Sample preparation and liquid chromatography-tandem mass spectrometry for the analysis of selected Pacific ciguatoxins in blood samples.

Consumption of ciguatoxin-contaminated seafood can lead to ciguatera poisoning (CP). The diagnosis of CP in humans is based on the clinical symptoms after eating the fish from tropical or subtropical areas because no confirmatory clinical tests are available. One of the challenges for ciguatoxin analysis is their extremely low but toxicologically relevant concentration in biological samples. We previously reported a method using acetonitrile to precipitate proteins and extract the ciguatoxins simultaneously in whole blood samples from animals for toxin quantification by N2A cell-based assay. However, a test method for unambiguous confirmation of exposure of marine animals or humans to ciguatoxins is still needed. In the present study, we adopted the acetonitrile extraction method and added sample clean-up in the sample preparation for the determination of Pacific ciguatoxins CTX1B (aka P-CTX-1), 52-epi-54-deoxyCTX1B (aka P-CTX-2), and CTX3C (aka P-CTX-3C) in blood plasma by LC-MS/MS. We investigated sample clean-up, LC mobile phases, LC solvent programming, and settings of the two mass spectrometers (4000 Q TRAP and AB SCIEX Triple Quad 5500) in order to improve the ability to detect the Pacific ciguatoxins at ppt level. Rat blood plasma was used for the method development. Average recoveries of the three toxins in the rat plasma samples ranged from 90% to 116% with relative standard deviations of less than 15%. The method detection limits were still not low enough for the determination of the Pacific ciguatoxins in individual blood samples from Hawaiian monk seals with the two LC-MS systems. The methods were applied to a pooled sample of blood plasma collected from Hawaiian monk seals for confirmation of toxin exposure. This study will benefit monitoring of Pacific ciguatoxins in marine mammals and potentially humans by LC-MS/MS.

Effects of Swimming Exercise on Serum Irisin and Bone FNDC5 in Rat Models of High-Fat Diet-Induced Osteoporosis.

This study aimed to investigate the expression of PGC-1α/FNDC5/irisin induced by attenuation of high-fat diet (HFD)-induced bone accrual and determine whether swimming exercise could improve attenuating bone accrual through this mechanism. Eight-week-old Sprague-Dawley rats were divided into two groups for the first 8 weeks: CD, control diet (n = 10); and HFD, high-fat diet (n = 20). HFD-fed rats were again divided into two groups for further 8 weeks treatment: HFD (n = 10) and HFD with swimming exercise (HEx, n = 10). During this time, the CD group continuously fed the normal diet. Throughout the 16 weeks study period, the rats were weighed once every week. Samples were collected for analysis after last 8 weeks of treatment in the 16 weeks. Morphological and structural changes of the femur and tibial bone were observed using micro-CT, and Osteocalcin, CTX-1 and irisin levels in the blood were measured by enzyme-linked immunosorbent assay. The expression of IL-1, ß-catenin, FNDC5 and PGC-1α, in the femur were evaluated by immunohistochemistry. Eight weeks of HFD increased body weight and epididymal fat mass and decreased bone mineral density (BMD). Subsequent 8 weeks of swimming exercise improved obesity, BMD, bone microstructure, and bone metabolic factors in the HEx group. The irisin levels in the blood and the expressions of FNDC5 and PGC-1α in the bone were significantly lower in the HFD group than in the CD group, but elevated in the HEx group than in the HFD group. Swimming exercise is effective in improving obesity-worsened bone health and increases blood irisin and bone PGC-1α and FNDC5 levels.

Simultaneous quantification of Pacific ciguatoxins in fish blood using liquid chromatography-tandem mass spectrometry.

