Brüggemann O, Meder M, Freitag R
Institut für Technische Chemie, Universität Hannover, Germany.
J Chromatogr A. 1996 Sep 13;744(1-2):167-76. doi: 10.1016/0021-9673(96)00173-2.
Over 90% of the lethal cases of mushroom toxin poisoning in man are caused by a species of amanita. The amatoxins (especially alpha- and beta-amanitin) found in amanita deserve special attention, because of their high pharmacological potency, their high natural concentration and their high chemical and thermal stability. Measures can be taken to improve the survival rates (aggressive gastroenteric decontamination, liver protection therapy) if the poisoning is diagnosed correctly and as early as possible. The standard assay for alpha-amanitin is a radioimmunoassay (RIA). Among other reagents, this assay uses 125I-labelled alpha-amaintin, which has a low shelf life. The assay is therefore not available at all hospitals and all year round. In this paper, a first attempt to employ capillary zone electrophoresis (CZE) to quantify amatoxins alpha- and beta-amanitin in urine samples of afflicted patients and in toadstool extracts is described. Diode array detection is used for identification of the resolved substances in the electropherogram. An analysis requires 20 min. The detection limit is 1 microgram/ml, i.e., 5 pg absolute. Relative standard deviations are between 1 and 2% for the calibration standards (peak height and area) and ca. 7.5% for the real samples. Advantages of the CZE over the RIA include lower cost, the possibility of quantifying several toxins in one analysis, less consumption of potentially harmful reagents (no radio-labelled substances, no addition of alpha-amanitin as reagent) and, most importantly, all-year-round availability of the assay. The detection limit is still somewhat high and does not cover the entire clinically relevant range. Attempts to lower the detection limit by the necessary order of magnitude are currently under way in our laboratory. These include application of laser-induced fluorescence detection, liquid chromatography-CZE and CZE-mass spectrometry techniques.
人类因蘑菇毒素中毒致死的病例中，超过90%是由一种鹅膏菌属物种引起的。鹅膏菌中发现的鹅膏毒素（尤其是α-和β-鹅膏毒肽）值得特别关注，因为它们具有很高的药理活性、很高的天然浓度以及很高的化学和热稳定性。如果中毒能被正确且尽早诊断，就可以采取措施提高存活率（积极的胃肠去污、肝脏保护治疗）。α-鹅膏毒肽的标准检测方法是放射免疫测定法（RIA）。该检测方法除其他试剂外，使用半衰期较短的125I标记的α-鹅膏毒肽。因此，并非所有医院全年都能进行该检测。本文描述了首次尝试采用毛细管区带电泳（CZE）对中毒患者尿液样本和毒蘑菇提取物中的α-和β-鹅膏毒肽进行定量分析。二极管阵列检测用于识别电泳图中分离出的物质。一次分析需要20分钟。检测限为1微克/毫升，即绝对量为5皮克。校准标准品（峰高和峰面积）的相对标准偏差在1%至2%之间，实际样品的相对标准偏差约为7.5%。CZE相对于RIA的优势包括成本更低、一次分析可对多种毒素进行定量、潜在有害试剂的消耗量更少（无放射性标记物质，无需添加α-鹅膏毒肽作为试剂），最重要的是该检测方法全年可用。其检测限仍然有点高，无法覆盖整个临床相关范围。我们实验室目前正在尝试将检测限降低到必要的数量级。这些尝试包括应用激光诱导荧光检测、液相色谱-CZE和CZE-质谱技术。
