saxitoxin and decarbamylsaxitoxin

To understand phycotoxin contamination in shellfish in the sub-Arctic and Arctic areas, scanning for the presence of 13 hydrophilic and lipophilic toxin components each was by liquid chromatography tandem quadrupole mass spectrometry analysis in shellfish samples collected from the Northern Bering Sea and the Chukchi Sea in 2014. The results showed that shellfish collected in both areas werecontaminated to different extents. Saxitoxin (STX), decarbamoylsaxitoxin (dcSTX) and decarbamoylneosaxitoxin (dcNEO) were the most frequently detected hydrophilic components, with maximum concentrations of 90.1 μg/kg, 112.25 μg/kg and 23.09 μg/kg, respectively. Although gonyautoxins (GTXs) were only detected in 3 samples, they were the main contributors to overall toxicity of high-latitude samples, especially GTX1. For lipophilic toxins, spirolide-1 (SPX1) and yessotoxin (YTX) were present in all samples at low levels (< 7 μg/kg and < 50 μg/kg, respectively). Only 5 samples showed evidence of okadaic acid (OA) and dinophysistoxin-2 (DTX-2) at low concentrations, ranging from 0.42 μg/kg to 7.23 μg/kg and 3.03 μg/kg to 30.59 μg/kg, respectively. Notably, a high level of pectenotoxin-1 (PTX-1) at 467.40 μg/kg was found in the shellfish collected at the northernmost station, exceeding the safety regulation standard by nearly 3 times. For both lipophilic and hydrophilic toxins, contamination in shellfish in the sub-Arctic and the Arctic area may be much more widespread and severe than was previously thought. This study highlighted the need to monitor toxins in a wider variety of shellfish, especially economic or commercial species, and across a wider range of sub-Arctic and Arctic waters, as well as the potential sources of these toxins.

Topics: Arctic Regions; Chromatography, Liquid; Food Contamination; Saxitoxin; Shellfish; Tandem Mass Spectrometry

The contents and composition of tetrodotoxin (TTX) and paralytic shellfish toxins (PSTs) in skin, muscle, and internal organs of two samples of marine puffer fish Canthigaster rivulata from Wakayama prefecture, Japan, were analyzed. Liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography with post-column derivatization and fluorescence detection (LC-FLD) were used for the analysis of TTX and PSTs, respectively. For both samples, TTX and two analogues of PSTs, saxitoxin (STX) and decarbamoyl STX (dcSTX), were detected at levels over the limit of quantization (LOQ) only in the skin. These toxins in the muscle and internal organs were at trace levels, or not detected (ND). TTX contents were 11,000 and 13,000 ng/g (or 35 and 41 nmol/g), while PSTs contents were 168 and 460 ng/g (or 0.63 and 1.72 nmol/g) in the two skin specimens. The compositions of total toxin content were 98.2 and 96.0 mol% TTX and 1.8 and 4.0 mol% PSTs, respectively. Thus, the main contributor to toxin content in C. rivulata skin was TTX and the levels of PSTs toxicity in C. rivulata were very low. When the PSTs contents were converted into mouse unit score from the LC-FLD results, the resulting values of 1.0 and 2.8 MU/g of PSTs in C. rivulata skin were similar to those in Takifugu poecilonotus and Takifugu vermicularis in Japan, as determined in previous studies.

Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Liquid; Japan; Mass Spectrometry; Saxitoxin; Spectrometry, Fluorescence; Tetraodontiformes; Tetrodotoxin

Toxins known to cause Paralytic Shellfish Poisoning (PSP) syndrome in humans that can have serious economic consequences for aquaculture were determined in ascidians of the genus Microcosmus. Significant concentrations of toxins were confirmed in all tested samples collected from the western coast of Istria Peninsula (Adriatic Sea, Croatia) when six people were poisoned following the consumption of fresh ascidians. Several species of bivalves that were under continuous monitoring had not accumulated PSP toxins although they were exposed to the same environmental conditions over the survey period. In the present study, HPLC-FLD with pre-column oxidation of PSP toxins has been carried out to provide evidence for the first human intoxication due to consumption of PSP toxic ascidians (Microcosmus vulgaris, Heller, 1877) harvested from the Adriatic Sea. Qualitative analysis established the presence of six PSP toxins: saxitoxin (STX), decarbamoylsaxitoxin (dcSTX), gonyautoxins 2 and 3 (GTX2,3), decarbamoylgonyautoxins 2 and 3 (dcGTX2,3), gonyautoxin 5 (GTX5) and N-sulfocarbamoylgonyautoxins 1 and 2 (C1,2), while quantitative analysis suggested STX and GTX2,3 as dominant toxin types and the ones that contribute the most to the overall toxicity of these samples with concentrations near the regulatory limit.

