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BackgroundGiven the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods.ObjectiveThe aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods.MethodsHomogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores.ResultsMethod performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC–MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC–MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC–MS/MS.ConclusionThe results from this study demonstrate that current LC–MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC–MS/MS protocol to further improve interlaboratory precision.HighlightsMultiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.

Naturally-occurring saxitoxins periodically accumulate in shellfish giving rise to Paralytic Shellfish Poisoning. For decades a mouse bioassay has been used for public health protection, but in 2019 a pre-column oxidation LC-FLD method became the official reference method in the European Union. Laboratories are now required to use this method for regulatory monitoring, which will prove challenging. This paper reviews the development, validation and implementation of the method, having been used for over twelve years, successfully providing effective controls against human poisonings without resorting to contingency bioassay. The review describes the work conducted to refine, semi-automate and standardise the method, as well as modifications to cope with new analogues and shellfish species. Approaches for qualitative screening, semi-quantitation and full quantitation are also highlighted, as well as faster chromatography, quality assurance tools and interlaboratory studies. Recommendations for those laboratories new to the method are also described.

A simple, rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed and optimized for the quantitation of a range of pharmaceuticals, metabolites, and related bioactive compounds in the bivalve mollusc species mussels (Mytilus edulis) and Pacific oysters (Crassostrea gigas). Shellfish tissues were extracted using a simple solvent-based extraction method prior to concentration and purification by pass-through solid-phase extraction and quantified using stable isotope dilution MS/MS. The analytes covered a range of therapeutic classes including antidepressants, anticonvulsants, beta-blockers, and antiplatelets. Of the 34 compounds included in the present study initially, 28 compounds were found to demonstrate acceptable performance. Performance was assessed by examining extraction efficiencies, matrix effects, sensitivity, and within- and between-batch precision. The results show that as indicated by acceptable HorRat and accuracy values, the method is fit for purpose. Application of this method to environmental mussel and oyster samples revealed the presence of 12 compounds at quantifiable concentrations, with the antidepressant sertraline being present at the highest level, reaching a concentration of 6.12 ng/g in mussel tissue. © 2021 Crown copyright. Environmental Toxicology and Chemistry 2021;40:3263-3274. © 2021 SETAC. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.

The IOC-ICES-PICES Harmful Algal Event Database (HAEDAT) was used to describe the diversity and spatiotemporal distribution of harmful algal events along the Atlantic margin of Europe from 1987 - 2018. The majority of events recorded are caused by Diarrhetic Shellfish Toxins (DSTs). These events are recorded annually over a wide geographic area from southern Spain to northern Scotland and Iceland, and are responsible for annual closures of many shellfish harvesting areas. The dominant causative dinoflagellates, members of the morphospecies ‘Dinophysis acuminata complex’ and D. acuta, are common in the waters of the majority of countries affected. There are regional differences in the causative species associated with PST events; the coasts of Spain and Portugal with the dinoflagellates Alexandrium minutum and Gymnodinium catenatum, north west France/south west England/south Ireland with A. minutum, and Scotland/Faroe Islands/Iceland with A. catenella. This can influence the duration and spatial scale of PST events as well as the toxicity of shellfish. The diatom Pseudo-nitzschia australis is the most widespread Domoic Acid (DA) producer, with records coming from Spain, Portugal, France, Ireland and the UK. Amnesic Shellfish Toxins (ASTs) have caused prolonged closures for the scallop fishing industry due to the slow depuration rate of DA. Amendments to EU shellfish hygiene regulations introduced between 2002 and 2005 facilitated end-product testing and sale of adductor muscle. This reduced the impact of ASTs on the scallop fishing industry and thus the number of recorded HAEDAT events. Azaspiracids (AZAs) are the most recent toxin group responsible for events to be characterised in the ICES area. Events associated with AZAs have a discrete distribution with the majority recorded along the west coast of Ireland. Ciguatera Poisoning (CP) has been an emerging issue in the Canary Islands and Madeira since 2004. The majority of aquaculture and wild fish mortality events are associated with blooms of the dinoflagellate Karenia mikimotoi and raphidophyte Heterosigma akashiwo. Such fish killing events occur infrequently yet can cause significant mortalities. Interannual variability was observed in the annual number of HAEDAT areas with events associated with individual shellfish toxin groups. HABs represent a continued risk for the aquaculture industry along the Atlantic margin of Europe and along the Atlantic margin of Europe and should be accounted for when considering expansion of the industry or operational shifts to offshore areas.