Ciguatera fish poisoning (CFP) is a food intoxication caused by exposure to ciguatoxins (CTXs) in coral reef fish. Rapid analytical methods have been developed recently to quantify Pacific-CTX-1 (P-CTX-1) in fish muscle, but it is destructive and can cause harm to valuable live coral reef fish. Also fish muscle extract was complex making CTX quantification challenging. Not only P-CTX-1, but also P-CTX-2 and P-CTX-3 could be present in fish, contributing to ciguatoxicity. Therefore, an analytical method for simultaneous quantification of P-CTX-1, P-CTX-2, and P-CTX-3 in whole blood of marketed coral reef fish using sonication, solid-phase extraction (SPE), and liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. The optimized method gave acceptable recoveries of P-CTXs (74-103 %) in fish blood. Matrix effects (6-26 %) in blood extracts were found to be significantly reduced compared with those in muscle extracts (suppressed by 34-75 % as reported in other studies), thereby minimizing potential for false negative results. The target P-CTXs were detectable in whole blood from four coral reef fish species collected in a CFP-endemic region. Similar trends in total P-CTX levels and patterns of P-CTX composition profiles in blood and muscle of these fish were observed, suggesting a relationship between blood and muscle levels of P-CTXs. This optimized method provides an essential tool for studies of P-CTX pharmacokinetics and pharmacodynamics in fish, which are needed for establishing the use of fish blood as a reliable sample for the assessment and control of CFP.

Outbreak of ciguatera fish poisoning on a cargo ship in the port of hamburg.

BACKGROUND: Ciguatera fish poisoning is a travel-related illness characterized by a combination of gastrointestinal and neurological symptoms in persons who eat ciguatoxic seafood in endemic areas. METHODS: In 2009, an outbreak of the disease on a refrigerator vessel in the port of Hamburg was investigated. The ship's crew fell ill after they ate fish from a catch in the Caribbean 2 weeks earlier. All 15 sailors on board were examined by port medical officers. Samples of blood and stool specimens were taken from symptomatic sailors. The frozen fish was secured for the prevention of further disease spreading and additional diagnostic tests. RESULTS: All but one sailor ate the fish. The intoxication resulted in gastrointestinal or neurological symptoms in all 14 sailors who consumed the fish and persisted in varying degrees in 93% of sailors over at least 14 days. No fatality occurred, but two seamen were "unfit for duty" on the ship due to severity of symptoms. The diagnosis was supported by the fact that all seafarers who consumed the same reef fish, experienced typical signs, symptoms, and time course consistent with ciguatera fish poisoning. The fish from the catch in the Caribbean was identified as Caranx sexfasciatus (Bigeye Trevally) and Cephalopholis miniata (Red Grouper). An experimental assay later confirmed presence of the ciguatoxin in the fish. CONCLUSIONS: Sailors are an occupational group at risk for ciguatera fish poisoning due to potentially unsafe food sources during international travel. Even if no fatality occurred, the disease affected marine operations due to high attack rates and chronicity of symptoms. Medical doctors must be aware that ciguatera fish poisoning is a risk for seafarers traveling in tropical and subtropical areas. Stocking of food in affected ports from safe sources, adequate training of ship cooks, and informing sailors about the risk of fishing are needed to prevent disease occurrence in seafarers in international trade and traffic.

Linking ciguatera poisoning to spatial ecology of fish: a novel approach to examining the distribution of biotoxin levels in the great barracuda by combining non-lethal blood sampling and biotelemetry.

Ciguatera in humans is typically caused by the consumption of reef fish that have accumulated Ciguatoxins (CTXs) in their flesh. Over a six month period, we captured 38 wild adult great barracuda (Sphyraena barracuda), a species commonly associated with ciguatera in The Bahamas. We sampled three tissues (i.e., muscle, liver, and blood) and analysed them for the presence of ciguatoxins using a functional in vitro N2A bioassay. Detectable concentrations of ciguatoxins found in the three tissue types ranged from 2.51 to 211.74pg C-CTX-1 equivalents/g. Blood and liver toxin concentrations were positively correlated (ρ=0.86, P=0.003), indicating that, for the first time, blood sampling provides a non-lethal method of detecting ciguatoxin in wild fish. Non-lethal blood sampling also presents opportunities to couple this approach with biotelemetry and biologging techniques that enable the study of fish distribution and movement. To demonstrate the potential for linking ciguatoxin occurrence with barracuda spatial ecology, we also present a proof-of-concept case study where blood samples were obtained from 20 fish before releasing them with acoustic transmitters and tracking them in the coastal waters using a fixed acoustic telemetry array covering 44km(2). Fish that tested positive for CTX may have smaller home ranges than non-toxic fish (median distance travelled, U=2.21, P=0.03). Results presented from this study may help identify high risk areas and source-sink dynamics of toxins, potentially reducing the incidence and human health risk of ciguatera fish poisoning. Moreover, development of the non-lethal sampling approach and measurement of ciguatera from blood provide future opportunities to understand the mechanistic relationship between toxins and the spatial ecology of a broad range of marine fish species.

