Vudathala Daljit, Smith Stephen, Khoo Lester, Kuhn David D, Mainous Mary E, Steadman James, Murphy Lisa
University of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology, New Bolton Center, Toxicology, 382 West Street Road, Kennett Square, PA, 19348, USA.
VA-MD College of Veterinary Medicine, Department of Biomedical Sciences & Pathobiology, Virginia Tech, 205 Duck Pond Drive, Blacksburg, VA, 24061, USA.
Toxicon. 2017 Nov;138:82-88. doi: 10.1016/j.toxicon.2017.08.005. Epub 2017 Aug 10.
Microcystins (MCs) are hepatotoxic cyanobacterial metabolites produced sporadically in aquatic environments under favorable environmental conditions. Affinity of these toxins to covalently bind with protein phosphatases poses a challenge in their detection. Lemieux oxidation to release 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), a common moiety to all MCs congeners, has been used in detection of these compounds, however a lack of sensitivity has limited the usefulness of the method. In this study, modifications of the oxidation and solid phase extraction procedures, combined with a sensitive LC/MS/MS (liquid chromatography/mass spectrometry) detection, have resulted in 25 ng/g method detection limits in both liver and plasma samples. Samples harvested from six fingerling channel catfish (Ictalurus punctatus) dosed intraperitoneally with a sublethal MC-LR dose of 250 μg/kg were analyzed, and microcystin concentrations ranging from 370 to 670 ng/g in plasma and 566-1030 ng/g in liver were detected. Similarly, 250 μg/kg nodularin-dosed channel catfish fish were found to contain 835-1520 ng/g in plasma and 933-1140 ng/g in liver. Detection of the toxins in serum and liver combined with the presence of histopathological lesions consistent with these hepatocellular toxin in exposed fish and no positive findings in the control fish demonstrates the usefulness of this analytical procedure for the diagnosis of suspected algal toxicity cases.
微囊藻毒素（MCs）是蓝藻产生的具有肝毒性的代谢产物，在有利的环境条件下于水生环境中偶尔产生。这些毒素与蛋白磷酸酶共价结合的亲和力给它们的检测带来了挑战。采用勒米厄氧化法释放2-甲基-3-甲氧基-4-苯基丁酸（MMPB），这是所有微囊藻毒素同系物的常见部分，已用于这些化合物的检测，然而灵敏度不足限制了该方法的实用性。在本研究中，对氧化和固相萃取程序进行了改进，并结合灵敏的液相色谱/串联质谱（LC/MS/MS）检测，在肝脏和血浆样品中均实现了25 ng/g的方法检测限。分析了从6条腹腔注射250 μg/kg亚致死剂量MC-LR的斑点叉尾鮰幼鱼采集的样品，检测到血浆中微囊藻毒素浓度为370至670 ng/g，肝脏中为566 - 1030 ng/g。同样，发现腹腔注射250 μg/kg节球藻毒素的斑点叉尾鮰血浆中含有835 - 1520 ng/g，肝脏中含有933 - 1140 ng/g。在血清和肝脏中检测到毒素，同时在暴露鱼中发现与这些肝细胞毒素一致的组织病理学损伤，而对照鱼无阳性结果，这证明了该分析程序在诊断疑似藻类中毒病例中的实用性。
