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Receptor binding assay and HPLC analysis of brevetoxins in organisms exposed to a Florida red tide
336
Abstracts
approximately 15-30 min after drug administration. The compound has a slightly shorter retention time than the parent 3,4-diaminopyridine in the high-performance liquid chromatographic (HPLC) assay used. Current isolation procedures involve microfiltration of the plasma followed by HPLC analysis of the filtrate. Nuclear magnetic resonance spectroscopy and gas chromatography/mass spectrometry of the metabolite will be discussed with respect to the toxicology of amino aromatics. 11-0x0 tetrodotoxin and a spect~cally labelled ‘H-TTX. B. Q. Wu,’ C. Y. Kao,’ M. Yotsu,Z T. YasumotoZ and S. R. Levinson3 (‘SUNY Downstate Medical Center, Brooklyn, NY 11203, U.S.A.; 2Tohoku University, Sendai, Japan; and ‘University of Colorado Medical Center, Denver, CO 80262, U.S.A.).
Currently used ligands for the voltage-gated sodium channel have sho~comings. A commer~ally available ‘H-saxitoxin has the ‘H on the C-l 1 methylene C from with it can readily exchange with solvent H. A diffusely labelled ‘H-TTX with limited specific activity is not available commercially. Other non-commercia! liaands are adducts of TTX, appreciably larger than TTX. We have made a stable specifically labelled 3H-‘ITX of high specific activity by reduction of 11-0x0 ‘ITX (U.S. patent 5288.870). This hvdrated aldehvde of TTX was discovered as a natural analogue by Khora and Yasumoto (1989). Citrate-free- TTX (Hebeij was oxidized by Fenton’s reagent (FeSO,) or P&zner-Moffat reaction (dicyclohexylcarbodiimide, DMSO). 11-0x0 TTX was isolated by HPLC, characterized by NMR and MS, and tested on the voltage-clamped frog skeletal muscle fiber. It blocked Z,, specitically at c. three times the potency of TTX (Wu et al., 1991). 11-0x0 TIX was then reduced to TTX with sodium ‘H-borohydride. The product required little work-up, had a specific activity of 3900 Ci/M (which could be increased), and bound specifically to the purified sodium channels of Electrophorus electricus. This marker could open new areas of studies on Na+ channels. Khora and Yasumoto (1989) Tetrahedron Lett. 30, 349. Wu et al. (1991) Biophys. J. 59, 261A. Supported by U.S. Army contract DAMDI7-87-C-7094 and NIH grant NS 14551. Receptor binding assay and HPLC
analysis of brevetoxins in organisms exposed to a Florida red tide. T. A. Leighfield,’ M. S. Henry,’ R. H. Pierce,z F. M. Van Dolah’ and J. Zhour (’ Marine Biotoxins Program, Charleston Laboratory, U.S. National Marine Fisheries Service, Charleston, SC 29422-2607, U.S.A.; and ZMote Marine Laboratory, Sarasota, Florida, U.S.A.).
The suitability of a microplate recgttor binding assay for the dete~ination of brevetoxins in field samples was assessed using samples obtained during a persistent bloom of Gymnodinium breve on the west coast of Florida from September 1994 to January 1995. Oysters (Crassostrea virginica), clams (Mercenaria mercenaria), sea squirt (Styela plicata), and mullet (Mugil cephalus) were collected throughout the bloom at Mote Marine Lab., Bay Dock. Brevetoxins were extracted with acetone followed by a two-step column fractionation through silica gel then alumina. A!! samples were evaporated to dryness and brought up in methanol prior to analysis. The brevetoxin receptor binding assay was based on competition between [‘H]PbTx-3 and PbTx-3 standards or unknown samples for binding to voltage-de~ndent sodium channels in rat brain synaptosomes. Binding competition was carried out in a microplate format and quantified using a microplate scintillation counter to permit high sample throughput. Receptor assay results were compared with HPLC analyses on Cl8 columns, B&J OD-5 with SSjlS methanol/water (Mote) or Rainin Microsorb with 80/20 methanol/water (NMFS), at a flow rate of 1m!/min with UV detection at 215 nm. Gymnodinium breve cell counts at Bay Dock were > 5000 cells/liter beginning 19 September, and were temporally variable with a maximum of 23,000,OOOcells/liter. Brevetoxin activity from oysters ranged from 0.05 to 0.38pg PbTx-3 equivalents/g, with a maximum concentration observed during late October. Good correlation was observed between values obtained with the receptor assay and HPLC analyses. Sea squirts analysed by receptor assay ranged from undetectable levels to 0.33 pg PbTx3 equivalents/g. Detectable levels of brevetoxins were not observed in clams or mullet tissue. Comparative response of human and murine cell lines by cell bioassay to sodium channel active marine toxins and extracts. R. L. Manger, L. S. Leja, S. Y. Lee, J. M. Hungerford and M. M. Wekell (U.S. FDA, Seafood Products
Research Center, Bothel!, WA 98021, U.S.A.). Cell-based bioassays offer a means for assessing the potency of marine toxins without the restrictions and inconvenience of animal test methods. Our focus has been the development of cell-based methods for the detection of some of the more problematic marine toxins that act at the level of the voltage-gated sodium channel, including the ciguatoxins, brevetoxins, and saxitoxins. Based upon the response of mouse neuroblastoma cells in vitro, this method has demonstrated high sensitivity and excellent correlation with animal toxicity studies, in particular the mouse bioassays currently used for monitoring. In the present study we have expanded upon our
Abstracts
approximately 15-30 min after drug administration. The compound has a slightly shorter retention time than the parent 3,4-diaminopyridine in the high-performance liquid chromatographic (HPLC) assay used. Current isolation procedures involve microfiltration of the plasma followed by HPLC analysis of the filtrate. Nuclear magnetic resonance spectroscopy and gas chromatography/mass spectrometry of the metabolite will be discussed with respect to the toxicology of amino aromatics. 11-0x0 tetrodotoxin and a spect~cally labelled ‘H-TTX. B. Q. Wu,’ C. Y. Kao,’ M. Yotsu,Z T. YasumotoZ and S. R. Levinson3 (‘SUNY Downstate Medical Center, Brooklyn, NY 11203, U.S.A.; 2Tohoku University, Sendai, Japan; and ‘University of Colorado Medical Center, Denver, CO 80262, U.S.A.).