Treatment of a major depression episode suppresses markers of bone turnover in premenopausal women.

UNLABELLED: Both decrease in bone mineral density and increase in bone turnover had been reported in patients with major depression compared to healthy controls. But the effect of antidepressant treatment on markers of bone turnover is not studied. The aim of this study was to investigate the effect of treatment of a major depressive episode with an SSRI antidepressant on bone turnover in premenopausal women. METHODS: Fifty premenopausal female patients with newly diagnosed major depression according to DSM IV-R criteria were included into the study. Before starting antidepressant therapy (escitalopram 10 mg/day) and three months later, blood samples were collected for the measurement of serum calcium, phosphorus, osteocalcin, ß-CTX and iPTH. Depressive status was determined with Hamilton Depression Scale. RESULTS: Treatment of depression did not create any change in laboratory levels of either calcium or phosphorus. Basal iPTH level was significantly decreased with the treatment. Treatment resulted in an increase in serum osteocalcin and decrease in ß-CTX levels. HAMD score was significantly correlated with both osteocalcin and ß-CTX. The decrease in ß-CTX and increase in osteocalcin levels were more prominent in patients with a HAMD score that remained below 15 than above 15 at the end of the study period. In conclusion, this study shows that with the treatment of depression bone formation increases and bone resorption decreases in premenopausal women with major depression.

Identification of ciguatoxins in Hawaiian monk seals Monachus schauinslandi from the Northwestern and Main Hawaiian Islands.

Ciguatoxins are potent algal neurotoxins that concentrate in fish preyed upon by the critically endangered Hawaiian monk seal (Monachus schauinslandi). The only report for Hawaiian monk seal exposure to ciguatoxins occurred during a 1978 mortality event when two seal liver extracts tested positive by mouse bioassay. Ciguatoxins were thus proposed as a potential threat to the Hawaiian monk seal population. To reinvestigate monk seal exposure to ciguatoxins we utilized more selective detection methods, the Neuro-2A cytotoxicity assay, to quantify ciguatoxin activity and an analytical method LC-MS/MS to confirm the molecular structure. Tissue analysis from dead stranded animals revealed ciguatoxin activity in brain, liver, and muscle, whereas analysis of blood samples from 55 free-ranging animals revealed detectable levels of ciguatoxin activity (0.43 to 5.49 pg/mL P-CTX-1 equiv) in 19% of the animals. Bioassay-guided LC fractionation of two monk seal liver extracts identified several ciguatoxin-like peaks of activity including a peak corresponding to the P-CTX-3C which was confirmed present by LC-MS/MS. In conclusion, this work provides first confirmation that Hawaiian monk seals are exposed to significant levels of ciguatoxins and first evidence of transfer of ciguatoxin to marine mammals. This threat could pose management challenges for this endangered marine mammal species.

Repeat exposure to ciguatoxin leads to enhanced and sustained thermoregulatory, pain threshold and motor activity responses in mice: relationship to blood ciguatoxin concentrations.