In early 2018, a large easterly storm hit the East Anglian coast of the UK, colloquially known as the &lsquo, Beast from the East&rsquo, which also resulted in mass strandings of benthic organisms. There were subsequent instances of dogs consuming such organisms, leading to illness and, in some cases, fatalities. Epidemiological investigations identified paralytic shellfish toxins (PSTs) as the cause, with toxins present in a range of species and concentrations exceeding 14,000 &micro, g STX eq./kg in the sunstar Crossaster papposus. This study sought to better elucidate the geographic spread of any toxicity and identify any key organisms of concern. During the summers of 2018 and 2019, various species of benthic invertebrates were collected from demersal trawl surveys conducted across a variety of locations in the North Sea. An analysis of the benthic epifauna using two independent PST testing methods identified a &lsquo, hot spot&rsquo, of toxic organisms in the Southern Bight, with a mean toxicity of 449 &micro, g STX eq./kg. PSTs were quantified in sea chervil (Alcyonidium diaphanum), the first known detection in the phylum bryozoan, as well as eleven other new vectors (&gt, 50 &micro, g STX eq./kg), namely the opisthobranch Scaphander lignarius, the starfish Anseropoda placenta, Asterias rubens, Luidia ciliaris, Astropecten irregularis and Stichastrella rosea, the brittlestar Ophiura ophiura, the crustaceans Atelecyclus rotundatus and Munida rugosa, the sea mouse Aphrodita aculeata, and the sea urchin Psammechinus miliaris. The two species that showed consistently high PST concentrations were C. papposus and A. diaphanum. Two toxic profiles were identified, with one dominated by dcSTX (decarbamoylsaxitoxin) associated with the majority of samples across the whole sampling region. The second profile occurred only in North-Eastern England and consisted of mostly STX (Saxitoxin) and GTX2 (gonyautoxin 2). Consequently, this study highlights widespread and variable levels of PSTs in the marine benthos, together with the first evidence for toxicity in a large number of new species. These findings highlight impacts to &lsquo, One Health&rsquo, with the unexpected sources of toxins potentially creating risks to animal, human and environmental health, with further work required to assess the severity and geographical/temporal extent of these impacts.

Previous studies have shown that mussels can pick up 17β-estradiol [E2] and testosterone [T] from water, metabolize them and conjugate them to fatty acids (esterification), leading to their accumulation in tissue. A key requirement for the esterification process is that a steroid must have a ‘reactive’ hydroxyl group to conjugate to a fatty acid (which in T, and probably E2, is the β-hydroxyl group on carbon 17). Progesterone (P) lacks any hydroxyl groups and theoretically cannot be esterified and hence should not accumulate in mussels in the same way as E2 or T. However, it is already known that mussels have an enzyme that can achieve 5α-reduction of the A ring of T and P and that there is also another reductase that can transform the 3-oxo group of the 5α-reduced A ring of T into a hydroxyl group. We hypothesized that, although intact P cannot be directly esterified, it might nevertheless be transformed into metabolites that can. To test this hypothesis, we investigated the rate and capacity of uptake, metabolism and potential depuration of tritiated P by the common mussel, Mytilus spp. We found that tritiated P was taken up from water at a similar rate to E2 and T (mean clearance rate 49 mL−1 animal−1 h−1) and that, as found with the other steroids, the rate of uptake could not be saturated by the addition of non-radioactive steroid (even at 7.6 μg L−1). We found that up to 66% of the radioactivity that was taken up was present in the ester fraction, suggesting that hydroxylation of the P must indeed have occurred. We then definitively identified two metabolites in the ester fraction: 5α-pregnane-3β,20β-diol and 3β-hydroxy-5α-pregnan-20-one. These same two steroids were also present in the free steroid fraction. Intact P was not detected in either of the fractions. When undergoing depuration (under semi-static conditions), the radioactivity in the ester fractions remained at the same concentration in the animals for at least 10 days. Our findings suggest that the lack of reactive hydroxyl groups on P does not preclude it from being taken up, metabolized and subsequently stored. Many questions remain, not least of which is why, when P seems to be so rapidly metabolized, two previous studies on mussels have reported concentrations of up to 30 ng g−1 wet weight of P in their flesh.

