Isolation of toxins

Boisier, P., et al.. Fatal mass poisoning in Madagascar following ingestion of a shark (Carcharhinus leucas) clinical and epidemiological aspects and isolation of toxins, Toxicon, 33, 10, 1359, 1995. 

Isolation of toxins. The digestive glands of shellfish were extracted with acetone at room temperature. After removal of the acetone by evaporation, the aqueous suspension was extracted with diethyl ether. The ether soluble residue was successively chromatographed twice over silicic acid columns with following solvents benzene to benzene-methanol (9 1), and diethyl ether to diethyl ether-methanol (1 1). To avoid degradation of dinophysistoxin-3 by contaminant acid, the silicic acid was washed with dilute sodium hydroxide solution and then with water prior to activation at 110 C. Toxic residue obtained in the second eluates was separated into two fractions... 

Figure 1. Flow diagram for the isolation of toxins Gambierdiscus toxicus.

Eotuhnum neurotoxins (EoNTs) are perhaps the most lethal toxins known. EoNTs are a set of seven serotypes (A, E, C, D, E, F and G) that are produced almost exclusively by the bacteria Clostridium botulinum. Serotypes C and D are found in birds and non-human mammals. Types A, E, E and F have been implicated in human cases of botuhsm. LD50 values for EoNTs range from 1.1 to 2.5 ng/kg body weight [7]. EoNTs have been associated with a variety of foods, including honey, chili, and hash browns. Isolation of toxins from the suspected food is the current means of diagnosis. 

As discussed above, there is the possibility that toxigenesis in Alexandrium is not intrinsic but due to symbionts. Whichever proves to be the case, the observed patterns of toxin composition, whether they are for the dinoflagellate itself or the isolated assemblage of dinoflagellate and symbiont, are a basis for recognizing and distinguishing the regional populations. 

Tetrodotoxin (TTX) and saxitoxin (STX) are potent sodium channel blockers that are found in phylogenetically diverse species of marine life. The wide distribution of TTX and STX has resulted in speculation that bacteria are the source of these toxins. Recently, investigators have reported isolation of marine bacteria, including Vibrio Alteromonas, Plesiomonas, and Pseudomonas species, that produce TTX and STX. This chapter details the methods and results of research to define bacterial sources of TTX and STX. 

Research in this area advanced in the 1970 s as several groups reported the isolation of potent toxins from P. brevis cell cultures (2-7). To date, the structures of at least eight active neurotoxins have been elucidated (PbTx-1 through PbTx-8) (8). Early studies of toxic fractions indicated diverse pathophysiological effects in vivo as well as in a number of nerve and muscle tissue preparations (reviewed in 9-11). The site of action of two major brevetoxins, PbTx-2 and PbTx-3, has been shown to be the voltage-sensitive sodium channel (8,12). These compounds bind to a specific receptor site on the channel complex where they cause persistent activation, increased Na flux, and subsequent depolarization of excitable cells at resting... 

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

Of particular interest in brevetoxin research are the diagnosis of intoxication and identification of brevetoxins and their metabolites in biological fluids. We are investigating the distribution and fate of radiolabeled PbTx-3 in rats. Three model systems were used to study the toxicokinetics and metabolism of PbTx-3 1) rats injected intravenously with a bolus dose of toxin, 2) isolated rat livers perfused with toxin, and 3) isolated rat hepatocytes exposed to the toxin in vitro. 

These studies represent the first report of the metabolism of brevetoxins by mammalian systems. PbTx-3 was rapidly cleared from the bloodstream and distributed to the liver, muscle, and gastrointestinal tract. Studies with isolated perfused livers and isolated hepatocytes conflrmed the liver as a site of metabolism and biliary excretion as an important route of toxin elimination. [ H]PbTx-3 was metabolized to several compounds exhibiting increased polarity, one of which appeared to be an epoxide derivative. Whether this compound corresponds to PbTx-6 (the 27,28 epoxide of PbTx-2), to the corresponding epoxide of PbTx-3, or to another structure is unknown. The structures of these metabolites are currently under investigation. 

