Whitney P L, Delgado J A, Baden D G
NIEHS Marine and Freshwater Biomedical Sciences Center, University of Miami RSMAS, Florida 33149, USA.
Nat Toxins. 1997;5(5):193-200. doi: 10.1002/nt.4.
Brevetoxins are produced by the marine dinoflagellate Ptychodiscus brevis, an organism linked to red tide outbreaks, and the accompanying toxicity to marine animals and to neurotoxic shellfish poisoning in humans. Brevetoxins bind with high affinity to voltage-sensitive sodium channels and cause increased sodium ion conductance and nerve cell depolarization. The brevetoxin competitive binding assay with tritium-labeled brevetoxin 3 (3H-PbTx-3) and rat brain synaptosomes is a sensitive and specific assay for pure brevetoxins. Here we report that extracts of manatee, turtle, fish, and clam tissues contain components that interfere with the assay by cooperative, noncompetitive inhibition of 3H-PbTx-3 specific binding and increased nonspecific binding to synaptosomes. By determining the "apparent" toxin concentration ("[Toxin]") in the extract at several assay concentrations, a reasonable correction for the complex inhibition could be made using a semilog plot to extrapolate [Toxin] to zero extract concentration to obtain [Toxin]0. Spiking 4 extracts with 60 nM PbTx-3 caused [Toxin]0 to increase by 41 +/- 8 nM, indicating that the noncompetitive components did not prevent the assay of toxin but did reduce the accuracy of the result. Fourfold repetition of the assay of 4 samples gave standard deviations of 25 to 60% of the value of [Toxin]0, so the error can be fairly large, especially for samples with little toxin. Purification of an extract with a 1 g sample prep column of C-18 decreased the complex inhibition by about 3-fold but did not eliminate interference in the assay.
短裸甲藻毒素由海洋双鞭毛藻短裸甲藻产生，这种生物与赤潮爆发有关，会对海洋动物造成毒性影响，并导致人类发生神经毒性贝类中毒。短裸甲藻毒素与电压敏感钠通道具有高亲和力结合，会导致钠离子电导增加和神经细胞去极化。用氚标记的短裸甲藻毒素3（3H-PbTx-3）和大鼠脑突触体进行的短裸甲藻毒素竞争性结合试验，是一种针对纯短裸甲藻毒素的灵敏且特异的试验。在此我们报告，海牛、海龟、鱼类和蛤类组织的提取物含有一些成分，这些成分通过对3H-PbTx-3特异性结合的协同、非竞争性抑制以及增加对突触体的非特异性结合来干扰该试验。通过在几种试验浓度下测定提取物中“表观”毒素浓度（“[毒素]”），可以使用半对数图对复杂抑制进行合理校正，将[毒素]外推至零提取物浓度以获得[毒素]0。用60 nM PbTx-3对4种提取物进行加标导致[毒素]0增加41±8 nM，表明非竞争性成分并未阻止毒素的检测，但确实降低了结果的准确性。对4个样品进行4次重复试验，得到的标准偏差为[毒素]0值的25%至60%，所以误差可能相当大，尤其是对于毒素含量低的样品。用1 g样品制备柱C-18对提取物进行纯化，可使复杂抑制降低约3倍，但并未消除试验中的干扰。
