LC–MS/MS Analysis of the Emerging Toxin Pinnatoxin-G and High Levels of Esterified OA Group Toxins in Galician Commercial Mussels

The occurrence of marine harmful algae is increasing worldwide and, therefore, the accumulation of lipophilic marine toxins from harmful phytoplankton represents a food safety threat in the shellfish industry. Galicia, which is a commercially important EU producer of edible bivalve mollusk have been subjected to recurring cases of mussel farm closures, in the last decades. This work aimed to study the toxic profile of commercial mussels (Mytilus galloprovincialis) in order to establish a potential risk when ingested. For this, a total of 41 samples of mussels farmed in 3 Rías (Ares-Sada, Arousa, and Pontevedra) and purchased in 5 local markets were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS). Chromatograms showed the presence of okadaic acid (OA), dinophysistoxin-2 (DTX-2), pectenotoxin-2 (PTX-2), azaspiracid-2 (AZA-2), and the emerging toxins 13-desmethyl spirolide C (SPX-13), and pinnatoxin-G (PnTX-G). Quantification of each toxin was determined using their own standard calibration in the range 0.1%–50 ng/mL (R2 > 0.99) and by considering the toxin recovery (62–110%) and the matrix correction (33–211%). Data showed that OA and DTX-2 (especially in the form of esters) are the main risk in Galician mollusks, which was detected in 38 samples (93%) and 3 of them exceeded the legal limit (160 μg/kg), followed by SPX-13 that was detected in 19 samples (46%) in quantities of up to 28.9 μg/kg. Analysis from PTX-2, AZA-2, and PnTX-G showed smaller amounts. Fifteen samples (37%) were positive for PTX-2 (0.7–2.9 μg/kg), 12 samples (29%) for AZA-2 (0.1–1.8 μg/kg), and PnTX-G was detected in 5 mussel samples (12%) (0.4 μg/kg–0.9 μg/kg). This is the first time Galician mollusk was contaminated with PnTX-G. Despite results indicating that this toxin was not a potential risk through the mussel ingestion, it should be considered in the shellfish safety monitoring programs through the LC–MS/MS methods.This research has received funding from the following FEDER co-funded grants. From Conselleria de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830, RTC-2016-5507-2, and ITC-20161072. From the European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX. This work was also supported by the program “Juan de la Cierva 2016” from the Spanish Government. Paz Otero is recipient of a Postdoctoral Funding (Ref. IJCI-2016-27774)

Paralytic Shellfish Toxins Occurrence in Non-Traditional Invertebrate Vectors from North Atlantic Waters (Azores, Madeira, and Morocco)

Paralytic shellfish toxins (PSTs) are potent alkaloids of microalgal and cyanobacterial origin, with worldwide distribution. Over the last 20 years, the number of poisoning incidents has declined as a result of the implementation of legislation and monitoring programs based on bivalves. In the summer of 2012 and 2013, we collected a total of 98 samples from 23 different species belonging to benthic and subtidal organisms, such as echinoderms, crustaceans, bivalves, and gastropods. The sampling locations were Madeira, São Miguel Island (Azores archipelago), and the northwestern coast of Morocco. The samples were analyzed using post-column oxidation liquid chromatography with a fluorescence detection method. Our main goal was to detect new vectors for these biotoxins. After reporting a total of 59 positive results for PSTs with 14 new vectors identified, we verified that some of the amounts exceeded the limit value established in the EU. These results suggest that routine monitoring of saxitoxin and its analogs should be extended to more potential vectors other than bivalves, including other edible organisms, for a better protection of public health.This research was partially funded by the Portuguese Fundation of Science and Technology (FCT) project UID/Multi/04423/2013 and by the projects ALERTOXNET (EAPA_317/2016), funded by the Interreg Atlantic program. The Spanish research leading to these results has received funding from the following European Fund for Economic and Regional Development (FEDER) cofunded-grants: Centro para el Desarrollo Tecnológico Industrial (CDTI) and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016; CDTI under India&Spain Innovating Program (ISIP) Programme, Spain, IDI-20130304 APTAFOOD; the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA. Acknowledment to project EMERTOX (grant 734748), funded by H2020-MSCA-RISE 2016

Determination of Gonyautoxin-4 in Echinoderms and Gastropod Matrices by Conversion to Neosaxitoxin Using 2-Mercaptoethanol and Post-Column Oxidation Liquid Chromatography with Fluorescence Detection

