Discovery of a Potential Human Serum Biomarker for Chronic Seafood Toxin Exposure Using an SPR Biosensor.

Domoic acid (DA)-producing harmful algal blooms (HABs) have been present at unprecedented geographic extent and duration in recent years causing an increase in contamination of seafood by this common environmental neurotoxin. The toxin is responsible for the neurotoxic illness, amnesic shellfish poisoning (ASP), that is characterized by gastro-intestinal distress, seizures, memory loss, and death. Established seafood safety regulatory limits of 20 µg DA/g shellfish have been relatively successful at protecting human seafood consumers from short-term high-level exposures and episodes of acute ASP. Significant concerns, however, remain regarding the potential impact of repetitive low-level or chronic DA exposure for which there are no protections. Here, we report the novel discovery of a DA-specific antibody in the serum of chronically-exposed tribal shellfish harvesters from a region where DA is commonly detected at low levels in razor clams year-round. The toxin was also detected in tribal shellfish consumers' urine samples confirming systemic DA exposure via consumption of legally-harvested razor clams. The presence of a DA-specific antibody in the serum of human shellfish consumers confirms long-term chronic DA exposure and may be useful as a diagnostic biomarker in a clinical setting. Adverse effects of chronic low-level DA exposure have been previously documented in laboratory animal studies and tribal razor clam consumers, underscoring the potential clinical impact of such a diagnostic biomarker for protecting human health. The discovery of this type of antibody response to chronic DA exposure has broader implications for other environmental neurotoxins of concern.

Detection and effects of harmful algal toxins in Scottish harbour seals and potential links to population decline.

Over the past 15 years or so, several Scottish harbour seal (Phoca vitulina) populations have declined in abundance and several factors have been considered as possible causes, including toxins from harmful algae. Here we explore whether a link could be established between two groups of toxins, domoic acid (DA) and saxitoxins (STXs), and the decline in the harbour seal populations in Scotland. We document the first evidence that harbour seals are exposed to both DA and STXs from consuming contaminated fish. Both groups of toxins were found in urine and faeces sampled from live captured (n = 162) and stranded animals (n = 23) and in faecal samples collected from seal haul-out sites (n = 214) between 2008 and 2013. The proportion of positive samples and the toxins levels measured in the excreta were significantly higher in areas where harbour seal abundance is in decline. There is also evidence that DA has immunomodulatory effects in harbour seals, including lymphocytopenia and monocytosis. Scottish harbour seals are exposed to DA and STXs through contaminated prey at potentially lethal levels and with this evidence we suggest that exposure to these toxins are likely to be important factors driving the harbour seal decline in some regions of Scotland.

A Rapid LC-HRMS Method for the Determination of Domoic Acid in Urine Using a Self-Assembly Pipette Tip Solid-Phase Extraction.

In this study, we developed a self-assembly pipette tip solid-phase extraction (PTSPE) method using a high molecular weight polymer material (PAX) as the adsorbent for the determination of domoic acid (DA) in human urine samples by liquid chromatography high-resolution mass spectrometry (LC-HRMS) analysis. The PTSPE cartridge, assembled by packing 9.1 mg of PAX as sorbent into a 200 µL pipette tip, showed high adsorption capacity for DA owing to the strong cationic properties of PAX. Compared with conventional SPE, the PTSPE is simple and fast, and shows some advantages in the aspects of less solvent consumption, low cost, the absence of the evaporation step, and short time requirement. All the parameters influencing the extraction efficiency such as pH, the amount of sorbent, the number of aspirating/dispensing cycles, and the type and volume of eluent in PTSPE were carefully investigated and optimized. Under the optimized conditions, the limit of detection (LOD) and limit of quantification (LOQ) values of DA were 0.12 µg/L and 0.37 µg/L respectively. The extraction recoveries of DA from the urine samples spiked at four different concentrations were in a range from 88.4% to 102.5%. The intra- and inter-day precisions varied from 2.1% to 7.6% and from 2.6% to 12.7%, respectively. The accuracy ranged from -1.9% to -7.4%.

