Kilcoyne Jane, McCarron Pearse, Twiner Michael J, Nulty Ciara, Crain Sheila, Quilliam Michael A, Rise Frode, Wilkins Alistair L, Miles Christopher O
Marine Institute, Rinville, Oranmore, County, Galway, Ireland.
Chem Res Toxicol. 2014 Apr 21;27(4):587-600. doi: 10.1021/tx400434b. Epub 2014 Feb 7.
Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-epi-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-epi-AZA2 and 37-epi-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-epi-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-epi-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an in vitro cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-epi-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the subnanomolar range. On the basis of EC50 comparisons, 37-epi-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes.
自1998年azaspiracid-1（AZA1）被鉴定以来，AZA类似物的数量已增加到30多种。使用中性流动相的液相色谱 - 质谱（LC-MS）方法的发展导致发现了AZA1、AZA2和AZA3的异构体，在浮游植物和贝类样品中，这些异构体的含量约为母体类似物的2 - 16%。在酸性流动相条件下，异构体及其母体无法分离。稳定性研究表明，这些异构体是自发差向异构化产物，加热会加速其形成。从受污染的贝类中分离出AZA1异构体，并通过核磁共振（NMR）光谱和化学分析鉴定为37-表-AZA1。类似分析表明，AZA2和AZA3的异构体分别对应于37-表-AZA2和37-表-AZA3。发现37-差向异构体在溶液中与母体化合物处于平衡状态。通过NMR对37-表-AZA1进行定量，并进行相对摩尔响应研究以确定AZA1和37-表-AZA1在LC-MS响应中的潜在差异。使用Jurkat T淋巴细胞作为体外细胞模型确定毒理学效应。采用基于代谢的染料（即MTS）的细胞毒性实验表明，37-表-AZA1引发了浓度和时间依赖性的致死反应，EC50值在亚纳摩尔范围内。基于EC50比较，37-表-AZA1的效力比AZA1高5.1倍。该数据表明，在为监管目的分析AZA时，应考虑海鲜产品中这些差向异构体的存在。
