Wong T K, Blanton T E, Hunnicutt C K, Everson R B
Placenta. 1985 Jul-Aug;6(4):297-310. doi: 10.1016/s0143-4004(85)80039-4.
Several issues regarding measurement of placental AHH and 7ECD activity were studied, and standardized procedures that appeared more suitable than previous assay procedures for measurement of MFO induction in epidemiological studies were adopted. In the AHH assay, deletion of the rat-liver supernatant eliminated a possible extraneous contribution to measurement of low levels of AHH activity and did not substantially affect measurement of higher levels of activity. Increasing the protein concentration of placentae homogenate from 2 to 6 mg, and the length of the incubation time from 20 to 60 min, allowed for accumulation of more BaP products, potentially maximizing the detection of low levels of AHH activity. Use of tissue homogenates made the procedure more convenient and did not appear to interfere with interindividual comparisons of activity. Assay of homogenates of fresh and frozen tissue from the same placenta gave similar results, so that frozen tissue was adopted for convenience and replicability. Although a potential problem for specimens with high AHH activity, degradation of product(s) was modest in AHH assays of human placenta. The efficiency of extraction of fluorescent products declined with increasing protein concentrations in the reaction mixture of AHH assays, but it was stable for a range of product concentrations, and could be controlled by the use of a constant amount of protein per assay. Recovery of product for the 7ECD assay was more complete and was not affected by protein concentration. Additionally, the 7ECD activity was easily detected in every placenta, regardless of smoking exposure. However, in this study the AHH assay appeared to be better at discriminating between non-smokers and smokers. These observations, and the potential differences in the spectrum of agents causing induction of mixed-function oxidases, suggest that both assays are potentially useful measures of human MFO induction in clinical or epidemiological studies.
对胎盘芳烃羟化酶（AHH）和7-乙氧基香豆素-O-脱乙基酶（7ECD）活性测量的几个问题进行了研究，并采用了比以往检测方法更适合流行病学研究中测量混合功能氧化酶（MFO）诱导的标准化程序。在AHH检测中，去除大鼠肝脏上清液消除了对低水平AHH活性测量可能的额外影响，并且对较高水平活性的测量没有实质性影响。将胎盘匀浆的蛋白质浓度从2mg增加到6mg，孵育时间从20分钟延长到60分钟，使得更多苯并[a]芘产物得以积累，可能最大限度地检测到低水平的AHH活性。使用组织匀浆使该程序更方便，并且似乎不干扰个体间活性的比较。对来自同一胎盘的新鲜和冷冻组织匀浆的检测给出了相似的结果，因此为方便和可重复性采用了冷冻组织。虽然对于具有高AHH活性的标本存在一个潜在问题，但在人胎盘的AHH检测中产物的降解程度适中。在AHH检测的反应混合物中，荧光产物的提取效率随着蛋白质浓度的增加而下降，但在一定范围的产物浓度内是稳定的，并且可以通过每次检测使用恒定数量的蛋白质来控制。7ECD检测中产物的回收率更完全，并且不受蛋白质浓度的影响。此外，无论是否接触吸烟，在每个胎盘中都很容易检测到7ECD活性。然而，在本研究中，AHH检测在区分非吸烟者和吸烟者方面似乎更好。这些观察结果以及导致混合功能氧化酶诱导的试剂谱的潜在差异表明，这两种检测方法在临床或流行病学研究中都是测量人类MFO诱导的潜在有用方法。