Extraction of domoic acid from seawater and urine using a resin based on 2-(trifluoromethyl)acrylic acid.

A new solid-phase extraction (SPE) matrix with high affinity for the neurotoxin domoic acid (DA) was designed and tested. A computational modelling study led to the selection of 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer capable of imparting affinity towards domoic acid. Polymeric adsorbents containing TFMAA were synthesised and tested in high ionic strength solutions such as urine and seawater. The TFMAA-based polymers demonstrated excellent performance in solid-phase extraction of domoic acid, retaining the toxin while salts and other interfering compounds such as aspartic and glutamic acids were removed by washing and selective elution. It was shown that the TFMAA-based polymer provided the level of purification of domoic acid from urine and seawater acceptable for its quantification by high performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) without any additional pre-concentration and purification steps.

Domoic acid transfer to milk: evaluation of a potential route of neonatal exposure.

Domoic acid (DA), produced by the diatom genus Pseudo-nitzschia, is a glutamate analog and a neurotoxin in humans. During diatom blooms, DA can contaminate filter-feeding organisms, such as shellfish, and can be transferred by ingestion to higher trophic levels. Several intoxication events involving both humans and various marine mammals have been attributed to DA. Affected organisms show neurological symptoms such as seizures, ataxia, headweaving, and stereotypic scratching, as well as prolonged deficits in memory and learning. Neonatal animals have been shown to be substantially more sensitive to DA than adults. However, it has not been demonstrated whether DA can be transferred to nursing young from DA-exposed mothers. This study demonstrates transfer of DA from spiked milk (0.3 and 1.0 mg/kg) to the plasma of nursing neonatal rats and an overall longer DA retention in milk than in plasma after 8 hr in exposed dams. DA was detectable in milk up to 24 hr after exposure (1.0 mg/kg) of the mothers, although the amount of DA transferred to milk after exposure was not sufficient to cause acute symptoms in neonates.

Rapid determination of domoic acid in serum and urine by liquid chromatography-electrospray tandem mass spectrometry.

A rapid, selective, and sensitive LC-MS/MS method was developed for the quantitative determination of domoic acid in serum and urine samples. Samples were prepared for analysis using an Oasis HLB SPE column. Determination was by a reversed phase HPLC using a mixture of methanol, acetonitrile, and water containing 1% acetic acid and an electrospray ionization (ESI) ion-trap mass spectrometer (Finnigan LCQ). The method was validated by analyzing five replicates each of negative control bovine serum or urine fortified with domoic acid at the 0.005 microg/g method detection limit (MDL) and at the 0.05 microg/g level. Recoveries ranged from 90 to 95% for fortifications at the MDL and from 92 to 98% for fortifications 10 times higher than the MDL. The diagnostic utility of the method was tested by analyzing samples from live animals showing clinical signs suggestive of domoic acid poisoning submitted to the veterinary toxicology laboratory.

Subchronic toxicity study of domoic acid in the rat.

Male and female Sprague-Dawley rats were dosed by gavage for 64 days with 0, 0.1 or 5 mg/kg/day domoic acid. Treated animals showed no clinical abnormalities. Terminal values in haematology and clinical chemistry did not reveal differences between treated and control groups. Findings in histopathology and immunohistochemistry were unremarkable. The 24-hr urinary excretion rate for domoic acid determined at three time points was approximately 1.8% of the dose and remained unchanged during the study.

Comparison of high-performance liquid chromatography with radioimmunoassay for the determination of domoic acid in biological samples.

A reversed-phase liquid chromatographic method employing UV absorption detection at 242 nm was compared to a radioimmunoassay technique for the determination of the marine toxin, domoic acid, in several types of seafood and biological samples. Agreement between the two methods for spiked samples of mussels and rat serum was very good over a range of concentrations of 0.15-7.3 micrograms/g domoic acid. Also, a very good correlation was observed between the two methods for naturally incurred residues of domoic acid in razor clams, anchovies and crab meat over a concentration range of 0.6-43 micrograms/g domoic acid.

