Rapid screening and multi-toxin profile confirmation of tetrodotoxins and analogues in human body fluids derived from a puffer fish poisoning incident in New Caledonia.

In August 2014, a puffer fish poisoning incidence resulting in one fatality was reported in New Caledonia. Although tetrodotoxin (TTX) intoxication was established from the patients' signs and symptoms, the determination of TTX in the patient's urine, serum or plasma is essential to confirm the clinical diagnosis. To provide a simple cost-effective rapid screening tool for clinical analysis, a maleimide-based enzyme-linked immunosorbent assay (mELISA) adapted for the determination of TTX contents in human body fluids was assessed. The mELISA was applied to the analysis of urine samples from two patients and a response for the presence of TTX and/or structurally similar analogues was detected in all samples. The analysis by LC-MS/MS confirmed the presence of TTX but also TTX analogues (4-epiTTX, 4,9-anhydroTTX and 5,6,11-trideoxyTTX) in the urine. A change in the multi-toxin profile in the urine based on time following consumption was observed. LC-MS/MS analysis of serum and plasma samples also revealed the presence of TTX (32.9 ng/mL) and 5,6,11-trideoxyTTX (374.6 ng/mL) in the post-mortem plasma. The results provide for the first time the TTX multi-toxin profile of human samples from a puffer fish intoxication and clearly demonstrate the implication of TTX as the causative agent of the reported intoxication case.

Assessment of deoxynivalenol exposure among Bangladeshi and German adults by a biomarker-based approach.

Deoxynivalenol (DON) is a frequent mycotoxin contaminant in cereal crops worldwide and can cause adverse health effects in exposed animals and humans. Since DON contamination in Bangladeshi food is unexplored, we conducted a biomonitoring study to assess DON exposure in the Bangladeshi population and compare it with that of German adults. In total 214 urines were collected, n=164 in Bangladesh and n=50 in Germany. In Bangladesh rural and urban residents of Rajshahi district provided urines in two seasons (n=69 in summer, n=95 in winter, with 62 participants enrolled in both periods). Urinary DON and its de-epoxy metabolite DOM-1 were measured by a previously validated sensitive LC-MS/MS method. In Bangladeshi urines, DON was detectable in 27% (range 0.16-1.78ng/mL) in summer and 31% (range 0.16-1.21ng/mL) in winter season. There was no significant difference at the mean DON level between season (summer 0.17±0.25ng/mL and winter 0.16±0.18ng/mL) and region (rural or urban residents). The metabolite DOM-1 was not detected in any urine from Bangladesh. In contrast, DON and DOM-1 were detected in 100% (range 0.16-38.44ng/mL) and 40% (range 0.10-0.73ng/mL), respectively, of the German urines. The mean DON level in German urines (9.02±6.84ng/mL) was about 53-fold higher than that found in Bangladeshi samples. This indicates a low and high dietary DON exposure among the adult population in Bangladesh and Germany, respectively. The biomarker concentrations found and published urinary excretion rates for DON then served to calculate the daily mycotoxin intake in both cohorts: the mean DON intake in Bangladesh being 6ng/kg b.w., and in Germany a mean of 268 and maximum intake of 975ng/kg b.w., values lower than the provisional maximum tolerable daily intake of 1µg/kg b.w. set by the WHO/JECFA.

Biomonitoring of concurrent mycotoxin exposure among adults in Sweden through urinary multi-biomarker analysis.

Mycotoxin producing moulds may contaminate numerous agricultural commodities either before harvest or during storage. A varied diet consisting of different foods may therefore be contaminated with a range of mycotoxins. The aim of the present study was to study concurrent exposure to mycotoxins through urinary multi-biomarker analysis, as well as its possible associations with the diet. Urinary samples from 252 adults, participating in the Swedish national dietary survey Riksmaten 2010-11, were collected together with a 4-day diet record. Concurrent mycotoxin exposure was studied using a multi-biomarker LC-MS/MS method. The results revealed that exposure to mycotoxins is common and concurrent exposure to more than one toxin was found in 69% of the study population. However, when comparing the number of toxins detected with the reported consumption data it was difficult to distinguish food patterns which would indicate an increased risk of exposure to many mycotoxins simultaneously. This is the first study to investigate concurrent mycotoxin exposure and urinary levels of fumonisin B1 (FB1), fumonisin B2 (FB2), nivalenol (NIV), ochratoxin A (OTA), zearalenone (ZEA), α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL) and de-epoxydeoxynivalenol (DOM-1) among adults in Sweden.

