RESUMO
[D-Leu1]MC-LY (1) ([M + H]+m/z 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from Microcystis aeruginosa strain CPCC464. The compound was characterized by 1H and 13C NMR spectroscopy, liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and UV spectroscopy. A calibration reference material was produced after quantitation by 1H NMR spectroscopy and LC with chemiluminescence nitrogen detection. The potency of 1 in a protein phosphatase 2A inhibition assay was essentially the same as for MCLR (2). Related microcystins, [D-Leu1]MC-LR (3) ([M + H]+m/z 1037.6041, Δ 1.0 ppm), [D-Leu1]MC-M(O)R (6) ([M + H]+m/z 1071.5565, Δ 2.0 ppm) and [D-Leu1]MC-MR (7) ([M + H]+m/z 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu1]MC-M(O2)R (8) ([M + H]+m/z 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC-HRMS/MS experiments. The relative abundances of 1, 3, 6, 7 and 8 in a freshly extracted culture in the positive ionization mode LC-HRMS were ca. 84, 100, 3.0, 11 and 0.05, respectively. These and other results indicate that [D-Leu1]-containing MCs may be more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC-MS/MS screening methods.
Assuntos
Microcistinas/isolamento & purificação , Microcystis , Cromatografia Líquida , Microcistinas/química , Proteína Fosfatase 2/antagonistas & inibidores , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas em TandemRESUMO
Many toxic secondary metabolites used for defense are also toxic to the producing organism. One important way to circumvent toxicity is to store the toxin as an inactive precursor. Several sulfated diesters of the diarrhetic shellfish poisoning (DSP) toxin okadaic acid have been reported from cultures of various dinoflagellate species belonging to the genus Prorocentrum. It has been proposed that these sulfated diesters are a means of toxin storage within the dinoflagellate cell, and that a putative enzyme mediated two-step hydrolysis of sulfated diesters such as DTX-4 and DTX-5 initially leads to the formation of diol esters and ultimately to the release of free okadaic acid. However, only one diol ester and no sulfated diesters of DTX-1, a closely related DSP toxin, have been isolated leading some to speculate that this toxin is not stored as a sulfated diester and is processed by some other means. DSP components in organic extracts of two large scale Prorocentrum lima laboratory cultures have been investigated. In addition to the usual suite of okadaic acid esters, as well as the free acids okadaic acid and DTX-1, a group of corresponding diol- and sulfated diesters of both okadaic acid and DTX-1 have now been isolated and structurally characterized, confirming that both okadaic acid and DTX-1 are initially formed in the dinoflagellate cell as the non-toxic sulfated diesters.
Assuntos
Ácido Okadáico/análise , Piranos/análise , Intoxicação por Frutos do Mar , Animais , Dinoflagellida/metabolismo , Toxinas Marinhas/análiseRESUMO
Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties.
Assuntos
Diarreia/complicações , Toxinas Marinhas/análise , Ácido Okadáico/análise , Piranos/análise , Padrões de Referência , Intoxicação por Frutos do Mar/complicações , Animais , Calibragem , Cromatografia Líquida/normas , Humanos , Espectroscopia de Ressonância Magnética/normas , Frutos do Mar , Espectrometria de Massas em Tandem/normasRESUMO
Spirolides are marine phycotoxins produced by the dinoflagellates Alexandrium ostenfeldii and A. peruvianum. Here we report that 13-desmethyl spirolide C shows little cytotoxicity when incubated with various cultured mammalian cell lines. When administered to mice by intraperitoneal (ip) injection, however, this substance was highly toxic, with an LD(50) value of 6.9 µg/kg body weight (BW), showing that such in vitro cytotoxicity tests are not appropriate for predicting the in vivo toxicity of this toxin. Four other spirolides, A, B, C, and 20-methyl spirolide G, were also toxic to mice by ip injection, with LD(50) values of 37, 99, 8.0 and 8.0 µg/kg BW respectively. However, the acute toxicities of these compounds were lower by at least an order of magnitude when administration by gavage and their toxic effects were further diminished when administered with food. These results have implications for future studies of the toxicology of these marine toxins and the risk assessment of human exposure.