Harmful Algal Blooms (HAB) are natural atypical proliferations of micro or macro algae in either marine or freshwater environments which have significant impacts on human, animal and ecosystem health. The causative HAB organisms are primarily dinoflagellates and diatoms in marine and cyanobacteria within freshwater ecosystems. Several hundred species of HABs, most commonly marine dinoflagellates affect animal and ecosystem health either directly through physical, chemical or biological impacts on surrounding organisms or indirectly through production of algal toxins which transfer through lower-level trophic organisms to higher level predators. Traditionally, a major focus of HABs has concerned their natural production of toxins which bioaccumulate in filter-feeding invertebrates, which with subsequent trophic transfer and biomagnification cause issues throughout the food web, including the human health of seafood consumers. Whilst in many regions of the world, regulations, monitoring and risk management strategies help mitigate against the impacts from HAB/invertebrate toxins upon human health, there is ever-expanding evidence describing enormous impacts upon invertebrate health, as well as the health of higher trophic level organisms and marine ecosystems. This paper provides an overview of HABs and their relationships with aquatic invertebrates, together with a review of their combined impacts on animal, human and ecosystem health. With HAB/invertebrate outbreaks expected in some regions at higher frequency and intensity in the coming decades, we discuss the needs for new science, multi-disciplinary assessment and communication which will be essential for ensuring a continued increasing supply of aquaculture foodstuffs for further generations.

A single laboratory method performance verification is reported for a rapid sensitive UHPLC-MS/MS method for the quantification of eight cyclic imine and two brevetoxin analogues in two bivalve shellfish matrices: mussel (Mytilus edulis) and Pacific oyster (Crassostrea gigas). Targeted cyclic imine analogues were from the spirolide, gymnodimine and pinnatoxin groups, namely 20-Me-SPX-C, 13-desMe-SPX-C, 13,19-didesMe-SPX-C, GYM-A, 12-Me-GYM, PnTx-E, PnTx-F and PnTx-G. Brevetoxin analogues consisted of the shellfish metabolites BTX-B5 and S-desoxy-BTX-B2. A rapid dispersive extraction was used as well as a fast six-minute UHPLC-MS/MS analysis. Mobile phase prepared using ammonium fluoride and methanol was optimised for both chromatographic separation and MS/MS response to suit all analytes. Method performance verification checks for both matrices were carried out. Matrix influence was acceptable for the majority of analogues with the MS response for all analogues being linear across an appropriate range of concentrations. In terms of limits of detection and quantitation the method was shown to be highly sensitive when compared with other methods. Acceptable recoveries were found with most analogues, with laboratory precision in terms of intra- and inter-batch precision deemed appropriate. The method was applied to environmental shellfish samples with results showing low concentrations of cyclic imines to be present. The method is fast and highly sensitive for the detection and quantification of all targeted analogues, in both mussel and oyster matrices. Consequently, the method has been shown to provide a useful tool for simultaneous monitoring for the presence or future emergence of these two toxin groups in shellfish.

The presence of the vertebrate steroids, testosterone (T) and 17β-estradiol in mollusks is often cited as evidence that they are involved in the control of their reproduction. In this paper, we show that a likely source of T in at least one species, the common mussel (Mytilus spp.), is from uptake from water. When mussels were exposed to waterborne tritiated T ([3H]-T) in a closed container, the radioactivity decreased rapidly and exponentially until, by 24h, approximately 35% remained in the water. The rate of uptake of radiolabel could not be saturated by concentrations as high as 16.5μgL-1 (mean measured) of non-radiolabeled T, showing that the animals have a very high capacity for uptake of T. At least 30% of the applied radioactivity could be extracted from the tissues of the animals with organic solvents and most of this (26% of the total applied radioactivity) was in the fatty acid ester fraction. Following alkaline hydrolysis, reverse phase HPLC and TLC, this fraction was shown to consist predominantly of 5α-dihydrotestosterone and 5α-androstane-3β,17β-diol, while T was a minor component. These steroids were definitively identified in the fatty acid ester fraction by mass spectrometry. Overall, less than 5% of the [3H]-T applied to the system remained untransformed at the end of exposure. After ten days of depuration there was no reduction in the total amount of radioactivity in the tissues, nor any changes in the ratio of the metabolites in the ester fraction. These findings show that any association between T presence and reproductive status or sex is confounded by their significant capacity for uptake, and that T undergoes extensive metabolism in mussels in vivo and therefore may not be representative of the androgenic burden of the animals. Consequently, measurements of T in mussel tissue offer little utility as an indicator of reproductive status or sex.

The data presented in this article primarily provide support for the research article entitled “Mussels (Mytilus spp.) display an ability for rapid and high capacity uptake of the vertebrate steroid, estradiol-17β from water” (T.I. Schwarz, I. Katsiadaki, B.H. Maskrey, A.P. Scott, 2016) [1]. Data are presented on the ability of mussels to absorb tritiated estradiol (E2) from water. The data indicate that most of the radioactivity remaining in the water is 1,3,5(10)-estratriene-3,17β-diol 3-sulfate (E2 3-S) and the radioactivity in the mussel tissue is mainly in the form of fatty acid esters. The latter, following saponification, were identified by ultra-high performance liquid chromatography in conjunction with tandem mass spectrometry (UHPLC-MS/MS) as intact E2. Data are included that indicate that the remaining radioactivity in the tissue is composed of E2 3-S and unidentified free metabolites. Experimental data included also relate to a) the efficiency of extraction of radioactivity from tissue, b) the efficiency of separation of free and esterified E2 using solvents and c) possible factors affecting the recovery of radioactivity. Finally, preliminary data are provided on concentrations of immunoreactive E2 in the free and ester fractions of tissue extracts from mussels caged in the field.