Ciguatera is a common illness in tropical and subtropical regions that manifests in complex and long-lived symptoms which are more severe in subsequent exposures. This study measures central and peripheral neurologic signs, in parallel with blood toxin levels, in mice exposed once or twice (at 3 days interval) to a sublethal dose of ciguatoxin P-CTX-1 (0.26ng/g via i.p.). Mice were implanted with radiotransmitters to monitor motor activity and core temperature. A single exposure to ciguatoxin elicited an immediate and transient decrease in motor activity and temperature, and subsequent long-lasting thermoregulatory dysfunction resulting in stabilized body temperature around 36.0 degrees C with no observable circadian rhythm. The hypothermic response and the reduced activity were enhanced with a second exposure with 30% of the mice dying within 7h. Measurement of the peripheral nervous system by the tail flick assay revealed increased latency with a single ciguatoxin exposure, and a greater effect following the second exposure. Toxin was measurable in blood up to 3 days following the first exposure; at the 1h time point the concentrations were significantly elevated after a second exposure. These findings indicate an early response to ciguatoxin manifest in a central response to lower body temperature and reduce motor activity and a more persistent effect on the peripheral system leading to spinal heat antinociception and delayed fever-like response. The greater neurological response to a second ciguatoxin exposure was associated with elevated concentrations of ciguatoxin in the blood solely over the first hour of exposure. In conclusion, a single exposure to toxin exerts a significant neurological response which may be enhanced with subsequent exposure.

Ciguatera fish poisoning: impact for the military health care provider.

Ciguatera toxin is a marine neurotoxin produced by microorganisms that becomes concentrated in predatory fish. Toxicity in humans results from the ingestion of contaminated fish harvested in tropical waters. Clinical manifestations of illness include the rapid onset of gastrointestinal symptoms and neurological abnormalities. Because of the rapid onset of symptoms and the potential for case clusters from a common source ingestion of contaminated fish, there is the potential that ciguatera poisoning may initially mimic illnesses caused by antipersonnel biological and chemical agents. We present data on an active duty soldier who presented to sick call for evaluation of new onset paresthesias and was diagnosed with ciguatera toxin poisoning. We also present a review of ciguatera poisoning literature with emphasis on the distinguishing features between ciguatoxin and other neurotoxins of military significance.

Optimization of ciguatoxin extraction method from blood for Pacific ciguatoxin (P-CTX-1).

Ciguatera diagnosis relies on clinical observations associated with a recent consumption of fish. Although needed, direct confirmation of exposure in subjects showing ciguatera disease symptoms is currently unavailable. We previously reported that ciguatoxins were measurable in the blood of mice exposed to extracts of Pacific ciguatoxins isolated from Gambierdiscus polynesiensis, and of Indian Ocean or Caribbean Sea ciguatoxins, isolated from fish. Although highly efficient for extracting spiked purified Caribbean-CTX-1, the methanolic extraction method previously described is found here to yield only 6% recovery of spiked Pacific-CTX-1 (P-CTX-1). We report in this short communication a substantially modified method for ciguatoxin extraction from both dried and fresh blood. With this method, toxin measurement is directly accomplished in acetonitrile deproteinated whole fresh blood or phosphate buffer solution (PBS) eluted dried blood using the N2A cell-based assay. Spike studies using increasing concentrations of purified ciguatoxins reveal linear (r2 above 0.87 for all toxins) and overall efficient toxin recoveries (62%, 96%, and 96% from fresh blood and 75%, 90%, and 74% from dried blood, for C-CTX-1, P-CTX-3C, and P-CTX-1, respectively). Comparative blood matrix analysis for P-CTX-1 recovery shows increased recovery of ciguatoxin activity from whole fresh blood than from dried blood, greater by 20% in P-CTX-1 spiked mice blood and by over 85% in P-CTX-1 exposed mouse blood. In conclusion, both Caribbean and Pacific ciguatoxins can be readily extracted from blood using this modified method; however, in the case of P-CTX-1 we find that fresh blood is optimal.

Serum cathepsin K concentrations reflect osteoclastic activity in women with postmenopausal osteoporosis and patients with Paget's disease.