Paralytic Shellfish Toxin blooms are common worldwide, which makes their monitoring crucial in the prevention of poisoning incidents. These toxins can be monitored by a variety of techniques, including mouse bioassay, receptor binding assay, and liquid chromatography with either mass spectrometric or pre- or post-column fluorescence detection. The post-column oxidation liquid chromatography with fluorescence detection method, used routinely in our laboratory, has been shown to be a reliable method for monitoring paralytic shellfish toxins in mussel, scallop, oyster and clam species. However, due to its high sensitivity to naturally fluorescent matrix interferences, when working with unconventional matrices, there may be problems in identifying toxins because of naturally fluorescent interferences that co-elute with the toxin peaks. This can lead to erroneous identification. In this study, in order to overcome this challenge in echinoderm and gastropod matrices, we optimized the conversion of Gonyautoxins 1 and 4 to Neosaxitoxin with 2-mercaptoethanol. We present a new and less time-consuming method with a good recovery (82.2%, RSD 1.1%, n = 3), requiring only a single reaction step.This research was partially funded by the Portuguese Fundation of Science and Technology (FCT) project UID/Multi/04423/2013 and by the projects MARBIOTECH (reference NORTE-07-0124-FEDER-000047) within the Scientific Resaerch and Technological Development (SR&TD) Integrated Program. MARVALOR—Building research and innovation capacity for improved management and valorizationof marine resources, supported by the Programa Operacional Regional do Norte (ON.2-O Novo Norte) and NOVOMAR (reference 0687-NOVOMAR-1-P), supported by the European Regional Development Fund. Marisa Silva also acknowledges FCT for the grant SFRH/BD/73269/2010. The spanish research leading to these results has received funding from the following European Fund for Economic and Regional Development (FEDER) cofunded-grants. From Centro para el Desarrollo Tecnológico Industrial (CDTI) and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under India&Spain Innovating Program (ISIP) Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA

Human Poisoning from Marine Toxins: Unknowns for Optimal Consumer Protection

Marine biotoxins are produced by aquatic microorganisms and accumulate in shellfish or finfish following the food web. These toxins usually reach human consumers by ingestion of contaminated seafood, although other exposure routes like inhalation or contact have also been reported and may cause serious illness. This review shows the current data regarding the symptoms of acute intoxication for several toxin classes, including paralytic toxins, amnesic toxins, ciguatoxins, brevetoxins, tetrodotoxins, diarrheic toxins, azaspiracids and palytoxins. The information available about chronic toxicity and relative potency of different analogs within a toxin class are also reported. The gaps of toxicological knowledge that should be studied to improve human health protection are discussed. In general, gathering of epidemiological data in humans, chronic toxicity studies and exploring relative potency by oral administration are critical to minimize human health risks related to these toxin classes in the near future.Support from the following FEDER cofunded-grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2, ITC-20161072. From European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, and H2020 778069-EMERTOX

Calculations of giant magnetoresistance in Fe/Cr trilayers using layer potentials determined from {\it ab-initio} methods

The ab initio full-potential linearized augmented plane-wave method explicitly designed for the slab geometry was employed to elucidate the physical origin of the layer potentials for the trilayers nFe/3Cr/nFe(001), where n is the number of Fe monolayers. The thickness of the transition-metal ferromagnet has been ranged from $n=1$ up to n=8 while the spacer thickness was fixed to 3 monolayers. The calculated potentials were inserted in the Fuchs-Sondheimer formalism in order to calculate the giant magnetoresistance (GMR) ratio. The predicted GMR ratio was compared with the experiment and the oscillatory behavior of the GMR as a function of the ferromagnetic layer thickness was discussed in the context of the layer potentials. The reported results confirm that the interface monolayers play a dominant role in the intrinsic GMR.Comment: 17 pages, 7 figures, 3 tables. accepted in J. Phys.: Cond. Matte

Pre-validación de un método de Cromatografía de Líquidos-Espectrometría de Masas para el análisis simultáneo de toxinas lipofílicas

