okadaic-acid and 9-diazomethylanthracene

A new analogue of okadaic acid (OA), the toxin mainly responsible for diarrhetic shellfish-poisoning (DSP) phenomena in Europe, has been isolated from toxic phytoplankton (Dinophysis acuta) collected in Irish waters. Fluorimetric LC analyses of the extracts of bulk phytoplankton samples using derivatisation with 9-anthryldiazomethane (ADAM) showed a complex toxin profile, with peaks corresponding to OA and dinophysistoxin-2 (DTX-2) as well as a third unidentified compound. This minor unidentified component was isolated by chromatographic techniques such as normal-phase chromatography, gel permeation on Sephadex, solid-phase extraction and reversed-phase separations. Ionspray mass spectrometry (MS) was used for structural investigation on this compound due to the very small amount of isolated material. Flow injection analysis (FIA)-MS of the isolated compound gave positive-ion mass spectrum dominated by the protonated molecule, [M + H]+, at signal m/z 805, whereas the deprotonated molecule [M - H]- was observed in the negative-ion spectrum at signal m/z 803, thus indicating the molecular weight of 804 for the new toxin, the same as OA and its known isomers, DTX-2 and DTX-2B. Collision-induced dissociation (CID) as obtained by positive and negative tandem mass spectrometry (MS-MS) showed a fragmentation pattern for the new compound which was very similar to that of OA, DTX-2 and DTX-2B. Ionspray microLC-MS of a mixture containing the compound under investigation together with OA analogues showed the compound eluted after OA, DTX-2, DTX-2B and before DTX-1. All the chromatographic and mass spectrometric data indicated the compound to be another OA isomer and it was therefore coded DTX-2C. To the best of our knowledge this is the first report on the isolation of a new compound related to DSP toxins from natural communities of toxic phytoplankton.

Topics: Anthracenes; Chromatography, High Pressure Liquid; Diarrhea; Fluorescent Dyes; Fluorometry; Food Analysis; Foodborne Diseases; Marine Toxins; Mass Spectrometry; Okadaic Acid; Phytoplankton; Pyrans

Okadaic acid (OA) is the main toxin produced by dinoflagellates, which can accumulate in the hepatopancreas of mussels and cause diarrhoetic shellfish poisoning in consumers. This toxin is also a tumour promoter and a specific potent inhibitor of protein phosphatases 1 and 2A. The results in this study show for the first time that this marine toxin is able to cross the transplacental barrier. Foetal tissue contains more okadaic acid than the liver or kidney: 5.60% compared to 1.90 and 2.55% respectively as measured by HPLC and fluorescent detection after derivatization with 9-Anthryldiazomethane (ADAM). In view of its adverse effects, okadaic acid might impair foetal development and promote tumours in neonates.

Topics: Animals; Anthracenes; Chromatography, High Pressure Liquid; Female; Fluorescent Dyes; Maternal-Fetal Exchange; Mice; Okadaic Acid; Pregnancy; Scintillation Counting; Tritium

Several derivatization reagents for the conversion of okadaic acid and related DSP toxins to fluorescent derivatives for analysis by liquid chromatography have been examined, viz: 9-anthryldiazomethane (ADAM), 1-pyrenyldiazomethane (PDAM), 4-diazomethyl-7-methoxycoumarin (DMMC), 4-bromomethyl-7-methoxycoumarin (BrMMC), 4-bromomethyl-7,8-benzcoumarin (BrMBC), 4-bromomethyl-7-acetoxycoumarin (BrMAC), and 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC). The ADAM reagent provides the greatest selectivity and sensitivity, but its application on a routine basis has been limited by its instability and cost. Improvement of this method was achieved through the production of ADAM in situ from the stable 9-anthraldehyde hydrazone. A detection limit of 30 ng/g hepatopancreas (equivalent to 6 ng/g whole tissue) was achieved. The other aryldiazomethane reagents were found to have insufficient reactivity. Of the bromomethylcoumarin reagents, BrDMC was found to have the greatest promise. The reagent is inexpensive and has excellent stability and purity. Quantitative derivatization may be achieved in a 2 hour reaction at 45 degrees C with N,N-diisopropylethylamine as a catalyst. Unfortunately, the lower reaction selectivity of BrDMC compared to that of ADAM limits its application to isolated toxins, plankton samples, and shellfish tissues with high levels of DSP toxins. The use of BrDMC for the determination of how toxin levels in shellfish tissues will require development of a more extensive clean-up prior to derivatization. Successful application of the ADAM and coumarin derivatization methods to real-world samples has been demonstrated.

Topics: Animals; Anthracenes; Chromatography, High Pressure Liquid; Coumarins; Dinoflagellida; Ethers, Cyclic; Fluorescent Dyes; Marine Toxins; Okadaic Acid; Pyrans; Pyrenes; Shellfish; Spectrometry, Fluorescence; Temperature; Umbelliferones

Diarrheic toxins, especially okadaic acid, are detected nearly every year in mussels on French coasts. The monitoring network determines the toxicity of these shellfish by using a mouse test now considered unsatisfactory from an ethical point of view. Two alternative methods have been investigated: the daphnia test, for which there is a standardized method used routinely in ecotoxicology, and a cytotoxicity test on the KB cell line developed for this study. Using the same samples, the results of these two tests were compared with those obtained by chemical analysis (HPLC okadaic acid assay) or the mouse test. Linear regression studies showed that results for the two bioassays were well correlated with those for HPLC or the mouse test.

Topics: Animals; Anthracenes; Biological Assay; Bivalvia; Carcinoma, Squamous Cell; Cell Survival; Chromatography, High Pressure Liquid; Daphnia; Ethers, Cyclic; Fluorescent Dyes; Foodborne Diseases; France; Humans; Linear Models; Male; Mice; Okadaic Acid; Reference Standards; Tumor Cells, Cultured

Okadaic acid and dinophysistoxin-1, the principal toxic components in diarrhetic shellfish poisoning, may be detected by high-performance liquid chromatography and fluorometric measurement as 9-anthrylmethyl esters. However, "greasy" samples may occur and the fluorescent reagent 9-anthryldiazomethane may decompose during storage, resulting in impurities that may seriously interfere with quantitative determination. Ultrasonic treatment of the samples during derivatization with 9-anthryldiazomethane was found to improve reproducibility. This may result from increased access to reactive sites on toxins by 9-anthryldiazomethane due to disruption of micelles formed by toxins and other partly hydrophobic compounds. A procedure for cleaning the derivatized samples, using a 0.1 g silica cartridge column and different eluent compositions from that reported by LEE et al. (1987), was found to facilitate chromatogram interpretation. Deoxycholic acid, a commercial available bile acid, was found to be an acceptable internal standard. The 9-anthrylmethyl esters of okadaic acid, dinophysistoxin-1 and deoxycholic acid, were stable at 4 degrees C for at least seven days when stored dry or in methanol.

Topics: Animals; Anthracenes; Bivalvia; Chromatography, High Pressure Liquid; Esters; Ethers, Cyclic; Fluorescent Dyes; Marine Toxins; Okadaic Acid; Pyrans; Reproducibility of Results; Ultrasonics