Topics: Animals; Chromatography, High Pressure Liquid; Croatia; Female; Food Contamination; Humans; Male; Marine Toxins; Saxitoxin; Seafood; Shellfish Poisoning; Urochordata

The mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins has been used in the AOAC Official Method and the official Japanese method. In the AOAC Official Method, the saxitoxin (STX) standard provided by the U.S. Food and Drug Administration (FDA) is used, but no standard is used in the official Japanese method. The objective of this study was to compare the toxicity of decarbamoyl STX (dcSTX), one of the derivatives of STX and a candidate standard for the MBA for PSP toxins in Japan, to that of FDA STX in the MBA platform. In this study, the toxicity of dcSTX was 918.0 ± 44.9 mouse units/µmol, and the relative toxicity ratio of dcSTX to FDA STX based on moles was 0.478.

Topics: Animals; Biological Assay; Japan; Mice; Reference Standards; Saxitoxin; Shellfish; Shellfish Poisoning; Specific Pathogen-Free Organisms; Tissue Extracts

The indirect identification and quantification of saxitoxin (STX) using other STX analogues by high-performance liquid chromatography with post-column oxidation and fluorescent detection (HPLC-FD) was investigated. Decarbamoylsaxitoxin (dcSTX) among the many STX analogues was selected as an external standard to identify and quantify STX. The retention time of STX in shellfish extracts by HPLC-FD was reproducibly estimated by using the retention time of dcSTX and the separation factor (α) between STX and dcSTX. Almost all of the columns tested to setup the method were useful to identify STX. Because a molar fluorescent coefficient of dcSTX was slightly different from that of STX, a factor used to correct the fluorescent coefficient in STX/dcSTX was determined to be 1.30. The indirect quantification of STX in scallop extracts by using the correction factor agreed to 80 - 100% precision with direct quantification using STX as an external standard.

Topics: Animals; Calibration; Chromatography, High Pressure Liquid; Molecular Structure; Oxidation-Reduction; Pectinidae; Reference Standards; Saxitoxin; Shellfish; Spectrometry, Fluorescence

Saxitoxin and its derivatives, the paralytic shellfish toxins (PSTs), are known to be toxic to humans, and maximum permitted levels in seafood have been established by regulatory authorities in many countries. Until recently, the mouse bioassay was the reference method for determining the levels of these toxins in seafood, but this has now been superseded by chemical methods of analysis. The latter methods are able to determine the levels of many PSTs in shellfish, but for risk assessment an estimate of the relative toxicities of the individual components of the PST mixture is required. The relative toxicities are expressed as "Toxicity Equivalence Factors" (TEFs). At present, TEFs are based on relative specific activities in the mouse bioassay, rather than on acute toxicity determinations, as measured by median lethal doses (LD50s). In the present study, the median lethal doses of saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and equilibrium mixtures of gonyautoxins 1&4 and gonyautoxins 2&3 have been determined by intraperitoneal injection, gavage and feeding. The results indicate that specific activities in the MBA do not consistently correlate with acute toxicities by any of the routes of administration, and TEFs, particularly for neosaxitoxin, require revision.

Topics: Administration, Oral; Animals; Injections, Intraperitoneal; Lethal Dose 50; Marine Toxins; Mice; Saxitoxin