Assuntos
Toxinas Marinhas/toxicidade , Compostos de Espiro/toxicidade , Animais , Linhagem Celular , Feminino , Humanos , Dose Letal Mediana , Camundongos , Medição de RiscoRESUMO
Azaspiracids are a group of lipophilic polyether toxins produced by the small dinoflagellate Azadinium spinosum. They may accumulate in shellfish and can result in illnesses when consumed by humans. Research into analytical methods, chemistry, metabolism, and toxicology of azaspiracids has been severely constrained by the scarcity of high-purity azaspiracids. Consequently, since their discovery in 1995, considerable efforts have been made to develop methods for the isolation of azaspiracids in sufficient amounts and purities for toxicological studies, in addition to the preparation of standard reference materials. A seven-step procedure was improved for the isolation of azaspiracids-1-3 (1, 2, and 3) increasing recoveries 2-fold as compared to previous methods and leading to isolation of sufficiently purified azaspiracid-6 (6) for structural determination by NMR spectroscopy. The procedure, which involved a series of partitioning and column chromatography steps, was performed on 500 g of Mytilus edulis hepatopancreas tissue containing ~14 mg of 1. Overall yields of 1 (52%), 2 (43%), 3 (43%), and 6 (38%) were good, and purities were confirmed by NMR spectroscopy. The structure of 6 was determined by one- and two-dimensional NMR spectroscopy and mass spectrometry. The stability of 6 relative to 1 was also assessed in three solvents in a short-term study that demonstrated the greatest stability in aqueous acetonitrile.
Assuntos
Fracionamento Químico/métodos , Dinoflagellida/metabolismo , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Mytilus edulis/química , Frutos do Mar/análise , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Toxinas Biológicas/química , Toxinas Biológicas/isolamento & purificação , Animais , Toxinas Marinhas/metabolismo , Espectrometria de Massas , Mytilus edulis/parasitologia , Frutos do Mar/parasitologia , Intoxicação por Frutos do Mar , Compostos de Espiro/metabolismo , Toxinas Biológicas/metabolismoRESUMO
The production and certification of a series of azaspiracid (AZA) calibration solution reference materials is described. Azaspiracids were isolated from contaminated mussels, purified by preparative liquid chromatography and dried under vacuum to the anhydrous form. The purity was assessed by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The final concentration of each AZA in a CD(3)OH stock solution was determined by quantitative NMR spectroscopy. This solution was then diluted very accurately in degassed, high purity methanol to a concentration of 1.47 ± 0.08 µmol/L for CRM-AZA1, 1.52 ± 0.05 µmol/L for CRM-AZA2, and 1.37 ± 0.13 µmol/L for CRM-AZA3. Aliquots were dispensed into argon-filled glass ampoules, which were immediately flame-sealed. The calibration solutions are suitable for method development, method validation, calibration of liquid chromatography or mass spectrometry instrumentation and quality control of shellfish monitoring programs.
Assuntos
Furanos/análise , Toxinas Marinhas/análise , Piranos/análise , Frutos do Mar , Compostos de Espiro/análise , Animais , Bivalves/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Soluções/químicaRESUMO
A new subclass of spirolide marine toxins, represented by spirolides H (1) and I (2), were isolated from the marine dinoflagellate Alexandrium ostenfeldii. Spirolides H and I are structurally distinct from other spirolides in that they contain a 5:6 dispiroketal ring system rather than the trispiroketal ring system characteristic of previously isolated spirolides. The structures were assigned using a combination of spectrometric and spectroscopic techniques. Previously isolated spirolides containing a cyclic imine moiety showed toxicity in the mouse bioassay. Spirolide H contains this cyclic imine moiety but does not show toxicity in the mouse assay, suggesting that the presence of the cyclic imine moiety is not the only structural requirement for toxicity.
Assuntos
Dinoflagellida/química , Toxinas Marinhas/isolamento & purificação , Toxinas Marinhas/toxicidade , Compostos de Espiro/isolamento & purificação , Compostos de Espiro/toxicidade , Animais , Toxinas Marinhas/química , Camundongos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
Biosynthetic origins of the cyclic imine toxin 13-desmethyl spirolide C were determined by supplementing cultures of the toxigenic dinoflagellate Alexandrium ostenfeldii with stable isotope-labeled precursors [1,2-13C2]acetate, [1-13C]acetate, [2-13CD3]acetate, and [1,2-13C2,15N]glycine and measuring the incorporation patterns by 13C NMR spectroscopy. Despite partial scrambling of the acetate labels, the results show that most carbons of the macrocycle are polyketide-derived and that glycine is incorporated as an intact unit into the cyclic imine moiety. This work represents the first conclusive evidence that such cyclic imine toxins are polyketides and provides support for biosynthetic pathways previously defined for other polyether dinoflagellate toxins.