INTRODUCTION: Cathepsin K, a cysteine protease, plays an essential role in osteoclast-mediated collagen degradation. Recently, an immunoassay to quantify cathepsin K in serum has been developed. We assessed the usefulness of serum cathepsin K as a marker of bone turnover in cross-sectional and longitudinal studies of patients with metabolic bone disease. METHODS: The study cohort consisted of 40 healthy subjects, 21 women with postmenopausal osteoporosis [66.1 +/- 7.9 yrs] and 10 patients with Paget's disease of bone [67.1 +/- 11.6 yrs]. All patients were started on oral or intravenous bisphosphonate treatment and were followed prospectively over 6 months. Circulating cathepsin K levels were determined by a specific sandwich enzyme immunoassay (Biomedica, Vienna, Austria). In addition, serum carboxyterminal cross-linked telopeptide of type I collagen (betaCTX-I) and bone-specific alkaline phosphatase (BALP) were measured for comparison. RESULTS: When compared to healthy subjects, mean serum cathepsin K levels were significantly elevated in women with postmenopausal osteoporosis (3.1 +/- 1.9 vs. 11.3 +/- 13.1 pmol/L, p = 0.01) and in patients with Paget's disease of bone (6.2 +/- 4.4 pmol/L, p = 0.04). In postmenopausal osteoporotic women, both oral and intravenous bisphosphonate treatment resulted in a significant reduction in serum cathepsin K levels (p = 0.03) with most of the effect occurring after one month (mean% change: -33%). In patients with mild Paget's disease, serum cathepsin K levels decreased during bisphosphonate treatment. CONCLUSIONS: Serum concentrations of cathepsin K appear to reflect osteoclastic activity in patients with postmenopausal osteoporosis and Paget's disease of bone and may hold promise as a marker of osteoclast activity.

Biomonitoring of ciguatoxin exposure in mice using blood collection cards.

Ciguatera is a human food poisoning caused by consumption of tropical and subtropical fish that have, through their diet, accumulated ciguatoxins in their tissues. This study used laboratory mice to investigate the potential to apply blood collection cards to biomonitor ciguatoxin exposure. Quantitation by the neuroblastoma cytotoxicity assay of Caribbean ciguatoxin (C-CTX-1) spiked into mice blood was made with good precision and recovery. The blood collected from mice exposed to a sublethal dose of Caribbean ciguatoxic extract (0.59 ng/g C-CTX-1 equivalents) was analyzed and found to contain detectable toxin levels at least 12 h post-exposure. Calculated concentration varied from 0.25 ng/ml at 30 min post-exposure to 0.12 ng/ml at 12 h. A dose response mice exposure revealed a linear dose-dependent increase of ciguatoxin activity in mice blood, with more polar ciguatoxin congeners contributing to 89% of the total toxicity. Finally, the toxin measurement in mice blood exposed to toxic extracts from the Indian Ocean or from the Pacific Ocean showed that the blood collection card method could be extended to each of the three known ciguatoxin families (C-CTX, I-CTX and P-CTX). The low matrix effect of extracted dried-blood samples (used at 1:10 or 1:20 dilution) and the high sensitivity of the neuroblastoma assay (limit of detection 0.006 ng/ml C-CTX-1), determined that the blood collection card method is suitable to monitor ciguatoxin at sublethal doses in mice and opens the potential to be a useful procedure for fish screening, environmental risk assessment or clinical diagnosis of ciguatera fish poisoning in humans or marine mammals.

[Detection of ciguatoxins: advantages and drawbacks of different biological methods]. / Detection des ciguatoxines: avantages et inconvénients des différentes méthodes biologiques utilisées.

Ciguatera is a seafood intoxication that results from ingestion of reef fish contaminated with ciguatoxins at levels orally toxic for humans. Precursors of those toxins, gambiertoxins, are produced by benthic dinoflagellates (genus Gambierdiscus), and then accumulated and biotransformed by herbivorous and carnivorous fishes into ciguatoxins, more toxic for humans. In the absence of specific treatment, that disease remains a health problem with otherwise adverse socio-economic impacts. Thus a cost-effective means of detecting ciguatoxins in fish has long been searched for. Many assays have been developed, including in vivo, in vitro, chemical or immunochemical approaches. This review focuses on some biological methods, from the well-standardised mouse assay to the specific radio-labelled ligand binding assay that is performed on rat brain synaptosomes. In addition to the mouse, the chick and the mongoose were still recently used, in particular for preliminary tests before ciguatoxin extraction from fish, since assays in these animals can directly assay the whole flesh. In contrast, various other in vivo methods, such as the kitten, mosquito and diptera larvae assays, were abandoned despite their interesting results. Finally, the mouse neuroblastoma and rat brain synaptosome assays, carried out in vitro as alternative approaches to animal-using assays, are highly sensitive and much more specific than the in vivo methods to detect ciguatoxins.