Graphical abstract Proposed mechanistic action of FEN in adipocytes. RA inhibits adipogenesis through early RAR activation, which PPARγ agonist ROSI cannot prevent. FEN inhibits adipogenesis with a delayed response in RAR signalling, however, inhibition is lost when combined with ROSI. The 4-OXO FEN catabolite cannot inhibit adipogenesis through RAR activation, but like FEN’s RAR-independent effects, displays increased phosphorylation of Akt, mild cellular stress/autophagy induction and decreased lipid accumulation. Moreover, FEN-mediated inhibition of DES-1 increases dihydroceramide levels in a RAR-independent manner, and is linked to a complete prevention of mitochondrial dysfunction and decreased adiposity in high-fat fed obese mice. Thus an alternative to the additive beneficial effects of FEN-mediated RAR-dependant and -independent signalling, 4-OXO FEN may be a novel therapeutic candidate to improve adipocyte hypertrophy via inhibition of ceramide biosynthesis and modulation of nutrient stress pathways., Fenretinide (FEN) is a synthetic retinoid that inhibits obesity and insulin resistance in high-fat diet (HFD)-fed mice and completely prevents 3T3-L1 pre-adipocyte differentiation. The aim of this study was to determine the mechanism(s) of FEN action in 3T3-L1 adipocytes and in mice. We used the 3T3-L1 model of adipogenesis, fully differentiated 3T3-L1 adipocytes and adipose tissue from HFD-induced obese mice to investigate the mechanisms of FEN action. We measured expression of adipogenic and retinoid genes by qPCR and activation of nutrient-signalling pathways by western blotting. Global lipid and metabolite analysis was performed and specific ceramide lipid species measured by liquid chromatography-mass spectrometry. We provide direct evidence that FEN inhibits 3T3-L1 adipogenesis via RA-receptor (RAR)-dependent signaling. However, RARα antagonism did not prevent FEN-induced decreases in lipid levels in mature 3T3-L1 adipocytes, suggesting an RAR-independent mechanism. Lipidomics analysis revealed that FEN increased dihydroceramide lipid species 5- to 16-fold in adipocytes, indicating an inhibition of the final step of ceramide biosynthesis. A similar blockade in adipose tissue from FEN-treated obese mice was associated with a complete normalisation of impaired mitochondrial β-oxidation and tricarboxylic acid cycle flux. The FEN catabolite, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-OXO), also decreased lipid accumulation without affecting adipogenesis. FEN and 4-OXO (but not RA) treatment additionally led to the activation of p38-MAPK, peIF2α and autophagy markers in adipocytes. Overall our data reveals FEN utilises both RAR-dependent and -independent pathways to regulate adipocyte biology, both of which may be required for FEN to prevent obesity and insulin resistance in vivo.

As the official control laboratory for marine biotoxins within Great Britain, the Centre for Environment, Fisheries and Aquaculture Science, in conjunction with the Scottish Association for Marine Science, has amassed a decade’s worth of data regarding the prevalence of the toxins associated with Amnesic Shellfish Poisoning within British waters. This monitoring involves quantitative HPLC-UV analysis of shellfish domoic acid concentration, the causative toxin for Amnesic Shellfish Poisoning, and water monitoring for Pseudo-nitzschia spp., the phytoplankton genus that produces domoic acid. The data obtained since 2008 indicate that whilst the occurrence of domoic acid in shellfish was generally below the maximum permitted limit of 20 mg/kg, there were a number of toxic episodes that breached this limit. The data showed an increase in the frequency of both domoic acid occurrence and toxic events, although there was considerable annual variability in intensity and geographical location of toxic episodes. A particularly notable increase in domoic acid occurrence in England was observed during 2014. Comparison of Scottish toxin data and Pseudo-nitzschia cell densities during this ten-year period revealed a complex relationship between the two measurements. Whilst the majority of events were associated with blooms, absolute cell densities of Pseudo-nitzschia did not correlate with domoic acid concentrations in shellfish tissue. This is believed to be partly due to the presence of a number of different Pseudo-nitzschia species in the water that can exhibit variable toxin production. These data highlight the requirement for tissue monitoring as part of an effective monitoring programme to protect the consumer, as well as the benefit of more detailed taxonomic discrimination of the Pseudo-nitzschia genus to allow greater accuracy in the prediction of shellfish toxicity.