Assuntos
Dinoflagellida/metabolismo , Compostos Macrocíclicos/metabolismo , Toxinas Marinhas/metabolismo , Compostos de Espiro/metabolismo , Animais , Isótopos de Carbono , Dinoflagellida/química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/isolamento & purificação , Espectroscopia de Ressonância Magnética/métodos , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Estrutura Molecular , Sensibilidade e Especificidade , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , EstereoisomerismoRESUMO
Using LC/MS methodology, spirolides were detected in two clonal isolates of Alexandrium ostenfeldii isolated from Limfjorden, Denmark. Examination of the LC/MS profiles of extracts from these Danish cultures revealed the presence of two dominant peaks representing two previously unidentified spirolide components and one minor peak identified as the previously reported desmethyl spirolide C (1). Culturing of these clonal strains, LF 37 and LF 38, of A. ostenfeldii resulted in the accumulation of sufficient cell biomass to allow for the isolation and structure elucidation of two new spirolides, 13,19-didesmethylspirolide C (2) and spirolide G (3). While 2 was found to differ from 1 only in that it contained one less methyl group, 3 was the first spirolide to be isolated that contained a 5:6:6-trispiroketal ring system. The effect of this new feature on the toxicity of 3 relative to other spirolides is presently being pursued.
Assuntos
Dinoflagellida/química , Iminas , Toxinas Marinhas , Compostos de Espiro , Animais , Dinamarca , Iminas/química , Iminas/isolamento & purificação , Iminas/farmacologia , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Toxinas Marinhas/farmacologia , Estrutura Molecular , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Compostos de Espiro/farmacologiaRESUMO
A new pectenotoxin, which has been named pectenotoxin-11 (PTX11), was isolated from the dinoflagellate Dinophysis acuta collected from the west coast of New Zealand. The structure of PTX11 was determined as 34S-hydroxypectenotoxin-2 by tandem mass spectrometry and UV and NMR spectroscopy. PTX11 appears to be only the third pectenotoxin identified as a natural biosynthetic product from algae after pectenotoxin-2 and pectenotoxin-12. The LD50 of PTX11 determined by mouse intraperitoneal injection was 244 microg/kg. The LD(min) of PTX11 in these experiments was 250 microg/kg. No signs of toxicity were recorded in mice following an oral dose of PTX11 at 5000 microg/kg. No diarrhea was observed in any of the animals administered with the test substance by either route of administration. Unlike pectenotoxin-2 (PTX2), PTX11 was not readily hydrolyzed to its corresponding seco acid by enzymes from homogenized green-lipped mussel (Perna canaliculus) hepatopancreas.
Assuntos
Dinoflagellida/química , Furanos/química , Furanos/isolamento & purificação , Toxinas Marinhas/química , Piranos/química , Piranos/isolamento & purificação , Animais , Injeções Intraperitoneais , Dose Letal Mediana , Macrolídeos , Espectroscopia de Ressonância Magnética , Toxinas Marinhas/administração & dosagem , Toxinas Marinhas/toxicidade , Camundongos , Modelos Moleculares , Conformação Molecular , Nova Zelândia , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , Estereoisomerismo , Fatores de TempoRESUMO
Mussels sampled in the spring of 2002 and 2003 from Skjer, a location in Sognefjord, Norway, tested positive in the mouse bioassay for lipophilic toxins. The symptoms, which included cramps, jumping, and short survival times (as low as 4 min), were not characteristic of toxins previously observed in Norway. A survey of the algae present at the aquaculture sites showed that the toxicity correlated with blooms of Alexandrium ostenfeldii. Up to 2200 cells/L were found at the peak of one bloom. In Canadian waters, this alga is known to be a producer of the cyclic imine toxins, spirolides. Analysis of mussel extracts from Skjer in the spring of 2002 and 2003, using liquid chromatography tandem mass spectrometry, revealed the presence of several new spirolides. The same compounds were also found in algal samples dominated by A. ostenfeldii, which had been sampled from Skjer in February 2003. A large-scale extraction of mussel digestive glands and chromatographic fractionation of the extracts allowed the isolation and structure elucidation of the main spirolide, 20-methyl spirolide G, with a molecular weight of 705.5. This is the first confirmed occurrence of spirolides in mussels and plankton from Norway.
Assuntos
Acetais/isolamento & purificação , Bivalves/química , Lactonas/isolamento & purificação , Toxinas Marinhas/isolamento & purificação , Plâncton/química , Frutos do Mar/análise , Compostos de Espiro/isolamento & purificação , Animais , Dinoflagellida/química , Feminino , Camundongos , NoruegaRESUMO
The biosynthetic origins of the diarrhetic shellfish poisoning toxins DTX-5a and DTX-5b have been elucidated by supplementing cultures of the producing organism Prorocentrum maculosum with stable isotope labeled precursors and determining the incorporation patterns by 13C NMR spectroscopy. The amino acid residue in the sulfated side chain is found to originate from glycine, and oxygen insertion in the chain is shown to occur after polyketide formation.