Late diagnosis of an outbreak of leanness-enhancing agent-related food poisoning.

INTRODUCTION: Ractopamine is a leanness-enhancing agent approved in the United States and 26 other countries to reduce body fat content, increase muscle mass, and improve growth rate of certain food-producing animals. Other ß-agonists with stronger pharmacologic effects, especially clenbuterol, had been illegally used as leanness-enhancing agents in the United States, China, and the European Union, and foodborne poisonings related to clenbuterol residue in meat or liver were rarely reported in the European Union and China. We describe an unusual outbreak of leanness-enhancing agent-related food poisoning in Taiwan and its associated diagnostic challenge. REPORT OF THE OUTBREAK: Twelve patients presented to the emergency department of a regional hospital after having dinner together. Their clinical manifestations included nausea, vomiting, palpitation, facial flush, trunk or limb numbness, tremor, headache, weakness, chill, and dyspnea. Laboratory workup revealed the presence of hypokalemia, leukocytosis, and hyperglycemia. Poisoning attributable to ß-agonists was suspected; however, the diagnosis of leanness-enhancing agent poisoning was delayed because there was no leftover meat for analysis and because the veterinary medicine was illegal in Taiwan. Clenbuterol and salbutamol were eventually detected in 10 patients' urine sample by using liquid chromatography-tandem mass spectrometry, and the concentrations ranged from 54 to 806 µg/L and from 0 to 4052 µg/L, respectively. CONCLUSION: ß-Agonist leanness-enhancing agent-related food poisonings are rarely encountered, especially in those countries where relevant veterinary medicines are banned, and may thus pose diagnostic challenge to both emergency physicians and clinical toxicologists.

Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS.

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient's signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient's urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.

Evaluation of fumonisin biomarkers in a cross-sectional study with two high-risk populations in China.

Levels of serum and urinary sphinganine (Sa) and sphingosine (So), the Sa/So ratio, and urinary-free fumonisin B(1) (FB(1)) were determined in a cross-sectional study consisting of 43 adults in Huaian and 34 adults in Fusui, China. Home-produced corn had 100% contamination with FB(1). There were 93.0% (40/43) of Huaian subjects and 52.9% (18/34) of Fusui subjects with daily FB(1) intakes greater than 2 microg kg(-1) body weight, which showed a significant difference (p < 0.01). Levels of sphinganine and sphingosine and the Sa/So ratio were not correlated with levels of dietary exposure. The median level of the serum Sa/So ratio in Huaian subjects (0.41, range = 0.14-0.85) was significantly lower than that in Fusui subjects (0.78, range = 0.57-1.08) (p < 0.01). The median level of the urinary Sa/So ratio was also significantly lower in Huaian subjects (0.31, range = 0.08-1.33) than in Fusui subjects (0.57, range = 0.03-2.52) (p < 0.01). Urinary-free FB(1) was detected in 83.7% (36/43) of Huaian samples and in 82.4% (28/34) of Fusui urine samples (p > 0.05). However, the median level of urinary-free FB(1) in Huaian subjects, 3.91 (range = 0.06-253.61) ng mg(-1) creatinine, was significantly higher than 0.39 (range = 0.01-3.72) ng mg(-1) creatinine found in Fusui subjects (p < 0.01). These results suggest that urinary-free FB(1) may be a potential biomarker for human fumonisin exposure, while further validation is needed in human epidemiological and intervention studies.

Rapid screening of tetrodotoxin in urine and plasma of patients with puffer fish poisoning by HPLC with creatinine correction.

