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We investigated the brevetoxin congener PbTx-3 to determine its distribution among carrier proteins, including albumin and blood lipoproteins. Using a radiolabeled brevetoxin tracer (PbTx-3), we found that 39% of the radiolabel remained associated with components in mouse plasma after > 15 kDa cutoff dialysis. Of this portion, only 6.8% was bound to serum albumin. We also examined the binding of brevetoxin to various lipoprotein fractions. Plasma, either spiked with PbTx-3 or from mice treated for 30 min with PbTx-3, was fractionated into different-sized lipoproteins by iodixanol gradient ultracentrifugation. Each fraction was then characterized and quantified by agarose gel electrophoresis and brevetoxin radioimmunoassay, respectively. In both the in vitro and in vivo experiments, the majority of brevetoxin immunoreactivity was restricted to only those gradient fractions that contained high-density lipoproteins (HDLs). Independent confirmation of brevetoxin binding to HDLs was provided by high molecular weight (100 kDa cutoff) dialysis of [3H]PbTx-3 from lipoprotein fractions as well as a scintillation proximity assay using [3H]PbTx-3 and purified human HDLs. This information on the association of brevetoxins with HDLs provides a new foundation for understanding the process by which the toxin is delivered to and removed from tissues and may permit more effective therapeutic measures to treat intoxication from brevetoxins and the related ciguatoxins.

There is a critical need to simply and reliably monitor brevetoxins routinely in the blood of humans and aquatic animals. We used striped mullet as laboratory test animals to better define the uptake and elimination kinetics of brevetoxin during an aqueous exposure to the brevetoxin-producing dinoflagellate Karenia brevis. Striped mullet were first exposed to sublethal densities of K. brevis (approximately 250,000 cells/L) for 1, 4, 8, 12, and 24 hr. No mortality was observed in the aquaria, and at each time point blood samples were taken and applied to blood collection cards for brevetoxin analysis using radioimmunoassay (RIA). The RIA indicated that blood levels of brevetoxin (PbTx-3) increased to values significantly different from that of the controls at all five time points during exposure (p < 0.05). Striped mullet were then exposed to a K. brevis culture with a known brevetoxin concentration of 0.5 ng/mL. Even after exposures at a low brevetoxin concentration, RIA was able to detect 2.25 +/- 0.62 ng/mL PbTx-3 equivalents in the blood of the mullet at 8 hr of exposure. When exposed to higher brevetoxin concentrations (3.5 and 5.4 ng/mL), blood brevetoxin increased to peak levels at 12 hr and then reached equilibrium after 24 hr in the continued presence of K. brevis. During this time of equilibrium, the mullet maintained brevetoxins with a blood:water coefficient of 2.2. To define the elimination of brevetoxin, striped mullet were next exposed for 8-10 hr and then transferred to fresh seawater containing no K. brevis for up to 116 hr. Blood brevetoxin levels remained elevated and decreased only by 50% 116 hr after transfer. The rate of elimination fit best to a two-phase exponential decay with a biologic half-life of 12 and 266 hr. This study, using RIA in conjunction with blood collection cards, demonstrates an effective means to monitor blood brevetoxin levels in finfish and provides a foundation to characterize biologically relevant levels of brevetoxin in other species impacted by red tide events.

We developed a radioimmunoassay (RIA) using a sheep anti-brevetoxin antiserum to evaluate detection of brevetoxin on blood collection cards from mice treated with the brevetoxin congener PbTx-3. The RIA has high affinity for PbTx-3 [half-maximal effective concentration (EC(50)) +/- SE = 1.2 +/- 0.2 nM; n = 10] and recognizes both type 1 and type 2 brevetoxins, but not ciguatoxin. Direct comparison of the RIA with a radiolabeled [(3)H]-PbTx-3 receptor-binding assay (RBA) revealed excellent sensitivity, congener selectivity, and minimal interference from blood matrix. We first analyzed blood samples from an acute time course exposure, using a maximal nonlethal dose [180 microg/kg body weight (bw)] for 0.5, 1, 2, 4, and 24 hr. Mean blood brevetoxin levels were 36 nM at 30 min and stayed above 20 nM during the 1-4 hr time points. We next analyzed blood brevetoxin levels after longer exposure (0.5, 1, 2, 3, 4, or 7 days). Mean blood brevetoxin levels were 26.0 nM at 0.5 days, decreased to 8.2 nM at 1.0 day, and maintained a significant level (p < 0.05) of 1.3 nM at day 2. We next determined the lowest measurable dose using increasing concentrations of PbTx-3 (10-300 micro g/kg bw). Analysis of the blood samples at 60 min revealed a linear relationship between administered and internal doses (r(2) = 0.993). All doses of brevetoxin administered were detectable at 1 hr, with significant levels found for the lowest administered dose of 10 micro g/kg bw--a dose that was 10-fold lower than the lowest observable effect level. This RIA provides an optimal first-tier detection of brevetoxin from blood collection cards and, used in combination with the RBA and liquid chromatography-mass spectrometry, should provide a complete panel of methods to biomonitor brevetoxin exposure.