Safety, Tolerability, Pharmacokinetics, and Concentration-QTc Analysis of Tetrodotoxin: A Randomized, Dose Escalation Study in Healthy Adults.

Tetrodotoxin (TTX) is a highly specific voltage-gated sodium channel (VGSC) blocker in clinical evaluation as a peripheral-acting analgesic for chronic pain. This study presents the first published results of the safety including cardiac liability of TTX at therapeutic-relevant concentrations in twenty-five healthy adults. Randomized, double-blind, placebo-, and positive- (moxifloxacin) controlled study evaluated single ascending doses of 15 µg, 30 µg, and 45 µg TTX over 3 periods with a 7-day washout between each period. Subcutaneous injections of TTX were readily absorbed, reaching maximum plasma concentration (Cmax) within 1.5 h. Both extent of exposure (AUC) and Cmax increased in proportion to dose. No QT prolongation was identified by concentration-QTc analysis and the upper bounds of the two-sided 90% confidence interval of predicted maximum baseline and placebo corrected QTcF (ΔΔQTcF) value did not exceed 10 ms for all tetrodotoxin doses, thereby meeting the criteria of a negative QT study. Safety assessments showed no clinically relevant changes with values similar between all groups and no subject withdrawing due to adverse events. Paresthesia, oral-paresthesia, headache, dizziness, nausea, and myalgia were the most common TEAEs (overall occurrence ≥5%) in the TTX treatment groups. TTX doses investigated in this study are safe, well-tolerated, and lack proarrhythmic proclivity.

Exonuclease I-assisted fluorescence aptasensor for tetrodotoxin.

A fluorescence aptasensor for the highly specific and sensitive determination of tetrodotoxin was established with tetrodotoxin-aptamer as the recognition unit, berberine as the signal reporter and exonuclease I as the elimination agent for the background. Berberine has a weak fluorescence emission at 540 nm, and it can form the tetrodotoxin-aptamer/berberine complex, resulted in an increased fluorescence. After introducing exonuclease I, it can degrade the single strand oligonucleotides of tetrodotoxin-aptamer into the single nucleotide in the absence of tetrodotoxin, which lead to dramatic fluorescence quenching, and reduce the background signal of sensing system. Once tetrodotoxin is in the presence, tetrodotoxin-aptamer is converted into the stable neck ring conformation, which resists the degradation of exonuclease I and provides a more rigid micro-environment for the excited state of berberine, and then the strong fluorescence is observed. Based on the above properties, an ultrasensitive label-free fluorescence aptasensor for tetrodotoxin is established. The fluorescence aptasensor shows good analytical performance with the linear increase of fluorescence intensity at the tetrodotoxin concentration from 0.030 nM to 6.0 × 103 nM. The detection limit of 11.0 pM is much lower than that of other reported sensor methods.

A simple, versatile, and automated pulse-diffusion-focusing strategy for sensitive milliliter-level-injection HILIC-MS/MS analysis of hydrophilic toxins.

The measurement of trace hydrophilic toxins in complex aqueous-rich matrices is a daunting challenge. To address this analytical bottleneck, pulse diffusion focusing (PDF), a novel sample injection technique for hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS), was developed. Theoretical and experimental investigations of the mechanism and key parameters revealed that the pulse-injection approach, assisted by solvent diffusion, efficiently solved the volume overload problem. This milliliter-level-injection HILIC-MS/MS technique was reported for the first time herein, and provided a significant enhancement in sensitivity compared to the conventional injection method, in addition to being an efficient approach to address the solvent incompatibility of off-line sample preparation and HILIC. The automated PDF-HILIC-MS/MS system was obtained by slightly modifying a commercial LC-MS/MS instrument in an easy and economical manner. The efficiency of the system was demonstrated through the detection of trace tetrodotoxin contents in plasma and urine samples. Low limits of detection (i.e., 0.65 and 2.2 ng·mL-1) were obtained using the simplified sample preparation method. The recoveries were in the range 91-113.3 % with intra-day and inter-day precisions of ≤9.6 %. Further experimental results proved the method to be versatile for various hydrophilic toxins.