A pilot study for the detection of acute ciguatera intoxication in human blood.

INTRODUCTION: Ciguatera fish poisoning arises from consumption of any of the 400 species of tropical marine reef fish containing polyether toxins. No laboratory method is available for clinical diagnosis of acute ciguatera poisoning. The objective of this pilot study was to ascertain the potential usefulness of a bioassay to detect ciguatoxins in humans suspected of acute intoxication. We analyzed plasma of healthy volunteers (asymptomatic negative controls), participants with gastrointestinal (GI) illness but without recent fish consumption (symptomatic negative controls), and participants with GI illness who had recently consumedfish. MATERIALS AND METHODS: Blood samples, questionnaires, and consent forms were collected from 11 symptomatic negative controls and 86 patients that visited emergency rooms in southern Puerto Rico over a 1-year period. Patients had consumed fish within 24 hour prior to the symptoms. Plasma samples were analyzed by a neuroblastoma cell bioassay that detects seafood toxins active at the sodium voltage-gated channel in a dose-dependent fashion. Concentrations were expressed in terms of brevetoxin-1 equivalents (ng PbTx-1 equiv/mL). RESULTS: The mean plasma concentration of 14 asymptomatic negative controls was 39.4 ng PbTx-1 equiv/mL (range 2-74). Of 86 potential ciguatoxic patients who reported fish consumption, 43 had values within the range of normal volunteers, and 9 had concentrations in the nondiagnostic range (73.9-100 ng). Thirty-four patients (40%) had concentrations 3 standard deviations above asymptomatic negative controls (>100 ng PbTx-1 equiv/mL). They had a mean concentration of 1,074 +/- 244.5 ng PbTx-1 equiv/mL (range 101-7,056ng). CONCLUSION: Preliminary findings of elevated PbTx-1 equivalents in 40% of the patients with both ciguatera symptomatology and fish consumption in a geographical area where ciguatera is common suggest that the neuroblastoma bioassay may be a potential diagnostic tool for acute ciguatera intoxication.

Ciguatera poisoning after ingestion of imported jellyfish: diagnostic application of serum immunoassay.

Ciguatera fish poisoning is an important public health problem wherever humans consume tropical and subtropical fish. It accounts for over half of fish-related poisonings in the United States but is uncommonly diagnosed and underreported. Produced by dinoflagellates, ciguatoxin accumulates up the food chain in herbivorous and carnivorous fishes. Cnidaria jellyfish and related invertebrates) have not previously been associated with direct ciguatera intoxication in humans. We report the first case of ciguatera fish poisoning associated with cnidarian ingestion. A 12-year-old Tongan female presented to our Emergency Department with mid-abdominal pain, nausea, change in mental status, and new-onset movement disorder after ingestion of jellyfish imported from American Samoa. Clinical diagnosis was confirmed by strongly positive serum identification of ciguatoxin and related polyether toxins (including okadaic acid) with a rapid extraction method (REM) and highly reliable solid-phase immunobead assay (S-PIA) performed by the Food Toxicology Research Group, University of Arizona. Ciguatera pathophysiology, clinical presentation, differential diagnosis (including consideration of palytoxin poisoning), and treatment are briefly reviewed. We emphasize the growing incidence of ciguatera fish poisoning outside "high-risk" areas. In regions with immigrant populations, privately imported exotic fish may be toxin vectors. Marine species other than carnivorous fish are now suspect in human ciguatera intoxication. Reliable tests can aid in premarket fish testing, diagnosis, and follow-up of ciguatera fish poisoning. The global prevalence of marine toxins demands fishermen, consumers, and physicians maintain a high index of suspicion for ciguatera fish poisoning.