A rapid and simple detection method for tetrodotoxin (TTX) in urine and plasma of patients with puffer fish poisoning was developed using commercially pre-packed solid-phase extraction (SPE) cartridges (C18 and weak cation exchange columns) and subsequent analyses by HPLC with UV detection. The detection limit of the standard TTX, TTX-spiked urine and plasma samples were all 10 ng/ml and the average TTX recovery in urine and plasma samples after SPE were 90.3 +/- 4.0 and 87.1 +/- 2.9%, respectively. It was noticed that the creatinine-adjusted urinary TTX levels obtained within the first 24 h of presentation apparently correlated much better with the severity of poisoning than the urinary TTX concentration without adjusting for variations in concomitant creatinine excretion.

[Evaluation of the endogenous intoxication syndrome in food toxic infections].

To study the endogenous intoxication syndrome in patients with food toxic infections is essential in revealing the pathogenetic mechanisms underlying this disease. For this, the authors measured the level of low and average molecular weight, as well as their protein component--oligopeptides in plasma, red blood cells, and urine in the course of the disease. There were increased levels of the study parameters, which depended on the stage and degree of a pathological process. The determination of the level of low and average molecular weight and oligopeptides in plasma, red blood cells, and urine may serve as a marker of the intoxication syndrome; the level of the study parameters may be used as additional criteria for the severity of the process, the prediction of disease development and comorbidity, and as a criterion for recovery completeness.

Unexpectedly dangerous escargot stew: oleandrin poisoning through the alimentary chain.

A female, aged 43 and a male, aged 66, experienced gastrointestinal and cardiovascular symptoms after a meal including snail stew. Twelve hours after the ingestion, they presented with nausea, vomiting, diarrhea, and cardiovascular symptoms typical of acute toxic digoxin ingestion and were hospitalized. The man's electrocardiogram was altered, and the woman's was normal. Serum digoxin levels, measured on a Roche COBAS Integra 800 with the Roche On-Line Digoxin reagent, were 1.14 and 1.00 nmol/L, respectively. Potassium levels were normal in both patients. The serum digoxin concentration decreased on the second day, and symptoms resolved on the third day with patients fully recovered (i.e., reversion to a normal sinus rhythm). Cardiac-glycoside-like intoxication symptoms follow the ingestion of leaves or flowers of Nerium oleander. The consumed snails were suspected to be responsible for the intoxication. In the homogenized snail tissue, the concentration expressed in digoxin equivalents was 0.282 nmol/g. The presence of oleandrin and oleandrigenin in the snails was confirmed by liquid chromatography-tandem mass spectrometry analysis, which was performed on a ionic-trap Finnigan LXQ instrument using an electrospray ionization interface. High-pressure liquid chromatographic separation was performed on a C18 column with a gradient of methanol/water. An extract of oleander leaves was used as reference.

Confirmed Datura poisoning in a horse most probably due to D. ferox in contaminated tef hay.

Two out of a group of 23 mares exposed to tef hay contaminated with Datura ferox (and possibly D. stramonium) developed colic. The 1st animal was unresponsive to conservative treatment, underwent surgery for severe intestinal atony and had to be euthanased. The 2nd was less seriously affected, responded well to analgesics and made an uneventful recovery. This horse exhibited marked mydriasis on the first 2 days of being poisoned and showed protracted, milder mydriasis for a further 7 days. Scopolamine was chemically confirmed in urine from this horse for 3 days following the colic attack, while atropine could just be detected for 2 days. Scopolamine was also the main tropane alkaloid found in the contaminating plant material, confirming that this had most probably been a case of D. ferox poisoning. Although Datura intoxication of horses from contaminated hay was suspected previously, this is the 1st case where the intoxication could be confirmed by urine analysis for tropane alkaloids. Extraction and detection methods for atropine and scopolamine in urine are described employing enzymatic hydrolysis followed by liquid-liquid extraction and liquid chromatography tandem mass spectrometry (LC/MS/MS).

[Determination of tetrodotoxin in puffer-fish tissues, and in serum and urine of intoxicated humans by liquid chromatography with tandem mass spectrometry].