Official control biotoxin testing of bivalve molluscs from Great Britain has been conducted by Cefas for over a decade. Reflecting the changes in legislation, bioassays were gradually replaced by analytical methods, firstly for analysis of Paralytic shellfish toxins, followed by introduction of liquid chromatography tandem mass spectrometric (LC MS/MS) method for lipophilic toxins (LTs) in 2011. Twelve compounds, representing three main groups of regulated lipophilic toxins, as well as two non-regulated cyclic imines were examined in over 20,500 samples collected between July 2011 and December 2016. The toxins belonging to Okadaic acid (OA) group toxins were the most prevalent and were quantified in 23% of samples, predominantly from Scotland. The temporal pattern of OA group occurrences remained similar each year, peaking in summer months and tailing off during autumn and winter, however their abundance and magnitude varied between years significantly, with concentrations reaching up to 4993 μg OA eq./kg. Three toxin profiles were identified, reflecting the relative contribution of the two main toxins, OA and dinophysis toxin-2 (DTX2). Dinophysis toxin-1 (DTX1) was less common and was never detected in samples with high proportions of DTX2. Inter-annual changes in profiles were observed within certain regions, with the most notable being an increase of DTX2 occurrences in north-west Scotland and England in the last three years of monitoring. In addition, seasonal changes of profiles were identified when OA, the dominant toxin in early summer, was replaced by higher proportions of DTX2 in late summer and autumn. The profile distribution possibly reflected the availability of individual Dinophysis species as a food source for shellfish, however persistence of DTX2 during autumn and winter in mussels might have also been attributed to their physiology. Mussels were the only species with higher average proportions of non-esterified toxins, while Pacific oysters, cockles, surf clams, razors and queen scallops contained almost exclusively ester forms. In addition, a temporal change in proportion of OA and DTX2 free form was observed in mussels. Pectenotoxin-2 (PTX2) was quantified only on rare occasions.

Within the last decade, multiple new medications have been licensed for treating type 2 diabetes. These agents are vastly more expensive than their predecessors and usage requires review in practice. This retrospective case series reviewed 55 patients to assess the effect of glucagon-like peptide-1 (GLP-1) mimetics as add-on to existing antidiabetic therapy, including insulin, in clinical practice. The primary outcome measures were change in HbA1c and weight. Analyses were also conducted to test the effect of: initial HbA1c; concomitant insulin use; GLP-1 prescribed; and duration of diabetes on the change in HbA1c and weight. Mean BMI at initiation was 39.7 kg/m2; duration of diabetes at initiation 8.2 years; duration of GLP-1 therapy at review 18.3 months. The mean changes in HbA1c (-15.9 mmol/mol) and weight (-5.5 kg) were both highly significant (p < 0.0001). NICE targets for HbA1c and weight reductions were met in 34.5%. Significant negative correlations were found between initial HbA1c and subsequent change in HbA1c (p = 0.0001) and between initial BMI and subsequent change in weight (p = 0.04), whereas a significant positive correlation was observed between initial HbA1c and subsequent change in weight (p = 0.01). No statistically significant changes in HbA1c or weight were observed in relation to: concomitant insulin use; GLP-1 prescribed; or duration of diabetes. This study confirms GLP-1 agents provide clinically useful reductions in HbA1c and weight. Further work is required to assess their effect if used earlier. A practical and pragmatic approach to assessment of the effectiveness of GLP-1 has been proposed. Copyright © 2014 John Wiley & Sons, Ltd.

