Paralytic Shellfish Poisoning Saxitoxin

A once exclusive instrumental technique for research, LC-MS is becoming ever more utilized in special applications. Since hospitals are requiring more robust, sensitive and specific assays for routine TDM analysis, it is inevitable, as demonstrated with the aforementioned NMBAs, that LC-MS methods will eventually become routine procedures in clinical settings. 

PSP has a rapid onset of symptoms occurring within 0.5-2 h after ingestion of the shellfish, depending on the amount of toxin consumed. Symptoms include 

Without clinical support, death may occur most commonly by respiratory paralysis or occasionally by cardiovascular collapse, despite respiratory support, caused by the weak hypotensive action of the toxin. With less serious cases (e.g. lower concentration of saxitoxin in shellfish or smaller amount of shellfish consumed), complete recovery with no lasting side-effects is expected if respiratory support transpires within 12h of exposure. In humans, 120-180 pg of STX can produce moderate symptoms such as tingling and numbness, 400-1060 pg of STX can cause deaths, and concentrations 2000 pg constitute a fatal dose.  

The mouse biosassay first introduced for detection of PSP toxins in 1937 by Sommer and Meyer was adopted by the Association of Official Analytical Chemists in 1984 as a non-selective bioassay with animal symptoms and time to death utilized as the measure of PSP toxicity. Its LOD of 40 pg per 100 g of tissue is adequate to determine if a tolerance level of 80 pg for humans has been reached during a PSP episode and whether actions should be taken to maintain public safety.  

HPLC is the most common chromatographic assay, predominantly utilizing fluorescence or mass spectrometric detection to measure presence of individual PSP toxins with a detection limit for saxitoxin of 20 fg per 100 g of tissue (0.2 ppm). For clinical purposes, serum and urine are the preferred matrices for analysis of saxitoxin. For postmortem analysis other organ tissues have been analyzed (e.g. heart, brain, liver, gastric, spleen). 

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Two other suspected alkaloid producing cyanobacteria strains, Anabaena flos-aquae NRC-525-17 and Aphanizomenon flos-aquae NH-5, are now being studied. The toxin of flos-aquae NRC-525-17 (anatoxin-a(s)) is thought to have CNS stimulating properties (7) and that of Aph. flos-aquae NH-5 (aphantoxin) is thought to produce the paralytic shellfish poisons saxitoxin and neosaxitoxin (Fig. 1)... 

Jellett, J.F. et al. Paralytic shellfish poison (saxitoxin family) bioassay Automated endpoint determination and standardization of the in vitro tissue culture bioassay, and comparison with the standard mouse bioassay. Toxicon, 30, 1143, 1992. 

This reaction can be generally applied to the preparation of enamino esters and /3-keto esters, which can be asymmetrically reduced to yield chiral /3-amino or hydroxyl esters and further hydrolyzed to /3-amino or hydroxyl acids and cyclized to form /3-lactams or lactones7 In addition, this reaction has been successfully used in the synthesis of the paralytic shellfish poisons, saxitoxin, and gonyautoxin. ... 

Radioactivity is a fading trend since it has been replaced by alternative technologies that pose a lower risk (fluorophores, antibodies, etc.). Still, there are several international projects to use radioreceptor assays for some marine toxins, notably PSP (paralytic shellfish poison, saxitoxin, etc.). Detection methods that use radioactive compounds use either antibodies (radioimmunoassay [32]) or receptors (radioreceptor assay [33]). 

Some dinoflagellates of the genus Alexandrium produce neurotoxic compounds known as paralytic shellfish poisoning (PSP) toxins. Because these toxins can contaminate filter-feeding shellfish they may threaten public health and create economic problems for fisheries. PSP-toxins include at least a dozen saxitoxins, neosaxitoxins, and gonyautoxins (Scheme 1). 

An interesting and important example of an animal poison is paralytic shellfish poison (PSP). This chemical, which is also known as saxitoxin and by several other names as well, is found in certain shellfish. But it is not produced by shellfish it is rather a metabolic product of certain marine microorganisms (Protista). These microorganisms are ingested by the shellfish as food, and their poison can remain behind in the shellfish s tissue. Paralytic shellfish poison is not a protein, but a highly complex organic chemical of most unusual molecular structure. 

Over the last eight years, most of the estimated cases of world-wide poisoning in humans due to the three major kinds of seafood toxins that are found in fresh and unspoiled marine organisms, namely, paralytic shellfish poison (PSP-saxitoxins/ gonyautoxins), ciguatoxin(s), and tetrodotoxin, (13-16) were caused by ciguatera (Table I). 

L/mole). It cross-reacts at >90% with saxitoxin but at <1% with neosaxitoxin. This antibody, when used in an anti-rabbit IgG "second antibody" radioimmunoassay format, can detect pmole quantities of saxitoxin. This assay has been shown to be a simple and efficient method for the analysis of saxitoxin in clam extracts. The lack of antibody cross-reactivity to the neosaxitoxin sub-group of the paralytic shellfish poisons limits the general utility of the assay to neurophysiology studies and to certain clam species which preferentially accumulate saxitoxin. However, the radioimmunoassay serves as a good precursor in the development of an enzyme immunoassay for the paralytic shellfish poisons. 

Preference is obviously for a simple chemical assay for PSP. Unfortunately the more specific the chemical test, the narrower is the window of compounds it can assay. The Paralytic Shellfish Poison is not just Saxitoxin (STX) as originally believed, but is a mixture of compounds closely related to STX Q) and the mix varies widely with location and with time ( ). It would seem, therefore that a chemical assay should determine at least the ratios of the several compounds, and that the relative toxicity of each of the compounds must be known. An effective assay must evaluate the actual biological toxicity of the shellfish being tested. For the chemical assay this requires the summated toxicity of all the... 