A simple and rapid method was developed for the analysis of tetrodotoxin in puffer-fish tissues, and in serum and urine of humans poisoned after consuming puffer-fish, by means of high-performance liquid chromatography with tandem mass spectrometry (LC/MS/MS). Tetrodotoxin was extracted with 2% acetic acid. The extracted solution from puffer-fish tissues was diluted with water, and the extracted solution from human serum and urine was cleaned up by LC/MS/MS with a methacrylate-styrenedivinylbenzene cartridge. The LC separation was performed on a C18 column (50 mm x 2.1 mm i.d.) using 10 mmol/L IPCC-MS7-methanol (65 : 35) as the mobile phase at a flow rate of 0.2 mL/min. The mass spectral acquisition was done in the positive ion mode by applying selected reaction monitoring (SRM). The recoveries of tetrodotoxin were 79-90% from puffer-fish tissues fortified at 0.1 microg/g and 1 microg/g, and 93-101% from human serum and urine fortified at 0.5 ng/mL and 5 ng/mL. The detection limits of tetrodotoxin were 0.01 microg/g in puffer-fish tissues and 0.1 ng/mL in human serum and urine. Thirty samples of puffer-fish from wholesale markets, and 7 serum and 5 urine samples of humans poisoned after consuming puffer-fish were analyzed by this method. Tetrodotoxin was detected in all puffer-fish tissues, and all serum and urine samples at the levels of 0.04-140 microg/g, 0.9-1.8 ng/mL and 15-150 ng/mL, respectively.

Analysis of ingested material and urine by GC-MS and 1H NMR spectroscopy: poisoning of an adult with adulterated soda.

The purpose of this work is to characterize chemical compounds added to an ingested soda by (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy and by gas chromatography-mass spectrometry in the electron impact mode. A second point was to highlight possible metabolic disturbances by considering urinary profile. Without any pretreatment, dimethylphtalate, 2-butanone, and 2,2,4-trimethylpentanediol diisobutyrate were found in the adulterated soda. Quantitative analysis was performed by relative integration of peak areas. Huge quantities of 2,2,4-trimethylpentanediol diisobutyrate and dimethylphtalate were found in the oily layer. 2-Butanone, which is miscible in water, was found in the two phases as well as small quantities of dimethylphtalate. The urine sample was collected on hospital admission and was also analyzed by (1)H NMR spectroscopy. The major abnormal compound found was 1,2-propanediol. Other disturbances concerned endogenous metabolites such as 2-ketoglutaric acid, lactic acid, and betaine.

Determination of tetramethylenedisulfotetramine in human urine with gas chromatograph-flame thermionic detection coupling with direct immersed solid-phase micro-extraction.

An analytical method for determination of tetramethylenedisulfotetramine (tetramine) in human urine by gas chromatography-flame thermionic detection (GC-FTD) coupling with a direct immersed solid-phase micro-extraction (DI-SPME) was developed. The enrichment effects of three fiber coatings of SPME for tetramine were compared. Results showed that the enrichment effect of polar 85 microm polyacrylate (PA) coating was better than that of apolar 100 and 7 microm polydimethylsiloxane (PDMS) coatings. Other experimental parameters, such as ionic strength, volume, temperature of sample solution and time for extraction, time and temperature for desorption, were also optimized. The correlation coefficient of the calibration curve was 0.9998 in the range of 0.082-41.0 ng/mL for tetramine. The limit of quantitation of tetramine in urine was 0.082 ng/mL. In this method, the sample pretreatment is simple and convenient. As a monitoring means, it has been successfully applied to detection of tetramine toxicosis in criminal cases, as well as clinical therapy of poisoned sufferer.

Acute tetrodotoxin-induced neurotoxicity after ingestion of puffer fish.