Paralytic Shellfish Toxins (PSTs) are highly toxic metabolic by-products of cyanobacteria and dinoflagellates. The filamentous cyanobacterium Lyngbya wollei produces a unique set of PSTs, including L. wollei toxins (LWT) 1-6. The accurate identification and quantification of PSTs from Lyngbya filaments is challenging, but critical for understanding toxin production and associated risk, as well as for providing baseline information regarding the potential for trophic transfer. This study evaluated several approaches for the extraction and analysis of PSTs from field-collected L. wollei dominated algal mats. Extraction of PSTs from lyophilized Lyngbya biomass was assessed utilizing hydrochloric acid and acetic acid at concentrations of 0.001-0.1 M. Toxin profiles were then compared utilizing two analysis techniques: pre-column oxidation (peroxide and periodate) High Performance Liquid Chromatography (HPLC) with Fluorescence (FL) detection and LC coupled with Mass Spectrometry (MS). While both acid approaches efficiently extracted PSTs, hydrochloric acid was found to convert the less toxic LWT into the more toxic decarbamoylgonyautoxins 2&3 (dcGTX2&3) and decarbamoylsaxitoxin (dcSTX). In comparison, extraction with 0.1 M acetic acid preserved the original toxin profile and limited the presence of interfering co-extractants. Although pre-chromatographic oxidation with HPLC/FL was relatively easy to setup and utilize, the method did not resolve the individual constituents of the L. wollei derived PST profile. The LC/MS method allowed characterization of the PSTs derived from L. wollei, but without commercially available LWT 1-6 standards, quantitation was not possible for the LWT. In future work, evaluation of the risk associated with L. wollei derived PSTs will require commercially available standards of LWT 1-6 for accurate determinations of total PST content and potency.

Topics: Biological Assay; Chromatography, High Pressure Liquid; Cyanobacteria; Dinoflagellida; Fluorescence; Marine Toxins; Mass Spectrometry; Saxitoxin; Shellfish

Eleven male and 14 female specimens of a marine puffer Arothron firmamentum were collected from Oita and Iwate Prefectures, Japan. The toxicity assay using mouse showed that only ovary and skin of the female specimens were toxic, the toxicity scores being 5-740 as paralytic shellfish poison and <5-30 MU/g as tetrodotoxin (TTX), respectively. The toxin extracts from the both tissues were then treated with cartridge columns, and subjected to high performance liquid chromatography and liquid chromatography-mass spectral analyses. In the analyses, saxitoxin (STX) and decarbamoylSTX (dcSTX) were identified as the major toxins in the ovary, while the skin contained only TTX.

Topics: Animals; Biological Assay; Chromatography, High Pressure Liquid; Female; Japan; Male; Mice; Ovary; Saxitoxin; Seawater; Skin; Tetraodontiformes; Tetrodotoxin; Tissue Distribution; Toxicity Tests, Acute

Saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) were determined by liquid chromatography with quadrupole time-of-flight mass spectrometry (Q-TOF MS). A shellfish tissue was extracted with 0.1 mol/l HCl under ultrasonication, and cleanup of extract was accomplished by solid-phase extraction with a C18 cartridge. Chromatographic separation was carried out on a C18 column (150 mm x 2.1 mm, 3.5 microm) with gradient elution of MeOH-H2O (20:80) containing 0.05% heptafluorobutyric acid and MeOH-H2O (15:85) containing 0.05% acetic acid. The protonated molecule [M + H]+ ions at m/z 257 for dcSTX and 300 for STX were selected in precursor ion scanning for Q-TOF MS in the positive electrospray ionizaion mode. Average recoveries and relative standard deviations, by analyzing samples spiked at a level of 0.1, 0.8 or 1.6 microg/g, were 84-92 and 8-14%, respectively. Identification of the presence of the toxins in shellfish tissues was based on the structural information offered by Q-TOF MS.

Topics: Animals; Mass Spectrometry; Reproducibility of Results; Saxitoxin; Shellfish

Paralytic shellfish poisoning toxins are produced by dinoflagellates. Shellfish filtering these unicellular algae will accumulate the toxins and pose a health risk when consumed by man. In the European Union, paralytic shellfish poisoning toxins in bivalve molluscs are regulated at a maximum content of 80 microg/100 g (91/492/EEC). The current reference method in the European Union is the mouse bioassay, but alternative methods including the liquid chromatography methodology are preferred for ethical reasons. Analyses of suspected shellfish batches revealed, however, unacceptable differences in results reported by a small group of Dutch laboratories all using liquid chromatography methods with precolumn derivatization, followed by fluorescence detection. Therefore, a series of proficiency studies were undertaken among these laboratories. In the first three studies, participants were more or less allowed their own choice of method execution details. This approach yielded unsatisfactory results. A fourth study was then initiated in which a standardized method was mandatory. Two types of test material were used in the fourth study: lyophilized Cardium tuberculatum material containing saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX), and lyophilized mussel material containing dc-STX. The latter material was investigated in an interlaboratory study involving 15 participants and was considered as the reference material. Among the four laboratories, coefficients of variation (ANOVA) for C. tuberculatum material were 10% (n = 11) and 9% (n = 12) for STX and dc-STX, respectively, and for the reference material was 8% (n = 12) for dc-STX. The joint efforts showed that variability in analysis results between laboratories that all apply more or less the same method can be drastically improved if the methodology is rigorously standardized.