[ESP] Las biotoxinas marinas de origen fitoplanctónico y de carácter lipofílico pueden acumularse en diferentes tipos de moluscos bivalvos, presentando un importante riesgo para la salud pública. La legislación de la UE, a través del Reglamento (CE) nº 2074/2005, establece los métodos de ensayos reconocidos para la detección de toxinas lipofílicas, señalando como método de referencia los ensayos biológicos e indicando posibles métodos alternativos al método de referencia, entre los que se encuentra la Cromatografía de líquidos acoplada a la Espectrometría de masas (LC-MS). Este Reglamento también indica la necesidad de sustituir lo antes posible los métodos biológicos por métodos de detección alternativos que hayan sido validados conforme a un protocolo acordado a nivel internacional. De acuerdo con esto, el Laboratorio Comunitario de Referencia de Biotoxinas Marinas (LCRBM) está actualmente coordinando, a nivel europeo, diversos estudios enfocados a la validación de un método de LC-MS para la determinación de toxinas lipofílicas. En el presente trabajo se describen los diferentes estudios realizados en la etapa de pre-validación del método, cuyo objetivo se centra en la optimización y estandarización de un protocolo para el análisis simultáneo de ácido ocadaico (AO) y dinofysistoxinas (DTXs), pectenotoxinas (PTXs), azaspirácidos (AZAs), yesotoxinas (YTXs) y espirólidos. A partir de los resultados obtenidos en esta etapa se elaboró un “Procedimiento Normalizado de Trabajo”, candidato a ser validado a través de un estudio colaborativo. Actualmente dicho procedimiento está en fase de evaluación y perfeccionamiento.Este trabajo ha sido financiado por DG SANCO (Comisión Europea, Bruselas), la Agencia Española de Seguridad Alimentaria y Nutrición (AESAN) y por el proyecto STREP FOOD-CT-2004-514055 (DETECTOX). Agradecer a INTECMAR (Instituto Tecnológico para o Control do Medio Mariño de Galicia) por facilitarnos muestras de moluscos naturalmente contaminados con toxinas lipofílicas

Costs and healthcare utilisation of patients with heart failure in Spain

BACKGROUND: Increasing the knowledge about heart failure (HF) costs and their determinants is important to ascertain how HF management can be optimized, leading to a significant decrease of HF costs. This study evaluated the cumulative costs and healthcare utilisation in HF patients in Spain. METHODS: Observational, retrospective, population-based study using BIG-PAC database, which included data from specialized and primary care of people >/=18 years, from seven autonomous communities in Spain, who received care for HF between 2015 and 2019. The healthcare and medication costs were summarized on a yearly basis starting from the index date (1st January 2015), and then cumulatively until 2019. RESULTS: We identified 17,163 patients with HF (year 2015: mean age 77.3 +/- 11.8 years, 53.5% men, 51.7% systolic HF, 43.6% on NYHA functional class II). During the 2015-2019 period, total HF associated costs reached 15,373 Euros per person, being cardiovascular disease hospitalizations the most important determinant (75.8%), particularly HF hospitalizations (51.0%). Total medication cost accounted for 7.0% of the total cost. During this period, there was a progressive decrease of cardiovascular disease hospital costs per year (from 2834 Euros in 2015 to 2146 Euros in 2019, P < 0.001), as well as cardiovascular and diabetic medication costs. CONCLUSIONS: During the 2015-2019 period, costs of HF patients in Spain were substantial, being HF hospitalizations the most important determinant. Medication costs represented only a small proportion of total costs. Improving HF management, particularly through the use of drugs that reduce HF hospitalization may be helpful to reduce HF burden

Comparison of Bisulfite Pyrosequencing and Methylation-Specific qPCR for Methylation Assessment

Different methodological approaches are available to assess DNA methylation biomarkers. In this study, we evaluated two sodium bisulfite conversion-dependent methods, namely pyrosequencing and methylation-specific qPCR (MS-qPCR), with the aim of measuring the closeness of agreement of methylation values between these two methods and its effect when setting a cut-off. Methylation of tumor suppressor gene p16/INK4A was evaluated in 80 lung cancer patients from which cytological lymph node samples were obtained. Cluster analyses were used to establish methylated and unmethylated groups for each method. Agreement and concordance between pyrosequencing and MS-qPCR was evaluated with Pearson's correlation, Bland-Altman, Cohen's kappa index and ROC curve analyses. Based on these analyses, cut-offs were derived for MS-qPCR. An acceptable correlation (Pearson's R2 = 0.738) was found between pyrosequencing (PYRmean) and MS-qPCR (NMP; normalized methylation percentage), providing similar clinical results when categorizing data as binary using cluster analysis. Compared to pyrosequencing, MS-qPCR tended to underestimate methylation for values between 0 and 15%, while for methylation >30% overestimation was observed. The estimated cut-off for MS-qPCR data based on cluster analysis, kappa-index agreement and ROC curve analysis were much lower than that derived from pyrosequencing. In conclusion, our results indicate that independently of the approach used for estimating the cut-off, the methylation percentage obtained through MS-qPCR is lower than that calculated for pyrosequencing. These differences in data and therefore in the cut-off should be examined when using methylation biomarkers in the clinical practice