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Fonfria E.S.,University of Santiago de Compostela | Vilarino N.,University of Santiago de Compostela | Molgo J.,French National Center for Scientific Research | Araoz R.,French National Center for Scientific Research | And 5 more authors.
Analytical Biochemistry | Year: 2010

Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with α-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata. The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 ± 1.9%. The quantification range for this toxin in mussel was 40-200 μg/kg of shellfish meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in shellfish as a screening assay. © 2010 Elsevier Inc.


Alonso E.,University of Santiago de Compostela | Otero P.,University of Santiago de Compostela | Vale C.,University of Santiago de Compostela | Alfonso A.,University of Santiago de Compostela | And 5 more authors.
Current Alzheimer Research | Year: 2013

Spirolides are marine toxins that are not currently in the routine monitoring assays. Nicotinic receptors seem to be the target of these compounds making them a promising pharmacological tool for related diseases as dementias as previously shown in vitro. In the present work, the bioavailability of 13-desMethyl spirolide C (13-desMeC) in the brain and in vivo effects were tested. Bioavailability was studied by ultra-performance liquid chromatography-mass spectrometry and its effect over Alzheimer hallmarks was studied by Proton magnetic resonance spectroscopy (H-MRS) and western blot. Only 2 minutes after its intraperitoneal injection it is found in brain and remains detectable even 24 hours post administration. Based on previous works that showed beneficial effects in an in vitro model of Alzheimer's disease (AD), we studied the effect in the same mice, 3xTg-AD, in vivo. We found that 13-desMeC (11.9 ug/kg, i.p.) induced positive effects on AD markers with an increase in N-acetyl aspartate (NAA) levels. These results were supported by an increase in synaptophysin levels and also a decrease in the intracellular amyloid beta levels in the hippocampus of treated 3xTg- AD versus non treated mice remarking the positive effects of this molecule in a well known model of AD. These data indicate for the first time that 13-desMeC cross the blood brain barrier and shows in vivo beneficial effects against AD after administration of low intraperitoneal doses of this marine toxin. This toxin may inspire a novel medical treatment of age-related diseases. © 2013 Bentham Science Publishers.


Rodriguez L.P.,University of Santiago de Compostela | Vilarino N.,University of Santiago de Compostela | Molgo J.,French National Center for Scientific Research | Araoz R.,French National Center for Scientific Research | And 3 more authors.
Analytical Chemistry | Year: 2011

The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the 13-desmethyl spirolide C with biotin-labeled α-bungarotoxin for binding to nicotinic acetylcholine receptors and the immobilization of the α-bungarotoxin-receptor complex on streptavidin-coated surfaces. The quantification of the immobilized receptor can be achieved using a specific antibody. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, three alternative substrates of this enzyme generate a chemiluminescent, fluorescent, or colorimetric signal. The assay performs well in shellfish extracts and the detection range is 5-150 nM of 13-desmethyl spirolide C in shellfish extracts, which is at least 5 times more sensitive than the existing fluorescence polarization assay. This assay can also detect gymnodimine, although with 10 times lower sensitivity than the spirolide. The detection of cyclic imines with microplate assays would be useful for screening purposes in order to reduce the number of samples to be processed by bioassays or analytical methods. © 2011 American Chemical Society.


Otero P.,University of Santiago de Compostela | Alfonso A.,University of Santiago de Compostela | Alfonso C.,Campus Universitario s n | Rodriguez P.,University of Santiago de Compostela | And 2 more authors.
Analytical Chemistry | Year: 2011

Chromatographic techniques coupled to mass spectrometry is the method of choice to replace the mouse bioassay (MBA) to detect marine toxins. This paper evaluates the influence of different parameters such as toxin solvents, mass spectrometric detection method, mobile-phase-solvent brands and equipment on okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) quantification. In addition, the study compares the results obtained when a toxin is quantified against its own calibration curve and with the calibration curve of the other analogues. The experiments were performed by liquid chromatography (LC) and ultraperformance liquid chromatography (UPLC) with tandem mass spectrometry detection (MS/MS). Three acetonitrile brands and two toxin solvents were employed, and three mass spectrometry detection methods were checked. One method that contains the transitions for azaspiracid-1 (AZA-1), azaspiracid-2 (AZA-2), azaspiracid-3(AZA-3), gimnodimine (GYM), 13-desmethyl spirolide C (SPX-1), pectenotoxin-2 (PTX-2), OA, DTX-1, DTX-2, yessotoxin (YTX), homoYTX, and 45-OH-YTX was compared in both instruments. This method operated in simultaneous positive and negative ionization mode. The other two mass methods operated only in negative ionization mode, one contains transitions to detect DTX-1, OA DTX-2, YTX, homoYTX, and 45-OH-YTX and the other only the transitions for the toxins under study OA, DTX-1, and DTX-2. With dependence on the equipment and mobile phase used, the amount of toxin quantified can be overestimated or underestimated, up to 44% for OA, 46% for DTX-1, and 48% for DTX-2. In addition, when a toxin was quantified using the calibration curve of the other analogues, the toxin amount obtained is different. The maximum variability was obtained when DTX-2 was quantified using either OA or a DTX-1 calibration curve. In this case, the overestimation was up to 88% using the OA calibration curve and up to 204% using the DTX-1 calibration curve. In summary, the correct quantification of DSP toxins by MS detection depends on multiple factors. Since these factors are not taken into account in a validated protocol, these results question the convenience of having MS/MS as a reference method for protecting consumers of marine toxins, moreover if toxicity of each group is considered independently and total toxicity is not summed anymore as it is in the MBA. © 2011 American Chemical Society.


Otero P.,University of Santiago de Compostela | Alfonso A.,University of Santiago de Compostela | Alfonso C.,CIFGA Laboratorio | Araoz R.,French National Center for Scientific Research | And 3 more authors.
Analytica Chimica Acta | Year: 2011

In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25nM for 13-desMeC and 150nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350μgkg -1 meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication. © 2011 Elsevier B.V.


Fonfria E.S.,University of Santiago de Compostela | Vilarino N.,University of Santiago de Compostela | Espina B.,University of Santiago de Compostela | Louzao M.C.,University of Santiago de Compostela | And 4 more authors.
Analytica Chimica Acta | Year: 2010

The detection of toxins in shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent α-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of α-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these shellfish extracts with the α-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6 ± 7.8% and 89.6 ± 3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 μg kg-1 and 85-700 μg kg-1 of shellfish meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in shellfish as a screening assay. © 2009 Elsevier B.V. All rights reserved.

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