Topics: Chromatography, Liquid; Food Analysis; Humans; Laboratories; Marine Toxins; Netherlands; Neurotoxins; Quality Control; Reproducibility of Results; Saxitoxin; Shellfish

Acanthocardia tuberculatum is a bivalve mollusc that presents recurrent problems of paralytic shellfish poisoning (PSP) contamination in the Mediterranean coasts of Spain and Morocco. Although not commercially exploited from the Portuguese south coast, it represents an alternative for reducing the harvest pressure on species presently exploited. Evaluation of accumulation of marine biotoxins was carried out by HPLC in this species, harvested during a campaign carried out in April 2001 aimed at evaluating bivalve's resources that covered the entire Portuguese south coast.PSP toxins were studied by automated pre-column oxidation. Toxins were found in specimens from all stations, but always under the regulatory limit of 80 microg STX eq./100g. Saxitoxin and decarbamoyl-saxitoxin were the only PSP toxins unambiguously identified. In commercially exploited species, saxitoxin and analogues were not detected. The amnesic toxin domoic acid was found, but in levels similar to those found in species commercially exploited, and always under the regulatory limit of 20 microg/g. A fast elimination, and not a prolonged retention, of domoic acid seems to occur in A. tuberculatum in view of the levels close to or higher than 20 microg/g found in commercial bivalves harvested the two preceding months. The diarrhoeic toxin okadaic acid was found at trace levels much under the allowable level, similar to what was happening with other species harvested during the same period.

Topics: Animals; Biological Assay; Chromatography, High Pressure Liquid; Eutrophication; Kainic Acid; Marine Toxins; Mollusca; Okadaic Acid; Portugal; Saxitoxin; Species Specificity

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.

Topics: Animals; Bivalvia; Certification; Food Contamination; Freeze Drying; Humans; Reference Standards; Saxitoxin; Spain

This paper describes the first part of a project undertaken to develop mussel reference materials for Paralytic Shellfish Poisoning (PSP) toxins. Two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX) in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the second part of the project: the certification exercise. In the first study, 18 laboratories were asked to measure STX and dc-STX in rehydrated lyophilized mussel material and to identify as many other PSP toxins as possible with a method of their choice. In the second interlaboratory study, 15 laboratories were additionally asked to determine quantitatively STX and dc-STX in rehydrated lyophilized mussel and in a saxitoxin-enriched mussel material. The first study revealed that three out of four post-column derivatization methods and one pre-column derivatization method sufficed in principle to determine STX and dc-STX. Most participants (13 of 18) obtained acceptable calibration curves and recoveries. Saxitoxin was hardly detected in the rehydrated lyophilized mussels and results obtained for dc-STX yielded a CV of 58% at a mass fraction of 1.86 mg/kg. Most participants (14 out of 18) identified gonyautoxin-5 (GTX-5) in a hydrolysed extract provided. The first study led to provisional criteria for linearity, recovery and separation. The second study revealed that 6 out of 15 laboratories were able to meet these criteria. Results obtained for dc-STX yielded a CV of 19% at a mass fraction of 3.49 mg/kg. Results obtained for STX in the saxitoxin-enriched material yielded a CV of 19% at a mass fraction of 0.34 mg/kg. Saxitoxin could not be detected in the PSP-positive material. Hydrolysis was useful to confirm the identity of GTX-5 and provided indicative information about C1 and C2 toxins in the PSP-positive material. The methods used in the second interlaboratory study showed sufficiently consistent analysis results to undertake a certification exercise to assign certified values for STX and dc-STX in lyophilized mussel.

