5 research outputs found
Improved Isolation Procedure for Azaspiracids from Shellfish, Structural Elucidation of Azaspiracid-6, and Stability Studies
Azaspiracids are a group of lipophilic polyether toxins
produced
by the small dinoflagellate <i>Azadinium spinosum</i>. 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 (<b>1</b>, <b>2</b>, and <b>3</b>) increasing recoveries 2-fold as compared to previous methods
and leading to isolation of sufficiently purified azaspiracid-6 (<b>6</b>) for structural determination by NMR spectroscopy. The procedure,
which involved a series of partitioning and column chromatography
steps, was performed on 500 g of <i>Mytilus edulis</i> hepatopancreas
tissue containing ∼14 mg of <b>1</b>. Overall yields
of <b>1</b> (52%), <b>2</b> (43%), <b>3</b> (43%),
and <b>6</b> (38%) were good, and purities were confirmed by
NMR spectroscopy. The structure of <b>6</b> was determined by
one- and two-dimensional NMR spectroscopy and mass spectrometry. The
stability of <b>6</b> relative to <b>1</b> was also assessed
in three solvents in a short-term study that demonstrated the greatest
stability in aqueous acetonitrile
Development of an ELISA for the Detection of Azaspiracids
Azaspiracids
(AZAs) are a group of biotoxins that cause food poisoning
in humans. These toxins are produced by small marine dinoflagellates
such as Azadinium spinosum and accumulate
in shellfish. Ovine polyclonal antibodies were produced and used to
develop an ELISA for quantitating AZAs in shellfish, algal cells,
and culture supernatants. Immunizing antigens were prepared from synthetic
fragments of the constant region of AZAs, while plate coating antigen
was prepared from AZA-1. The ELISA provides a sensitive and rapid
analytical method for screening large numbers of samples. It has a
working range of 0.45–8.6 ng/mL and a limit of quantitation
for total AZAs in whole shellfish at 57 μg/kg, well below the
maximum permitted level set by the European Commission. The ELISA
has good cross-reactivity to AZA-1–10, -33, and -34 and 37-<i>epi</i>-AZA-1. Naturally contaminated Irish mussels gave similar
results whether they were cooked or uncooked, indicating that the
ELISA also detects 22-carboxy-AZA metabolites (e.g., AZA-17 and AZA-19).
ELISA results showed excellent correlation with LC-MS/MS analysis,
both for mussel extract spiked with AZA-1 and for naturally contaminated
Irish mussels. The assay is therefore well suited to screening for
AZAs in shellfish samples intended for human consumption, as well
as for studies on AZA metabolism
Epimers of Azaspiracids: Isolation, Structural Elucidation, Relative LC-MS Response, and <i>in Vitro</i> Toxicity of 37-<i>epi</i>-Azaspiracid‑1
Since
azaspiracid-1 (AZA1) was identified in 1998, the number of
AZA analogues has increased to over 30. The development of an LC-MS
method using a neutral mobile phase led to the discovery of isomers
of AZA1, AZA2, and AZA3, present at ∼2–16% of the parent
analogues in phytoplankton and shellfish samples. Under acidic mobile
phase conditions, isomers and their parents are not separated. Stability
studies showed that these isomers were spontaneous epimerization products
whose formation is accelerated with the application of heat. The AZA1
isomer was isolated from contaminated shellfish and identified as
37-<i>epi</i>-AZA1 by nuclear magnetic resonance (NMR) spectroscopy
and chemical analyses. Similar analysis indicated that the isomers
of AZA2 and AZA3 corresponded to 37-<i>epi</i>-AZA2 and
37-<i>epi</i>-AZA3, respectively. The 37-epimers were found
to exist in equilibrium with the parent compounds in solution. 37-<i>epi</i>-AZA1 was quantitated by NMR, and relative molar response
studies were performed to determine the potential differences in LC-MS
response of AZA1 and 37-<i>epi</i>-AZA1. Toxicological effects
were determined using Jurkat T lymphocyte cells as an <i>in vitro</i> cell model. Cytotoxicity experiments employing a metabolically based
dye (i.e., MTS) indicated that 37-<i>epi</i>-AZA1 elicited
a lethal response that was both concentration- and time-dependent,
with EC<sub>50</sub> values in the subnanomolar range. On the basis
of EC<sub>50</sub> comparisons, 37-<i>epi</i>-AZA1 was 5.1-fold
more potent than AZA1. This data suggests that the presence of these
epimers in seafood products should be considered in the analysis of
AZAs for regulatory purposes
Structure Elucidation, Relative LC–MS Response and In Vitro Toxicity of Azaspiracids <b>7</b>–<b>10</b> Isolated from Mussels (Mytilus edulis)
Azaspiracids
(AZAs) are marine biotoxins produced by dinoflagellates that can accumulate
in shellfish, which if consumed can lead to poisoning events. AZA7–10, <b>7</b>–<b>10</b>, were isolated from shellfish and
their structures, previously proposed on the basis of only LC–MS/MS
data, were confirmed by NMR spectroscopy. Purified AZA4–6, <b>4</b>–<b>6</b>, and <b>7</b>–<b>10</b> were accurately quantitated by qNMR and used to assay cytotoxicity
with Jurkat T lymphocyte cells for the first time. LC–MSÂ(MS)
molar response studies performed using isocratic and gradient elution
in both selected ion monitoring and selected reaction monitoring modes
showed that responses for the analogues ranged from 0.3 to 1.2 relative
to AZA1, <b>1</b>. All AZA analogues tested were cytotoxic to
Jurkat T lymphocyte cells in a time- and concentration-dependent manner;
however, there were distinct differences in their EC<sub>50</sub> values,
with the potencies for each analogue being: AZA6 > AZA8 > AZA1
> AZA4 ≈ AZA9 > AZA5 ≈ AZA10. This data contributes
to the understanding of the structure–activity relationships
of AZAs
Isolation, Structure Elucidation, Relative LC-MS Response, and in Vitro Toxicity of Azaspiracids from the Dinoflagellate <i>Azadinium spinosum</i>
We identified three new azaspiracids
(AZAs) with molecular weights
of 715, 815, and 829 (AZA33 (<b>3</b>), AZA34 (<b>4</b>), and AZA35, respectively) in mussels, seawater, and <i>Azadinium
spinosum</i> culture. Approximately 700 μg of <b>3</b> and 250 μg of <b>4</b> were isolated from a bulk culture
of <i>A. spinosum</i>, and their structures determined by
MS and NMR spectroscopy. These compounds differ significantly at the
carboxyl end of the molecule from known AZA analogues and therefore
provide valuable information on structure–activity relationships.
Initial toxicological assessment was performed using an in vitro model
system based on Jurkat T lymphocyte cytotoxicity, and the potencies
of <b>3</b> and <b>4</b> were found to be 0.22- and 5.5-fold
that of AZA1 (<b>1</b>), respectively. Thus, major changes in
the carboxyl end of <b>1</b> resulted in significant changes
in toxicity. In mussel extracts, <b>3</b> was detected at low
levels, whereas <b>4</b> and AZA35 were detected only at extremely
low levels or not at all. The structures of <b>3</b> and <b>4</b> are consistent with AZAs being biosynthetically assembled
from the amino end