Cefas has been responsible for the delivery of official control biotoxin testing of bivalve molluscs from Great Britain for just over a decade. Liquid chromatography tandem mass spectrometric (LC–MS/MS) methodology has been used for the quantitation of lipophilic toxins (LTs) since 2011. The temporal and spatial distribution of okadaic acid group toxins and profiles in bivalves between 2011 and 2016 have been recently reported. Here we present data on the two other groups of regulated lipophilic toxins, azaspiracids (AZAs) and yessotoxins (YTXs), over the same period. The latter group has also been investigated for a potential link with Protoceratium reticulatum and Lingulodinium polyedra, both previously recognised as YTXs producing phytoplankton. On average, AZAs were quantified in 3.2% of all tested samples but notable inter-annual variation in abundance was observed. The majority of all AZA contaminated samples were found between July 2011 and August 2013 in Scotland, while only two, three-month long, AZA events were observed in 2015 and 2016 in the south-west of England. Maximum concentrations were generally reached in late summer or early autumn. Reasons for AZAs persistence during the 2011/2012 and 2012/2013 winters are discussed. Only one toxin profile was identified, represented by both AZA1 and AZA2 toxins at an approximate ratio of 2 : 1, suggesting a single microalgal species was the source of AZAs in British bivalves. Although AZA1 was always the most dominant toxin, its proportion varied between mussels, Pacific oysters and surf clams. The YTXs were the least represented group among regulated LTs. YTXs were found almost exclusively on the south-west coast of Scotland, with the exception of 2013, when the majority of contaminated samples originated from the Shetland Islands. The highest levels were recorded in the summer months and followed a spike in Protoceratium reticulatum cell densities. YTX was the most dominant toxin in shellfish, further strengthening the link to P. reticulatum as the YTX source. Neither homo-YTX, nor 45−OH homo-YTX were detected throughout the monitored period. 45−OH YTX, thought to be a shellfish metabolite associated with YTX elimination, contributed on average 26% in mussels. Although the correlation between 45−OH YTX abundance and the speed of YTX depuration could not be confirmed, we noted the half-life of YTX was more than two-times longer in queen scallops, which contained 100% YTX, than in mussels. No other bivalve species were affected by YTXs. This is the first detailed evaluation of AZAs and YTXs occurrences and their profiles in shellfish from Great Britain over a period of multiple years.

The beneficial health properties of dietary omega-3 polyunsaturated fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have long been known and their metabolic dysfunction has been linked to a range of diseases including various inflammatory disorders, cardiovascular diseases, and cancer. However, the molecular mechanisms underlying their health benefits have remained unclear. Recent technological advances in lipidomic analytical strategies have resulted in the discovery of a range of bioactive mediators derived from EPA and DHA that possess potent anti-inflammatory and pro-resolving properties and that may be responsible, at least in part, for the beneficial effects observed. These mediators include resolvins, protectins and maresins, as well as EPA derivatives of classical arachidonic acid derived eicosanoids, such as prostaglandin E3 . The aim of this review is to provide an overview of the biosynthetic pathways and biological properties of these omega-3 mediators, with a particular focus on the emerging importance of the counter-regulatory role of omega-3 and -6 fatty acids in the spatial and temporal regulation of the inflammatory response. It will also provide an insight into a range of lipidomic approaches, which are currently available to analyse these fatty acids and their metabolites in biological matrices.

Alternatively activated macrophages (AAMφ) are a major component of the response to helminth infection; however, their functions remain poorly defined. To better understand the helminth-induced AAMφ phenotype, we performed a systems-level analysis of in vivo derived AAMφ using an established mouse model. With next-generation RNA sequencing, we characterized the transcriptomes of peritoneal macrophages from BALB/c and IL4Rα−/− mice elicited by the nematode Brugia malayi, or via intraperitoneal thioglycollate injection. We defined expression profiles of AAMφ-associated cytokines, chemokines, and their receptors, providing evidence that AAMφ contribute toward recruitment and maintenance of eosinophilia. Pathway analysis highlighted complement as a potential AAMφ-effector function. Up-regulated mitochondrial genes support in vitro evidence associating mitochondrial metabolism with alternative activation. We mapped macrophage transcription start sites, defining over-represented cis-regulatory motifs within AAMφ-associated promoters. These included the binding site for PPAR transcription factors, which maintain mitochondrial metabolism. Surprisingly PPARγ, implicated in the maintenance of AAMφ, was down-regulated on infection. PPARδ expression, however, was maintained. To explain how PPAR-mediated transcriptional activation could be maintained, we used lipidomics to quantify AAMφ-derived eicosanoids, potential PPAR ligands. We identified the PPARδ ligand PGI2 as the most abundant AAMφ-derived eicosanoid and propose a PGI2-PPARδ axis maintains AAMφ during B malayi implantation.