This study documents the effects of puffer-fish poisoning on peripheral nerve. Excitability measurements investigated membrane properties of sensory and motor axons in four patients. The median nerve was stimulated at the wrist, with compound muscle potentials recorded from abductor pollicis brevis and compound sensory potentials from digit 2. Stimulus-responses, strength-duration time constant (tau(SD)), threshold electrotonus, and current-threshold relations were recorded. The urine of each patient tested positive for tetrodotoxin. Compared with controls, axons were of higher threshold, compound muscle action potentials and compound sensory nerve action potentials were reduced in amplitude, latency was prolonged, and tau(SD) was reduced. In recovery cycles, refractoriness, superexcitability, and late subexcitability were decreased. Threshold electrotonus of motor axons exhibited distinctive abnormalities with less threshold decline than normal on depolarization and greater threshold increase on hyperpolarization (p < 0.0005 for each patient). The changes in excitability were reproduced in a mathematical model by reducing sodium (Na(+)) permeabilities by a factor of two. This study confirms that the neurotoxic effects of puffer-fish poisoning can be explained by tetrodotoxin blockade of Na(+) channels. It demonstrates the ability of noninvasive nerve excitability studies to detect Na(+) channel blockade in vivo and also the utility of mathematical modeling to aid interpretation of altered excitability properties in disease.

Use of high performance liquid chromatography to measure tetrodotoxin in serum and urine of poisoned patients.

Tetrodotoxin (TTX) poisoning is an infrequent but important problem in South-eastern Asia. Despite it being a considerable public health issue in some countries and its potential lethality there continues to be no readily available method for measuring TTX in urine or serum. Previously published methods have used immunoaffinity chromatography, or the conversion of TTX to its C9-base derivative for measurement by mass spectrometry. A simple and reproducible method was developed using solid phase extraction cartridges to clean up serum and urine samples from TTX-poisoned patients, and the subsequent analysis of the samples by high performance liquid chromatography with post-column derivatisation and fluorescence detection. Minimum quantifiable concentrations of TTX were 5 and 20 ng/ml for serum and urine, respectively. Precision and accuracy of the assay were 13 and 15%, respectively. The standard curves were linear in the range of 20-300 ng/ml for urine and 5-20 ng/ml for serum. TTX was quantified in six samples of urine and six samples of serum from seven patients who ingested common toadfish and had clinical effects consistent with TTX poisoning. TTX was detected in all urine samples but in only one serum sample. Using this method confirmation of TTX poisoning will be far simpler and readily available. A 24 h urine collection in the period immediately following poisoning is likely to be the most sensitive test for TTX poisoning. With appropriately collected and timed serum and urine specimens it will be possible to properly define the pharmacokinetics of TTX in humans.

Occurrence of a food poisoning incident by palytoxin from a serranid Epinephelus sp. in Japan.

Between October 30 and November 4, 2000, eleven persons were intoxicated due to ingestion of a serranid fish Epinephelus sp. in Kochi Prefecture, Japan. Their symptoms were mainly featured by severe muscle pain, low back pain, and discharge of black urine. Serum creatine phosphokinase (CPK) levels of victims were higher (700-23,800 IU/l) than normal values, and their recovery times were more than one month. Immediately after the incident, the leftovers were collected for investigation. The causative agent was identified as palytoxin (PTX) on the basis of delayed haemolytic activity which was inhibited by an anti-PTX antibody and ouabain (g-strophanthin). To our knowledge, this is the first report on palytoxin poisoning with serranid fish.

Clinical and pharmacological profile in a clenbuterol epidemic poisoning of contaminated beef meat in Italy.

Long-acting beta adrenergic agonists, such as clenbuterol accumulate in the liver, but not meat of treated farm animals, and result in epidemic poisonings in consumers. We describe an outbreak of poisoning in 15 people, following the consumption of meat. Clinical symptoms (distal tremors, palpitations, headache, tachipnoea-dyspnoea, and also moderate hyperglycaemia, hypokalemia and leucocytosis) were seen in nine hospitalised patients, starting about 0.5-3 h after poisoning, and disappearing within 3-5 days later. Clenbuterol was found in the urine of all the symptomatic patients, at higher levels than pharmacokinetic computing (mean level 28 ng/ml, 36 h after ingestion), based on the levels found in the meat (1140-1480 ng/g edible tissue). Thus, epidemic poisoning can be produced following the consumption of contaminated meat. The need for a better definition of pharmaco- and toxico-kinetics, not only for drugs ingested as parent drug, but also when ingested as residues with animal tissues, is recommended.

Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients.

Immunoaffinity chromatography using the monoclonal antibody (Tl-1) specific for tetrodotoxin (TTX) has been developed for isolating TTX from urine samples. By combining immunoaffinity chromatography with fluorometric high performance liquid chromatography (HPLC), it has become possible to detect a small amount of TTX in urine samples. The detection limit of TTX in urine was 2 ng/ml. By this combined method, TTX was detected in all the urine samples that were collected from poisoned patients during the week following TTX ingestion. The combination of immunoaffinity chromatography with HPLC was very useful in detecting TTX from the urine samples of poisoned patients for diagnosis of TTX-food poisoning.

A receptor binding assay for paralytic shellfish poisoning toxins: recent advances and applications.

We recently described a high throughput receptor binding assay for paralytic shellfish poisoning (PSP) toxins, the use of the assay for detecting toxic activity in shellfish and algal extracts, and the validation of 11-[3H]-tetrodotoxin as an alternative radioligand to the [3H]-saxitoxin conventionally employed in the assay. Here, we report a dramatic increase in assay efficiency through application of microplate scintillation technology, resulting in an assay turn around time of 4 h. Efforts are now focused on demonstrating the range of applications for which this receptor assay can provide data comparable to the more time consuming, technically demanding HPLC analysis of PSP toxins, currently the method of choice for researchers. To date, we have compared the results of both methods for a variety of sample types, including different genera of PSP toxin producing dinoflagellates (e.g. Alexandrium lusitanicum, r2 = 0.9834, n = 12), size-fractioned field samples of Alexandrium spp. (20-64 microm; r2 = 0.9997, n = 10) as well as its associated zooplankton grazer community (200-500 microm: r2 = 0.6169, n = 10; >500 microm: r2 = 0.5063, n = 10), and contaminated human fluids (r2 = 0.9661, n = 7) from a PSP outbreak. Receptor-based STX equivalent values for all but the zooplankton samples were highly correlated and exhibited close quantitative agreement with those produced by HPLC. While the PSP receptor binding assay does not provide information on toxin composition obtainable by HPLC, it does represent a robust and reliable means of rapidly assessing PSP-like toxicity in laboratory and field samples. Moreover, this assay should be effective as a screening tool for use by public health officials in responding to suspected cases of PSP intoxication.

[Collective scombroid fish poisoning following tuna consumption in Castellon]. / Escombrointoxicación colectiva por consumo de atún en Castellón.

BACKGROUND: To investigate and control of a collective scombroid-fish poisoning (SFP) outbreak, that took place in Castellón, Spain. METHODS: Description of the outbreak and case-control study in order to identify risk factors. Active surveillance of SFP cases, and inspection of implicated markets. Histamine determination in urine of cases and foods. RESULTS: During June 1994, 15 cases of SFP were found out, with 12 cases occurred on June, 28 and 29. Five families were affected (attack rate 68.2%). The median incubation period was 45 minutes. Disease symptoms included facial or general flushing, headache, diarrhea, nausea, abdominal pain, and peppery taste. Implicated food was fresh tuna, bought in a hypermarket of Castellón (odds ratio = 26.4, 95% confidence intervals: 1.05-666.8), adjusted by age and sex using logistic regression analysis. Four samples of urine from cases presented histamine concentration above 35 micrograms/l. Three samples of tuna consumed by cases and one sample of tuna from the hypermarket had histamine concentration above a 200 ppm. Considering the situation of risk, remaining suspected tuna was confiscated from the hypermarket. Rest unknown if the descompositions of tuna occurred in the hypermarket or during capture and distribution. CONCLUSIONS: SFP was caused by fresh tuna ingestion with epidemiologic and analytic confirmation. Determination of histamine in urine of patients could permit to confirm SFP.