Detection of gymnodimine-A and 13-desmethyl C spirolide phycotoxins by fluorescence polarization.
The gymnodimines and spirolides are phycotoxins classified into a heterogeneous group of marine biocompounds called cyclic imines. Although there is no clear evidence of their toxicity to humans, gymnodimines and spirolides are highly toxic to rodents and constitute a source of false positives in lipophilic toxin detection by the mouse bioassay. Using nicotinic acetylcholine receptor-enriched membranes of Torpedo, and fluorescent alpha-bungarotoxin, we developed a fluorescence polarization assay to detect and quantify gymnodimine-A and 13-desmethyl C spirolide. The presence of these cyclic imines in solution inhibited the interaction of fluorescent-labeled alpha-bungarotoxin with nicotinic acetylcholine receptors in a concentration-dependent manner. The sensitivity of the assay is in the order of nanomolar concentrations of gymnodimine and 13-desmethyl C spirolide. Okadaic acid, yessotoxin, and brevetoxin-2, three lipophilic marine toxins, did not interfere with this assay. A suitable extraction method in shellfish was also developed. The gymnodimine-A and 13-desmethyl C spirolide recovery rates of mussel matrix extraction with acetone/chloroform were 63.6% +/- 3.5% and 87.4% +/- 5.3%, respectively. In summary, this inhibition assay is capable of gymnodimine-A and 13-desmethyl C spirolide detection in mussel extracts with enough sensitivity and specificity to quantify these toxins in the range of 50-2000 microg/kg and 70-700 microg/kg of shellfish meat, respectively.
Vilariño
Natalia
N
Departamento de Farmacología, Facultad de Veterinaria, Campus Universitario, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
Fonfría
Eva S
ES
Molgó
Jordi
J
Aráoz
Rómulo
R
Botana
Luis M
LM
D000818:Animals; D002038:Bungarotoxins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D005454:Fluorescence Polarization; D006575:Heterocyclic Compounds, 3-Ring / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity*; D006844:Hydrocarbons, Cyclic / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity*; D007097:Imines / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity*; D007783:Lactones / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity*; D011978:Receptors, Nicotinic / Q000378:metabolism; D012758:Shellfish; D013997:Time Factors; D014101:Torpedo / Q000378:metabolism
C429214:13-desmethyl-C; D002038:Bungarotoxins; D006575:Heterocyclic Compounds, 3-Ring; D006844:Hydrocarbons, Cyclic; D007097:Imines; D007783:Lactones; D011978:Receptors, Nicotinic; C407350:gymnodimine