Six experiments were carried out to define the optimum conditions for investigating the dynamics of uptake and metabolism of tritiated E 2 from water by adult blue mussels, Mytilus spp. Optimum uptake was achieved using 400 mL aerated sea water animal −1 and an incubation period of no more than 24 h. The pattern of disappearance conformed closest to an inverse hyperbolic curve with the percentage of radiolabel that could be measured in the water reaching an asymptote that was on average 50% of the original. This apparent inability of the animals to absorb all the radiolabel was investigated further. Solvent partition and chromatography revealed that, after 24 h, c . 60% of the radiolabel still present in the water was composed of water soluble conjugates, c . 25% was composed of tritiated water and only 15% ran on and around the chromatographic position of E 2 . The major water soluble constituent was identified by chromatography and mass-spectrometry as 1,3,5(10)-estratriene-3,17β-diol 3-sulfate (estradiol 3-S). The clearance rate of radiolabel was 46.9 ± 1.8 mL animal −1 h −1 . This was not significantly affected by the addition of as much as 25 μg L −1 cold E 2 to the water, demonstrating that mussels have a large capacity for E 2 uptake. A new procedure involving solvent partition was developed for separating the free, esterified and sulfated forms of E 2 present in the flesh of mussels. This involved extracting the soft tissue with organic solvents and then treating a portion of dried extract with a combination of heptane (dissolved fatty acid esters of E 2 ) and 80% ethanol (dissolved free and sulfated E 2 ). The latter fraction was further partitioned between water (sulfate) and diethyl ether (free steroid). This procedure was much cheaper and less time-consuming than chromatography. Approximately 80% of the radioactivity that was taken up by the animals was present in the form of ester. Moreover, E 2 was the only steroid identified after saponification of these esters. Of the remaining radioactivity, c . 10% was in the form of unidentified free steroids and c . 10% was estradiol 3-S. In order to determine how rapidly mussels were able to depurate tritiated E 2 and its metabolites, two experiments were carried out. Animals from the first experiment purged up to 63% of radioactivity in 20 days under flow-through conditions; whereas animals from the second experiment released only 16% of radioactivity in 10 days under semi-static conditions. The ratios of the different forms of E 2 did not change substantially during the course of depuration.

BACKGROUND:There is no consensus and a limited evidence base for choice of contrast agents (CA) in angiography. This study evaluated the impact of iohexol and iodixanol CA on fibrinolytic factors (tissue plasminogen activator [t-PA] and plasminogen activator inhibitor-1 [PAI-1]), as well as platelet-monocyte conjugates in cardiac patients undergoing elective angiography in a double-blind, randomised parallel group study. METHODS:Patients (men, 50-70 years old; n = 12) were randomised to receive either iohexol (Omnipaque; n = 6) or iodixanol (Visipaque; n = 6) during elective angiography at Raigmore Hospital, Inverness, UK. Arterial and venous blood samples were drawn prior to CA delivery and following angiography. Assessment of platelet-monocyte conjugation, t-PA and PAI-1 antigen and activity was conducted in samples pre- and post-angiography. OUTCOME:Plasma t-PA antigen was depressed equally in the study groups after angiography, but there was a greater reduction in PAI-1 antigen in the group receiving iodixanol. These findings corresponded to a substantial reduction in t-PA activity in patients receiving iohexol, with no change in those receiving iodixanol (P = 0.023 between the CA groups). Both CAs caused a reduction in platelet-monocyte conjugation, with no difference between the groups. No adverse events were reported during the trial. CONCLUSION:Avoiding reduced plasma t-PA activity might be an important consideration in choosing iodixanol over iohexol in patients at risk of thrombosis following angiography. The trial is registered on the ISRCTN register (ISRCTN51509735) and funded by the Coronary Thrombosis Trust and National Health Service (Highland) R&D Endowments. The funders had no influence over study design or reporting. TRIAL REGISTRATION:Controlled-Trials.com ISRCTN51509735.

The inflammatory response is an integral part of the innate immune mechanism that is triggered in response to a real or perceived threat to tissue homeostasis, with a primary aim of neutralizing infectious agents and initiating repair to damaged tissue. By design, inflammation is a finite process that resolves as soon as the threat of infection abates and sufficient repair to the tissue is complete. Resolution of inflammation involves apoptosis and subsequent clearance of activated inflammatory cells – a tightly regulated event. Chronic inflammation is a characteristic feature in virtually all inflammatory diseases, including atherosclerosis, and it is becoming increasingly clear that derangement of the processes usually involved in resolution of inflammation is an underlying feature of chronic inflammatory conditions. This review will draw on evidence from a range of diseases in which dysregulated inflammation is important, with particular emphasis on cardiovascular disease.

The peritoneal macrophage (Mφ) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11bhigh cells, with the remaining 5% being 12/15-LOX−. 12/15-LOX+ cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX− cells are a dendritic cell population. Resident peritoneal Mφ numbers were significantly increased in 12/15-LOX−/− mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mφ from 12/15-LOX−/− mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX−/− mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX−/− adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX+ cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX−/− mice showed elevated TGF-β1, along with increased immigration of monocytes/Mφ, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-α. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MΦ population and regulates both the recruitment of monocytes/Mφ and cytokine response to bacterial products in vivo.

