title: Complex behavior of marine animal tissue extracts in the competitive binding assay of brevetoxins with rat brain synaptosomes.
abstract: 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.
authors.affiliation: NIEHS Marine and Freshwater Biomedical Sciences Center, University of Miami RSMAS, Florida 33149, USA.
authors.forename: P L
authors.initials: PL
authors.lastname: Whitney
authors.forename: J A
authors.initials: JA
authors.lastname: Delgado
authors.forename: D G
authors.initials: DG
authors.lastname: Baden
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D049872:Bivalvia; D001921:Brain / Q000378:metabolism; D005399:Fishes; D066298:In Vitro Techniques; D016018:Least-Squares Analysis; D008110:Liver Extracts / Q000737:chemistry / Q000378:metabolism; D008322:Mammals; D008387:Marine Toxins / Q000032:analysis* / Q000302:isolation & purification; D009498:Neurotoxins / Q000032:analysis* / Q000302:isolation & purification; D035964:Oxocins; D051381:Rats; D015203:Reproducibility of Results; D013574:Synaptosomes / Q000378:metabolism*; D014020:Tissue Extracts / Q000737:chemistry / Q000378:metabolism; D014426:Turtles; D000095702:Polyether Toxins
chemical_list: D008110:Liver Extracts; D008387:Marine Toxins; D009498:Neurotoxins; D035964:Oxocins; D014020:Tissue Extracts; C053342:brevetoxin; D000095702:Polyether Toxins