Project description:Being able to quantify ichthyotoxic metabolites from microalgae allows for the determination of ecologically-relevant concentrations that can be simulated in laboratory experiments, as well as to investigate bioaccumulation and degradation. Here, the ichthyotoxin karmitoxin, produced by Karlodinium armiger, was quantified in laboratory-grown cultures using high-performance liquid chromatography (HPLC) coupled to electrospray ionisation high-resolution time-of-flight mass spectrometry (HRMS). Prior to the quantification of karmitoxin, a standard of karmitoxin was purified from K. armiger cultures (80 L). The standard was quantified by fluorescent derivatisation using Waters AccQ-Fluor reagent and derivatised fumonisin B₁ and fumonisin B₂ as standards, as each contain a primary amine. Various sample preparation methods for whole culture samples were assessed, including six different solid phase extraction substrates. During analysis of culture samples, MS source conditions were monitored with chloramphenicol and valinomycin as external standards over prolonged injection sequences (>12 h) and karmitoxin concentrations were determined using the response factor of a closely eluting iturin A2 internal standard. Using this method the limit of quantification was 0.11 μg·mL-1, and the limit of detection was found to be 0.03 μg·mL-1. Matrix effects were determined with the use of K. armiger cultures grown with 13C-labelled bicarbonate as the primary carbon source.
Project description:The karlotoxins are a family of amphidinol-like compounds that play roles in avoiding predation and in prey capture for the toxic dinoflagellate Karlodinium veneficum. The first member of the toxin group to be reported was KmTx 1 (1), and here we report an additional five new members of this family (3-7) from the same strain. Of these additional compounds, KmTx 3 (3) differs from KmTx 1 (1) in having one less methylene group in the saturated portion of its lipophilic arm. In addition, 64-E-chloro-KmTx 3 (4) and 10-O-sulfo-KmTx 3 (5) were identified. Likewise, 65-E-chloro-KmTx 1 (6) and 10-O-sulfo-KmTx 1 (7) were also isolated. Comparison of the hemolytic activities of the newly isolated compounds to that of KmTx 1 shows that potency correlates positively with the length of the lipophilic arm and is disrupted by sulfonation of the polyol arm.
Project description:Marine algae from the genus Karlodinium are known to be involved in fish-killing events worldwide. Here we report for the first time the chemistry and bioactivity of a natural product from the newly described mixotrophic dinoflagellate Karlodinium armiger. Our work describes the isolation and structural characterization of a new polyhydroxy-polyene named karmitoxin. The structure elucidation work was facilitated by use of 13C enrichment and high-field 2D NMR spectroscopy, where 1H-13C long-range correlations turned out to be very informative. Karmitoxin is structurally related to amphidinols and karlotoxins; however it differs by containing the longest carbon-carbon backbone discovered for this class of compounds, as well as a primary amino group. Karmitoxin showed potent nanomolar cytotoxic activity in an RTgill-W1 cell assay as well as rapid immobilization and eventual mortality of the copepod Acartia tonsa, a natural grazer of K. armiger.