Eicosanoids are oxidized arachidonate-derived lipids products generated by lipoxygenase, cyclo-oxygenase and cytochrome P450. Their bioactivity is mediated via receptor-dependent mechanisms and they are involved in a diverse array of processes in both health and disease. For many years, GC–MS was the method of choice for eicosanoid analysis, but, more recently, the availability of high-sensitivity electrospray LC (liquid chromatography)–MS/MS (tandem MS) has provided a new approach for quantification, while minimizing sample preparation. The present review summarizes the various methods available for routine quantification of eicosanoids, focusing ultimately on their analysis using a hybrid Q-Trap mass spectrometer.

Background Aspirin is one of the most widely used non-steroidal anti-inflammatory drugs (NSAIDs). It is also a commonly used anti-platelet drug, which inhibits the formation of the platelet activator, thromboxane A2 (TxA2) via inhibition of cyclooxygenase-1 (COX-1). However, the presence of a patient subset that fails to respond to aspirin despite reduced TxA2 concentrations suggests that the effect of aspirin might be more complex than exclusive COX-1 inhibition. Methods In this study we evaluated the impact of in vivo oral administration of a standard anti-platelet dose (75 mg) of aspirin in healthy volunteers on the acute impact of in vitro collagen-mediated platelet aggregation and generation of platelet-derived TxA2 and the 12-lipoxygenase (LOX) metabolite 12-hydroxyeicosatetraenoic acid (12-HETE). The eicosanoids were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results Low-dose aspirin administration not only inhibited TxA2 generation but also decreased the production of 12-HETE. Furthermore, a significant correlation was observed between the levels of 12-HETE and collagen-induced platelet aggregation. Pre-treatment of platelets with the 12-LOX inhibitor, baicalein, prior to activation attenuated platelet aggregation. Conclusions These findings support a role for 12-HETE as a pro-aggregatory eicosanoid in platelet function and suggest a role for 12-HETE in variable sensitivity to aspirin. The study also highlights a potentially important mechanism by which aspirin impacts upon eicosanoid generation.

The metabolism of bromine in marine brown algae remains poorly understood. This contrasts with the recent finding that the accumulation of iodide in the brown alga Laminaria serves the provision of an inorganic antioxidant - the first case documented from a living system. The aim of this study was to use an interdisciplinary array of techniques to study the chemical speciation, transformation, and function of bromine in Laminaria and to investigate the link between bromine and iodine metabolism, in particular in the antioxidant context. First, bromine and iodine levels in different Laminaria tissues were compared by inductively coupled plasma MS. Using in vivo X-ray absorption spectroscopy, it was found that, similarly to iodine, bromine is predominantly present in this alga in the form of bromide, albeit at lower concentrations, and that it shows similar behaviour upon oxidative stress. However, from a thermodynamic and kinetic standpoint, supported by in vitro and reconstituted in vivo assays, bromide is less suitable than iodide as an antioxidant against most reactive oxygen species except superoxide, possibly explaining why kelps prefer to accumulate iodide. This constitutes the first-ever study exploring the potential antioxidant function of bromide in a living system and other potential physiological roles. Given the tissue-specific differences observed in the content and speciation of bromine, it is concluded that the bromide uptake mechanism is different from the vanadium iodoperoxidase-mediated uptake of iodide in L. digitata and that its function is likely to be complementary to the iodide antioxidant system for detoxifying superoxide. published

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.

While investigating new mechanisms by which the dietary omega-3 fatty acids regulate inflammation, the authors have identified a new step in the regulation of neutrophil migration across vascular endothelial cells., Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-α, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D2 (PGD2) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD3. This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD2 receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD2 signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs., Author Summary Inflammation is a physiological response to tissue trauma or infection. Neutrophils, which circulate in the blood stream, are the first inflammatory cells to be recruited to a site of tissue inflammation. In response to recruitment signals provided by chemotactic peptides called chemokines, neutrophils traverse the endothelial cell lining of blood vessels. This process involves a multistep cascade of neutrophil adhesion and activation events on the endothelial barrier. While investigating the anti-inflammatory functions of the omega-3 fatty acid , EPA, which is found, for instance, in dietary fish oil, we identified an additional unexpected lipid-derived signal that is essential for neutrophil migration across the endothelium. Our experiments show that a chemokine delivered the first signal needed to bind neutrophils firmly to the endothelial surface. However, in order to traverse the endothelium, a subsequent signal delivered by prostaglandin-D2 (PGD2), a lipid derived from the omega-6 fatty acid arachidonic acid, was essential. When EPA, was introduced into the experiment, it was used to form PGD3. This alternative lipid blocked interactions between PGD2 and its receptor on neutrophils, preventing the process of migration across the endothelial barrier. Thus, we reveal a new step in the recruitment of neutrophils during inflammation, and a novel anti-inflammatory mechanism of action of dietary EPA.