Currently used ligands for the voltage-gated sodium channel have sho~comings. A commer~ally available ‘H-saxitoxin has the ‘H on the C-l 1 methylene C from with it can readily exchange with solvent H. A diffusely labelled ‘H-TTX with limited specific activity is not available commercially. Other non-commercia! liaands are adducts of TTX, appreciably larger than TTX. We have made a stable specifically labelled 3H-‘ITX of high specific activity by reduction of 11-0x0 ‘ITX (U.S. patent 5288.870). This hvdrated aldehvde of TTX was discovered as a natural analogue by Khora and Yasumoto (1989). Citrate-free- TTX (Hebeij was oxidized by Fenton’s reagent (FeSO,) or P&zner-Moffat reaction (dicyclohexylcarbodiimide, DMSO). 11-0x0 TTX was isolated by HPLC, characterized by NMR and MS, and tested on the voltage-clamped frog skeletal muscle fiber. It blocked Z,, specitically at c. three times the potency of TTX (Wu et al., 1991). 11-0x0 TIX was then reduced to TTX with sodium ‘H-borohydride. The product required little work-up, had a specific activity of 3900 Ci/M (which could be increased), and bound specifically to the purified sodium channels of Electrophorus electricus. This marker could open new areas of studies on Na+ channels. Khora and Yasumoto (1989) Tetrahedron Lett. 30, 349. Wu et al. (1991) Biophys. J. 59, 261A. Supported by U.S. Army contract DAMDI7-87-C-7094 and NIH grant NS 14551. Receptor binding assay and HPLC
analysis of brevetoxins in organisms exposed to a Florida red tide. T. A. Leighfield,’ M. S. Henry,’ R. H. Pierce,z F. M. Van Dolah’ and J. Zhour (’ Marine Biotoxins Program, Charleston Laboratory, U.S. National Marine Fisheries Service, Charleston, SC 29422-2607, U.S.A.; and ZMote Marine Laboratory, Sarasota, Florida, U.S.A.).
The suitability of a microplate recgttor binding assay for the dete~ination of brevetoxins in field samples was assessed using samples obtained during a persistent bloom of Gymnodinium breve on the west coast of Florida from September 1994 to January 1995. Oysters (Crassostrea virginica), clams (Mercenaria mercenaria), sea squirt (Styela plicata), and mullet (Mugil cephalus) were collected throughout the bloom at Mote Marine Lab., Bay Dock. Brevetoxins were extracted with acetone followed by a two-step column fractionation through silica gel then alumina. A!! samples were evaporated to dryness and brought up in methanol prior to analysis. The brevetoxin receptor binding assay was based on competition between [‘H]PbTx-3 and PbTx-3 standards or unknown samples for binding to voltage-de~ndent sodium channels in rat brain synaptosomes. Binding competition was carried out in a microplate format and quantified using a microplate scintillation counter to permit high sample throughput. Receptor assay results were compared with HPLC analyses on Cl8 columns, B&J OD-5 with SSjlS methanol/water (Mote) or Rainin Microsorb with 80/20 methanol/water (NMFS), at a flow rate of 1m!/min with UV detection at 215 nm. Gymnodinium breve cell counts at Bay Dock were > 5000 cells/liter beginning 19 September, and were temporally variable with a maximum of 23,000,OOOcells/liter. Brevetoxin activity from oysters ranged from 0.05 to 0.38pg PbTx-3 equivalents/g, with a maximum concentration observed during late October. Good correlation was observed between values obtained with the receptor assay and HPLC analyses. Sea squirts analysed by receptor assay ranged from undetectable levels to 0.33 pg PbTx3 equivalents/g. Detectable levels of brevetoxins were not observed in clams or mullet tissue. Comparative response of human and murine cell lines by cell bioassay to sodium channel active marine toxins and extracts. R. L. Manger, L. S. Leja, S. Y. Lee, J. M. Hungerford and M. M. Wekell (U.S. FDA, Seafood Products
Research Center, Bothel!, WA 98021, U.S.A.). Cell-based bioassays offer a means for assessing the potency of marine toxins without the restrictions and inconvenience of animal test methods. Our focus has been the development of cell-based methods for the detection of some of the more problematic marine toxins that act at the level of the voltage-gated sodium channel, including the ciguatoxins, brevetoxins, and saxitoxins. Based upon the response of mouse neuroblastoma cells in vitro, this method has demonstrated high sensitivity and excellent correlation with animal toxicity studies, in particular the mouse bioassays currently used for monitoring. In the present study we have expanded upon our