a set of quality control (QC) criteria were proposed for the in-vitro test: the concentration of the target toxicant extracted from the reagent blank should not contribute 10% or more to the sample tests, an average ABF of 100 ± 10 % for SRM for both phases, and an average RBFG of 35 ± 6 % and RBFI of 30 ± 4 % for the NIST soil. |
