Bovee T F, Hoogenboom L A, Hamers A R, Traag W A, Zuidema T, Aarts J M, Brouwer A, Kuiper H A
State Institute for Quality Control of Agricultural Products (RIKILT-DLO), Wageningen, The Netherlands.
Food Addit Contam. 1998 Nov-Dec;15(8):863-75. doi: 10.1080/02652039809374723.
There is a strong need for the development of relatively cheap and rapid bioassays for the determination of dioxins and related compounds in food. A newly developed CALUX (Chemical-Activated LUciferase gene eXpression) bioassay was tested for its possible use to determine low levels of dioxins in bovine milk. Data show that this mammalian cell-based test is very sensitive for 2,3,7,8-substituted dioxins and related PCBs, thereby reflecting the relative potencies of these compounds in comparison to TCDD (TEF-values). The limit of detection was about 50 fg of TCDD. Furthermore, the response obtained with a mixture of dioxins was additive, in accordance with the TEF-principle. Milk fat was isolated by centrifugation followed by clean-up of the fat with n-pentane, removal of the fat on a 33% H2SO4 silica column, and determination of Ah receptor agonist activity with the CALUX-bioassay. An equivalent of 67 mg fat was tested per experimental unit, resulting in a limit of quantification around 1 pg i-TEQ/g fat. To investigate the performance of the method, butter fat was cleaned and spiked with a mixture of 17 different 2,3,7,8-substituted PCDD and PCDF congeners at 1, 3, 6, 9, 12 and 15 pg TEQ/g fat, as confirmed by GC/MS. In this concentration range, the method showed a recovery of TEQs around 67% (58-87%). The reproducibility, determined in three independent series showed a CV varying between 4% and 54%, with the exception of the sample spiked at 1 pg i-TEQ (CV 97%). The repeatability determined with the sample spiked at 6 pg i-TEQ/g showed a CV of 10%. Testing of 22 bovine milk samples, taken at different sites in The Netherlands, in the CALUX-assay showed combined dioxin and dioxin-like PCB levels equivalent to 1.6 pg TCDD/g fat (range 0.2-4.6). GC/MS analysis of these samples revealed an average level of 1.7 pg i-TEQ/g fat, varying between 0.5 and 4.7 pg i-TEQ/g fat. All five samples showing a GC/MS determined dioxin content of more than 2 pg i-TEQ/g fat gave a response in the CALUX-assay corresponding to more than 2 pg TCDD/g fat. These data clearly show that the CALUX-bioassay is a promising method for the rapid and low cost screening of dioxins in bovine milk.
迫切需要开发相对廉价且快速的生物测定方法来测定食品中的二恶英及相关化合物。对一种新开发的CALUX（化学激活荧光素酶基因表达）生物测定法进行了测试，以确定其是否可用于测定牛奶中的低水平二恶英。数据表明，这种基于哺乳动物细胞的测试对2,3,7,8 - 取代二恶英和相关多氯联苯非常敏感，从而反映了这些化合物相对于2,3,7,8 - 四氯二苯并 - 对 - 二恶英（TCDD，毒性当量因子值）的相对效力。检测限约为50 fg的TCDD。此外，根据毒性当量因子原理，二恶英混合物获得的响应是相加的。通过离心分离牛奶脂肪，然后用正戊烷净化脂肪，在33%硫酸硅胶柱上去除脂肪，并使用CALUX生物测定法测定芳烃受体激动剂活性。每个实验单位测试相当于67 mg的脂肪，定量限约为1 pg国际毒性当量/克脂肪。为了研究该方法的性能，对黄油脂肪进行净化，并添加17种不同的2,3,7,8 - 取代多氯二苯并二恶英和多氯二苯并呋喃同系物的混合物，添加水平为1、3、6、9、12和15 pg毒性当量/克脂肪，经气相色谱/质谱法确认。在此浓度范围内，该方法的毒性当量回收率约为67%（58 - 87%）。在三个独立系列中测定的重现性显示变异系数在4%至54%之间，除了添加1 pg国际毒性当量的样品（变异系数97%）。对添加6 pg国际毒性当量/克的样品测定的重复性显示变异系数为10%。对在荷兰不同地点采集的22份牛奶样品进行CALUX测定表明，二恶英和二恶英类多氯联苯的总含量相当于1.6 pg TCDD/克脂肪（范围为0.2 - 4.6）。对这些样品的气相色谱/质谱分析显示平均水平为1.7 pg国际毒性当量/克脂肪，在0.5至4.7 pg国际毒性当量/克脂肪之间变化。所有五个气相色谱/质谱测定的二恶英含量超过2 pg国际毒性当量/克脂肪的样品在CALUX测定中的响应对应于超过2 pg TCDD/克脂肪。这些数据清楚地表明，CALUX生物测定法是一种用于快速、低成本筛查牛奶中二恶英的有前景的方法。