Comparison of UV absorption and electrospray mass spectrometry for the high-performance liquid chromatographic determination of domoic acid in shellfish and biological samples.

Domoic acid, a neurotoxic amino acid produced by the marine diatom Nitchia pungens multiseries, was determined in samples of anchovies, razor clams, mussels, crab, rat serum, urine and feces by HPLC with UV absorption and electrospray (ESI) mass spectrometric (MS) detection. Shellfish samples were extracted with methanol-water followed by clean-up of the extracts with solid-phase extraction cartridges (strong anion or strong cation exchange). An aliquot of the fraction containing the domoic acid was analysed by HPLC. HPLC column size, mobile phase composition and flow-rate were selected so that essentially the same conditions could be used for both HPLC-UV and HPLC-ESI-MS with selected ion monitoring (SIM) determinations. These included the use of acetonitrile-water-formic acid as the mobile phase, at a flow-rate of 0.2 ml/min (split 13:1 for HPLC-ESI-MS-SIM, 10 microliters/min to the mass spectrometer). The results indicated that extracts found positive by the HPLC-UV method could be readily confirmed directly by HPLC-ESI-MS-SIM without additional sample treatment down to levels of 0.1 micrograms/g of domoic acid. This study demonstrates the use of HPLC-ESI-MS-SIM for the routine confirmation of domoic acid in a wide variety of samples.

Renal clearance of domoic acid in the rat.

The renal clearance (Clr) of the seafood toxin domoic acid (DA) was investigated in the rat. Following cannulation of the right femoral artery, the left femoral vein and the bladder of anaesthetized rats, a single bolus injection of either [3H]DA, [14C]p-aminohippuric acid (PAH) or [3H]inulin was administered through the venous cannula. Blood samples were taken from the arterial cannula at 1, 2, 10, 30, 50, 70, 90, 110 and 130 min following injection, and urine samples were collected at 20-min intervals starting from the time of bolus injection. Based on plasma concentration-time profiles, the total clearances (Clt) for DA, PAH and inulin were 9.12, 33.17 and 7.50 ml/min/kg body weight, respectively. The Clr calculated from urinary excretion rates were not significantly different from the Clt. Probenecid significantly reduced the Clr of PAH but did not affect that of DA. When DA was given at doses of 0.5 ng, 0.5 mg and 2.0 mg/kg body weight, the pharmacokinetic parameters Clt, Clr, elimination-rate constant and apparent volume of distribution at steady state were not statistically different between doses. The entire dose of 3H was recovered in the urine by 160 min after dosing, and analysis of urine samples by HPLC confirmed that the radiolabel (3H) was associated predominantly with the parent form of DA. The results of the present study demonstrate that DA is cleared from plasma primarily through the kidneys. DA clearance occurs primarily by renal glomerular filtration since its Clt is comparable with that of inulin, is less than that of PAH and is not affected by probenecid.

Determination of domoic acid in seafoods and in biological tissues and fluids.

Domoic acid is extracted from mussel tissue using the Association of Official Analytical Chemists procedure for paralytic shellfish toxins. This involves boiling the sample for 5 min with O.1N HCl then cooling and centrifuging. An aliquot of the supernatant is diluted 10 to 100 times with water, filtered and analyzed by reversed-phase liquid chromatography with a mobile phase of acetonitrile-water (12:88) at pH 2.5 and absorption detection at 242 nm. The detection limit is about 1 mg/kg domoic acid in seafood samples. The method was successfully used in collaborative studies and a survey of 44 different commercially purchased shellfish products from areas outside of Prince Edward Island showed no domoic acid greater than 1 mg/kg. The same method was applied to urine and feces from monkeys and blood (serum) from humans. The method was unsuccessful for urine and blood which required additional cleanup before analysis. The method worked well for feces at domoic acid levels greater than 1 mg/kg.