Topics: Animals; Bivalvia; Freeze Drying; Laboratories; Marine Toxins; Molecular Structure; Neurotoxins; Reference Standards; Saxitoxin; Shellfish

Paralytic shellfish poisoning (PSP) toxins are produced by certain dinoflagellate species such as Gymnodinium catenatum and Alexandrium tamarensis, during certain periods of the year influenced by several environmental factors, affecting the aquaculture industry and mainly bivalve molluscs. HPLC with fluorescence detection is a powerful analytical technique for the analysis of such toxins; several HPLC alternatives have been developed in order to improve the liquid chromatographic analysis, but due to the complexity of the sample matrix, important work has been focused recently on the clean-up of samples prior to HPLC analysis. Solid-phase extraction procedures offer advantages for this clean-up. In this work we focus on the study of three different clean-up methods prior to HPLC with fluorescence detection analysis of PSP toxin present in contaminated mussel samples; by spiking uncontaminated mussel samples with two different PSP toxin standards and by calculating the recovery values for these experiments. These recoveries must be taken into account in order to quantify the exact amount of PSP toxins present in the contaminated samples.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Food Analysis; Saxitoxin

Two species of freshwater puffer fish, Tetraodon cutcutia and Chelonodon patoca, collected from several locations in Bangladesh, showed lethal potency in mice ranging from 2.0 to 40.0 MU/g tissue as paralytic shellfish poison. In both species, toxicity of the skin was generally higher than the other tissues examined (muscle, liver and ovary). Water-soluble toxins from T. cutcutia were partially purified by activated charcoal treatment followed by column chromatographies using Bio-Gel P-2 and Bio-Rex 70. Analyses by cellulose acetate membrane electrophoresis and high-performance liquid chromatography with fluorometric detection demonstrated that the toxins were composed of saxitoxin, decarbamoylsaxitoxin, gonyautoxins 2 and 3, decarbamoylgonyautoxins 2 and 3, and three unidentified components which are possibly related to paralytic shellfish poison.

Topics: Animals; Bangladesh; Chromatography, High Pressure Liquid; Electrophoresis, Cellulose Acetate; Female; Fish Venoms; Mice; Reference Standards; Saxitoxin

Screening tests were carried out on the toxicity of freshwater puffers Tetraodon leiurus complex and Tetraodon suvatii collected from Udonthani province, north-eastern Thailand. Toxicity was highest in the liver and varied according to the location and season of fish catch. Fish which were reared in tap water for 3 months reduced the toxicity substantially. Partial purification was achieved by an ultrafiltration technique. Toxin components were consequently identified by high-performance liquid chromatography. It was found that toxins separated from the eggs, liver, skin and muscle of these puffers were composed of saxitoxin, neosaxitoxin and decarbamoylsaxitoxin.

Topics: Animals; Biological Assay; Chromatography, High Pressure Liquid; Fishes, Poisonous; Marine Toxins; Neuromuscular Blocking Agents; Saxitoxin; Shellfish; Thailand

The potencies of synthetic saxitoxin (+/- STX) and six of its synthetic analogues, including the enantioselectively synthesized unnatural (-)enantiomer of decarbamoyl saxitoxin (dcSTX), were measured and compared to those of natural saxitoxin [(+)STX]. The analogues, all of which were racemic (+/-) mixtures except for dcSTX, varied in the substituents at the C6 position, the carbamoyl 'moeity', and the C12 position, the hydrated ketone. The ability of the toxins to inhibit the compound action potential (AP) and to displace radiolabeled natural saxitoxin (3H-STX) from nerve membranes at equilibrium were both used as potency assays. Biological activity of both (+)- and (-)dcSTX was analyzed by the kinetics of block of single Na+ channels reconstituted in planar lipid bilayer membranes, where it was demonstrated that only (+)dcSTX had biological activity. The potency of STX analogues fell markedly as the substituent at the C6 position became smaller; Ki values from the binding competition assay (at 4 degrees C) are: (+/-)6-methanolic-STX, 5 x 10(-10) M; (+/-)6-methyl-STX, 1 x 10(-6) M; (+/-)6-dihydro-STX, 3.5 x 10(-5) M. Replacement of the ketone at the C12 position by a methylene group was accomplished in two derivatives, although both also had substituents at the C6 position. The compound (+/-)6-methyl-12-deoxy-STX was about 0.03 as potent as (+/-)6-methyl-STX and only 10(-5) as potent as racemic (+/-)STX. In synthetic compounds where the benzyloxymethyl (-CH2OCH2C6H5) substituent occurred at the C6 position, the C12-methylene derivative still displayed some binding activity (Ki = 6 x 10(-4) M). However, when the same C6 derivatized compounds also contained a 6-membered heterocyclic group (-C3H8S2-) conjugated to carbon 12, the measured binding affinity was even further decreased (Ki = 2 x 10(-3) M). The findings show that substitutions on the carbon 6 position of STX have stronger effects on STX potency than previously believed, and that the toxin may form a hydrogen bond with the sodium channel at this site. Furthermore, the total removal of oxygen from the C12 position does not completely abolish the binding activity of the molecule.