Data from both in vivo and in vitro systems showed PbTx-3 to have an intermediate extraction ratio, indicating in vivo clearance of PbTx-3 was equally dependent upon liver blood flow and the activity of toxin-metabolizing enzymes. Studies on the effects of varying flow rates and metabolism on the total body clearance of PbTx-3 are planned. Finally, comparison of in vivo metabolism data to those derived from in vitro metabolism in isolated perfused livers and isolated hepatocytes suggested that in vitro systems accurately reflect in vivo metabolic processes and can be used to predict the toxicokinetic parameters of PbTx-3. 

Chemical techniques for the isolation, purification and elucidation of the structure of toxins have evolved to the extent that it is frequently a routine procedure to identify the chemical nature of a newly discovered toxin once it has been purified, although difficulties arise when the toxin is a very large polypeptide, protein, or a very complex organic molecule. However, it is sometimes found that a toxin becomes progressively more labile and stabilizing contaminants are removed by the purification processes. An example of this is Cyanea toxic material which becomes increasingly labile with each purification step 111). 

With the extraction procedure we employed (22), ferulic acid was isolated as the most inhibitory component in wheat straw. There could also be other unknown compounds in the straw which would not be evident with this procedure. In addition, we ignored the possible influence of toxin-producing microorganisms. Microorganisms may have influenced the phytotoxicity exhibited by the aqueous wheat extract in Table IX. Although the present study was not concerned with the phytotoxic effects of microbially decomposed wheat straw, an influence of microbial activity on ferulic acid phytotoxicity was observed. From the results shown in Table XI, it appears that the presence of the prickly sida seed carpel enhanced the inhibitory effects of ferulic acid. In addition to ferulic acid in test solutions containing prickly sida seeds with carpels, a second compound, 4-hydroxy-3-methoxy styrene, was also found to be present. This compound is formed by the decarboxylation of ferulic acid and was produced by a bacterium present on the carpel of prickly sida seed. The decarboxylation of ferulic acid was detected in aqueous solutions of ferulic acid inoculated with the bacterium isolated from the carpels of prickly sida seed. No conversion occurred when the bacterium was not present. 

Foster G, Ross H M, Pennycott T W, Hopkins G F and McLaren I M (1998), Isolation of Escherichia coli 086 K61 producing cyto-lethal distending toxin from wild birds of the finch family , Letters in Appl. Microbiology, 26, 395-398. 

A-chain immunotoxins, however, may not be quite as cytotoxic as conjugates formed from intact toxin molecules (Manske et al., 1989). In an alternative approach to A chain use, the intact toxin of two-subunit proteins is directly conjugated to a monoclonal without isolation of the A chain. Conjugation of an antibody with intact A-B chain toxins can be done without a cleavable linker, as long as the A chain can still separate from the B chain once it is internalized. Therefore, it is important to avoid intramolecular crosslinking during the conjugation process which can prevent release of the A-B complex. In addition, since the B chain... 

React for 18 hours at room temperature to form the final conjugate. Isolation of the ideal 1 1 or 1 2 antibody-toxin conjugate can be done through gel filtration separation using a column of Sephacryl S-300 or the equivalent. 

Figure 21.8 SMPT may be used to form immunotoxin conjugates by activation of the antibody component to form a thiol-reactive derivative. Reduction of an A-B toxin molecule with DTT can facilitate subsequent isolation of the A chain containing a free thiol. Mixing the A-chain containing a sulfhydryl group with the SMPT-activated antibody causes immunotoxin formation through disulfide bond linkage. The hindered disulfide of an SMPT crosslink has been found to survive in vivo for longer periods than conjugates formed with SPDP.

CDC Case Definition An illness characterized by diarrhea and/or vomiting severity is variable. Laboratory criteria for diagnosis is (1) isolation of toxigenic (i.e., cholera toxin-producing) V. cholerae Ol or0139 from stool orvomitus or (2) serologic evidence of recent infection. 

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