Proposal for a simple and sensitive haemolytic assay for palytoxin: Toxicological dynamics, kinetics, ouabain inhibition and thermal stability

Benthic dinoflagellates of the genus Ostreopsis are important components of subtropical and tropical marine coral reef–lagoonal environments. Currently, as a result of global warming and trade globalization, they are also distributed worldwide. These microalgae are shown to produce palytoxin, one of the most potent non-protein marine toxins known.The haemolytic assay is a very easy, rapid and sensitive method to determine palytoxin. However, under the conditions reported in previous works this assay is inadequate for a rigorous dose–response treatment, since: (1) it produces degenerate sigmoidal profiles, with a pronounced slope which makes the calculation of the ED50 very sensitive to the experimental error; (2) at the usual work temperature, the in vitro stability of the system is low, which accentuates the variability and ambiguity of the response. To resolve these problems haemolysis of sheep erythrocytes is studied, including it's toxicological dynamics, kinetics, inhibition by ouabain and response to temperature. The results show that, to obtain a smoother, more stable and reproducible response, it is necessary to apply two resources simultaneously: operation at a moderate temperature and partial inhibition of the palytoxin by ouabain. It also produces highly reliable parameters and allows strict equivalencies to be established with the mouse bioassays, a traditional reference point, though bioethically questionable and 20 times less sensitive than the bioassay proposed here.