Dello Iacovo, Emma
Chemical analysis of toxic microalgae from Mediterranean Sea.
[Tesi di dottorato]
Marine plankton is constituted by microalgae existing either in unicellular forms or in colonies of cells. These invisible organisms play a key role in the aquatic ecosystem as they are the producers of organic material thus being the first ring of the aquatic food chain.
Algal proliferation in the plankton, commonly referred to as algal bloom, is a periodic phenomenon occurring in many countries across the world usually in coincidence with particular climatic and environmental events. Blooms due to microalgae producing biotoxins are known as “harmful algal blooms”, as they pose serious threats to human health. In fact, oysters, mussels, clams and in general bivalve organisms, while feeding by filtering seawater, can accumulate toxins in their edible tissues sometimes to such a level to harass unaware consumers. Hence it can be stated that bivalves constitute a crucial ring of the food chain responsible for transferring toxicity from the plankton onto humans.
During my PhD within Prof. Ciminiello’s research group, I investigated the toxic profile of the Mediterranean Sea where over the past decades a number of toxic microalgae have bloomed. In particular, I focused my attention on toxic outbreaks caused by microalgae belonging to the Ostreopsis genus, quite rife along the Italian coasts.
Harmful algal blooms linked to Ostreopsis spp. reached alarming proportions in the late July of 2005 and 2006, when many people required extended hospitalization for respiratory distress after exposure to marine aerosol along the beach and promenade of Genoa. During these toxic episodes Prof. Ciminiello’s research group succeeded in identifying a putative palytoxin - the most potent non-proteic toxin so far known (DL50 <100 nanograms/Kg) - as well as a new palytoxin analog, ovatoxin-a, as the two main compounds responsible for the human poisoning. This was achieved by setting up a new analytical method for detecting palytoxin based on association of liquid chromatography with mass spectrometry (LC-MS). In the wake of this research on Ostreopsis spp. I carried out in-depth studies on O. ovata toxic profile succeeding in identifying four new palytoxin-like compounds, named ovatoxin-b, -c, -d, and, -e by High Resolution (HR)LC-MS and MS/MS experiments on a hybrid Linear Ion Trap (LTQ) Orbitrap XLTM FTMS operating up to 100,000 of resolution power. On the basis of the MS data in our hands we could also give some preliminary information about their structures; however NMR mono- and bi-dimensional experiments are needed to definitively assign their chemical architecture.
Since over the last years the restating of the O. ovata summer blooms has raised serious concerns to both human health and economy, from October 2006 a commission appointed by the Ministero della Salute has been in charge of investigating and monitoring Ostreopsis phenomenon along the whole Italian coasts to the aim of evaluating risks to human health and preventing any possible human intoxication. So, from the summer of 2007 on, a program of monitoring in the frame of the project “Monitoraggio Ostreopsis ovata litorale costiero Regione Campania” was started by ARPA Campania in collaboration with Stazione Zoologica A. Dohrn, Istituto Zooprofilattico Sperimentale del Mezzogiorno, and with Dipartimento di Chimica delle Sostanze Naturali, where I carried out my PhD. Therefore, every summer from 2007 to 2010, during the O. ovata blooms I carried out LC-MS analyses on samples of both mussels and sea-urchins, collected along the Campania coasts, once proven positive to the mouse bioassay. For many Campania sites, for which our analyses showed toxin contents above the EFSA (European Food Safety Authority)-established tolerance limits (30 μg/Kg), mussel harvesting was banned. Such a ban was lifted only when our analyses highlighted toxin amounts in the investigated samples well below the above official limits.
Considering the deep sanitary and economic impact due to the Ostreopsis outbreaks, I also tried to set up a suitable purification procedure for ovatoxin-a – which accounts for 50% of the total toxin content - with the purpose of obtaining sufficient amount of pure compound for investigating its toxicology as well as its mechanism of action. The best results were obtained by extracting ovatoxin-a from algal pellets with methanol and methanol/water 1:1, followed by partition of the water extract with chloroform, and then by a medium pressure chromatographic separation employing a reverse stationary phase and a UV instrument as detector, and eventually by a high pressure chromatographic separation using LTQ Orbitrap XL FTMS as detector. In this frame I also analyzed O. ovata cultures coming from the Adriatic (Ancona) and the Tyrrhenian Seas (Latina) in collaboration with the University of Bologna. Pellet samples and culture mediums were collected in both exponential and stationary cellular growth phases, extracted, and finally analyzed by LC-MS to the aim of evaluating the toxins production in the different Italian strains and at the different growth levels. Our studies showed that the Adriatic strain is richer in toxins than the Tyrrhenian one, and the toxins content during the stationary growth phase is higher than that of the exponential phase.
In the field of this research on palytoxins, I also studied extracts from soft corals belonging to the Palythoa genus collected in Hawaii. In particular, I carried out LC-MS analyses on a triple quadrupole mass spectrometer at unit resolution, with addiction of NaCl and KCl to the mobile phase to induce adduct ions production. LC-MS experiments were also recorded on a time of flight instrument, operating at high resolution. The whole of the above studies allowed us to individuate a new palytoxin analogue whose molecular formula (C129H224N3O55) appeared to contain an oxygen atom more than palytoxin (C129H224N3O54). NMR mono- and bi-dimensional experiments on the new palytoxin analogue identified it as 42-OH palytoxin.
Another subject of my research was the investigation of the toxin profile of Alexandrium. ostenfeldii, a dinoflagellate producing spirolides, fast acting toxins with an unusual 7-membered spiro-linked cyclic imine moiety. Beyond the fact that once injected intraperitoneally into a mouse spirolides give rise to neurologic symptoms, their human toxicity is largely unknown. A. ostenfeldii samples collected in the Adriatic Sea were extracted and analyzed by LC-MS/MS that highlighted the presence of some unreported spirolides on the basis of their characteristic fragmentation pattern. Finally, NMR studies allowed to characterize two of the new spirolides as 27-OH-13-desmetil spirolide C and 27-oxo-13,19-didesmetil spirolide C.
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