The main genera responsible for freshwater toxic blooms are Microcystis, Anabaena, Aphanizomenon and Oscillatoria. Toxins produced include 1. anatoxins, alkaloids and peptides of Anabaena 2. the peptide microcystin and related peptides of Microcystis 3. aphantoxins, compounds of Aphanizomenon with properties similar to some paralytic shellfish poisons. Properties of Oscillatoria toxin suggest they are peptides similar to those of Microcystis. Microcystis toxins are peptides (M.W. approx. 1200) which contain three invariant D-amino acids, alanine, erythro-3-methyl aspartic and glutamic acids, two variant L-amino acids, N-methyl dehydro alanine and a 3-amino acid. Individual toxic strains have one or more multiples of this peptide toxin. The one anatoxin characterized is a bicylic secondary amine called anatoxin-a (M.W. 165). The aphantoxin isolated in our laboratory contains two main toxic fractions. On TLC and HPLC the fractions have the same characteristics as saxitoxin and neosaxitoxin. 

Earlier research had already suggested that certain blooms Aph. flos-aquae could produce paralytic shellfish poisons. These studies used water blooms collected from Kezar Lake, New Hampshire (25,30). In 1980 Carmichael isolated a neurotoxic strain of Aph. flos-aquae from a small pond in New Hampshire. These strains have also been shown to produce toxins similar to saxitoxin and neo- axitoxin (23) and are the ones used in the studies presented here. 

The neurotoxins isolated from Aph. flos-aquae were shown to have similar chemical and biological properties to paralytic shellfish poisons (PSP) (25,29,38) Sawyer et al. in 1968 (25) were the first to demonstrate that the crude preparation of aphantoxins behave like saxitoxin, the major paralytic shellfish poison. They showed that the toxins had no effect on the resting membrane potential of frog sartorius muscle blocked action potential on de-sheathed frog sciatic nerve and also abolished spontaneous contractions in frog heart. Sasner et al. (1981) (29) using the lab cultured strain reported similar results. 

Cyanobacterial toxins (both marine and freshwater) are functionally and chemically a diverse group of secondary chemicals. They show structure and function similarities to higher plant and algal toxins. Of particular importance to this publication is the production of toxins which appear to be identical with saxitoxin and neosaxitoxin. Since these are the primary toxins involved in cases of paralytic shellfish poisons, these aphantoxins could be a source of PSP standards and the study of their production by Aphanizomenon can provide information on the biosynthesis of PSP s. The cyanobacteria toxins have not received extensive attention since they have fewer vectors by which they come in contact with humans. As freshwater supplies become more eutrophicated and as cyanobacteria are increasingly used as a source of single cell protein toxic cyanobacteria will have increased importance (39). The study of these cyanobacterial toxins can contribute to a better understanding of seafood poisons. 

Paralytic Shellfish Poisoning (PSP) was first determined to be a problem in 1942 after three people and many seabirds died from eating shellfish on the west coast of the United States, near the Columbia River. It is caused by the saxitoxin family (saxitoxin + 18 related compounds) produced by several species of Alexandrium dinoflagellates. The main contamination problems include mussels, clams, crabs, and fish of the Pacific Northwest and Northeast Atlantic. 

Kvitek, R. G. and Beitler, M. K., Relative insensitivity of butter clam neurons to saxitoxin a preadaptation for sequestering paralytic shellfish poisoning toxins as a chemical defense, Mar. Ecol. Prog. Ser., 69, 47, 1991. 

Saxitoxin (32) is listed in Schedule 1 of the CWC. It is a polar, cationic, relatively low molecular mass toxin and is one of 18 structurally related neurotoxins collectively known as paralytic shellfish poisoning (PSP) toxins. Analogues are formed by addition of sulfate, A-sulfo and A-hydroxyl groups, and by decarbamylation. They block neuronal sodium channels, and thereby neurotransmission, death resulting from respiratory paralysis. Saxitoxin is produced by dinoflagellate species (and by some freshwater cyanobacteria), and accumulates in shellfish. The cationic nature of saxitoxin makes capillary electrophoresis combined with... 

Almost all Schedule 1 chemicals are soluble in the organic NMR solvents used in verification (see Section 3.1) and can be analyzed by NMR spectroscopy. Saxitoxin (1.A.7) and ricin (1.A.8) differ from the others. Both are derived from natural sources - the former is a paralytic shellfish poison and the latter, a glycoprotein toxin (40). Analytical methods (ROPs) have not been established for either chemical. NMR data... 

Saxitoxin (STX) is a potent neurotoxin that can cause paralytic shellfish poisoning (PSP). Produced by certain strains of dinoflagellates, saxitoxin leads to the contamination of commercial shellfish and cause severe outbreaks of seafood poisoning. The public health problems caused by these outbreaks have led to significant interest in the development of analytical methods for the analysis of saxitoxin in environmental and biological samples. Saxitoxin is also one of a series of several closely related... 

CE analysis with direct UV absorbance detection at 200 nm has been described for the separation and detection of underivatized toxins, including saxitoxin, associated with paralytic shellfish poisoning (27). Confirmation of the electrophoretic peaks was made by CE/ESI/MS. Saxitoxin and neosaxi-toxin (NEO) were separated using a 20 mM sodium citrate buffer at pH 2.1 yielding a mass LOD of 15 pg (5 xM) for saxitoxin. 

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