Topics: Action Potentials; Animals; Binding, Competitive; Brain Chemistry; Electrophysiology; In Vitro Techniques; Kinetics; Magnetic Resonance Spectroscopy; Neurons; Patch-Clamp Techniques; Rabbits; Rana pipiens; Rats; Saxitoxin; Sciatic Nerve; Sodium Channels; Stereoisomerism; Synaptosomes

Toxin production of a Malaysian isolate of the toxic red tide dinoflagellate Pyrodinium bahamense var. compressum was investigated at various stages of the batch culture growth cycle and under growth conditions affected by temperature, salinity, and light intensity variations. In all the experiments conducted, only 5 toxins were ever detected. Neosaxitoxin (NEO) and gonyautoxin V (GTX5) made up 80 mole percent or more of the cellular toxin content and saxitoxin (STX), GTX6 and decarbamoylsaxitoxin (dcSTX) made up the remainder. No gonyautoxins I-IV or C toxins were ever detected. In nutrient-replete batch cultures, toxin content rapidly peaked during early exponential phase and just as rapidly declined prior to the onset of plateau phase. Temperature had a marked effect on toxin content, which increased 3-fold as the temperature decreased from the optimum of 28 degrees C to 22 degrees C. Toxin content was constant at salinities of 24% or higher, but increased 3-fold at 20%. Toxin content decreased 2-fold and chlorophyll content increased 3-fold when light intensity was reduced from 90 to 15 microE m-2 s-1. This accompanied a 30% decrease in growth rate. Toxin composition (mole % individual toxin cell-1) remained constant throughout the course of the nutrient-replete culture and during growth at various salinities, but varied significantly with temperature and light intensity changes. At 22 degrees C, GTX5 was 25 mole % and NEO was 65 mole %, while at 34 degrees C, GTX5 increased to 55 mole % and NEO decreased proportionally to 40 mole %. When light intensity was reduced from 90 to 15 microE m-2 s-1, NEO decreased from 55 to 38 mole %, while GTX5 increased from 40 to 58 mole %. These data suggest that low light and high temperature both somehow enhance sulfo-transferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Count; Chromatography, High Pressure Liquid; Culture Media; Dinoflagellida; Fluorometry; Light; Malaysia; Marine Toxins; Neuromuscular Blocking Agents; Saxitoxin; Temperature

1. The actions of three saxitoxin (STX) analogues have been studied on the frog sartorius muscle fibre and the squid giant axon. One--neosaxitoxin--is a natural analogue, and two--decarbamylsaxitoxin and reduced saxitoxin--are synthetic. 2. The maximum dV/dt of the action potential in paired-muscle protocol is reduced by the analogues with relative potencies: STX (1), tetrodotoxin (1), neo-STX (1), decarbamyl-STX (0.2) and reduced-STX (0.01). 3. In constant-current studies on frog muscle fibres and in voltage-clamp studies on squid axons, all three analogues block only the sodium channel without affecting the potassium channel. 4. All three analogues bind to the same site as does STX in a competitive manner. 5. The experimental results suggest that the active groups in STX are the 7,8,9 guanidinium and the C-12 hydroxy groups. The carbamyl group contributes to, but is not essential for activity. 6. Stereospecific groups in the tetrodotoxin (TTX) molecule are the 1,2,3 guanidinium and the C-9, C-10 hydroxy groups. C-4 and C-8 groups are also important. 7. As new view is proposed in which STX and TTX can bind to a receptor located in the outside surface of the membrane very close to the orifice of the sodium channel.

Topics: Action Potentials; Animals; Axons; Binding Sites; Decapodiformes; Dose-Response Relationship, Drug; Drug Interactions; In Vitro Techniques; Ion Channels; Membrane Potentials; Muscles; Rana pipiens; Saxitoxin; Sodium; Structure-Activity Relationship; Tetrodotoxin