Highly sensitive analysis of tetrodotoxin based on free-label fluorescence aptamer sensing system.

Tetrodotoxin (TTX) specifically can bind to its nucleic acid aptamer (TTX-aptamer) and cause the conformation of TTX-aptamer to be switched from the single-strand random coiling form to the compact neck ring structure. Based on the microenvironment difference of the fluorescence reporter, berberine in between the single-stranded coil oligonucleotides and the structure of the neck ring, a simple, rapid and sensitive label-free fluorescence aptamer sensing system for detection of TTX was developed. Various factors affecting the analysis of TTX were optimized, including the concentration of berberine, ion strength, pH, reaction time, the concentration of TTX-aptamer. Under the optimal experimental conditions, the fluorescence intensity of the sensing system and the concentration of TTX showed a good linear relationship in the range of 0.1 nM to 500 nM, with the detection limit of 0.074 nM. The standard recovery test result exhibited that the recoveries of TTX in serum samples were 96.54%-106.40%. The established method has the advantages of high specificity, good sensitivity, quickness and convenience, low cost, and can be used for the detection of TTX in serum samples.

Pufferfish Saxitoxin and Tetrodotoxin Binding Protein (PSTBP) Analogues in the Blood Plasma of the Pufferfish Arothron nigropunctatus, A. hispidus, A. manilensis, and Chelonodon patoca.

Pufferfish saxitoxin and tetrodotoxin (TTX) binding protein (PSTBP) is a glycoprotein that we previously isolated from the blood plasma of the pufferfish Takifugu pardalis; this protein was also detected in seven species of the genus Takifugu. We proposed that PSTBP is a carrier protein for TTX in pufferfish; however, PSTBP had not yet been found in genera other than Takifugu. In this study, we investigated the presence of PSTBP-like proteins in the toxic pufferfish Arothron nigropunctatus, A. hispidus, A. manilensis, and Chelonodon patoca. On the basis of ultrafiltration experiments, TTX was found to be present and partially bound to proteins in the plasma of these pufferfish, and Western blot analyses with anti-PSTBP antibody revealed one or two bands per species. The observed decreases in molecular mass following deglycosylation with glycopeptidase F suggest that these positive proteins are glycoproteins. The molecular masses of the deglycosylated proteins detected in the three Arothron species were larger than that of PSTBP in the genus Takifugu, whereas the two bands detected in C. patoca had molecular masses similar to that of tributyltin-binding protein-2 (TBT-bp2). The N-terminal amino acid sequences of 23⁻29 residues of these detected proteins were all homologous with those of PSTBP and TBT-bp2.

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.

RETRACTED: Facilely self-assembled magnetic nanoparticles/aptamer/carbon dots nanocomposites for highly sensitive up-conversion fluorescence turn-on detection of tetrodotoxin.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor following concerns raised by a reader. The article uses two electron micrographs which have been used in other publications as well, denoting different samples. Fig. 1A is identical to Fig. 3E in RSC Adv., 2013,3, 20959-20969 doi: 10.1039/C3RA43120G despite describing different samples. Fig. 2A is identical to Fig. 1B in Sensors & Actuators B: Chemical, vol 245, pp 386-394 https://doi.org/10.1016/j.snb.2017.01.166 and Fig. 1A in Materials Letters vol 195 pp 131-135 https://doi.org/10.1016/j.matlet.2017.02.119 despite describing different samples. These problems with the data presented cast doubt on all the data, and accordingly also the conclusions based on that data, in this publication. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process., http://dx.doi.org/10.1016/j.talanta.2017.08.043.

A Study of 11-[³H]-Tetrodotoxin Absorption, Distribution, Metabolism and Excretion (ADME) in Adult Sprague-Dawley Rats.

Tetrodotoxin (TTX) is a powerful sodium channel blocker that in low doses can safely relieve severe pain. Studying the absorption, distribution, metabolism and excretion (ADME) of TTX is challenging given the extremely low lethal dose. We conducted radiolabeled ADME studies in Sprague-Dawley rats. After a single dose of 6 µg/(16 µCi/kg) 11-[³H]TTX, pharmacokinetics of plasma total radioactivity were similar in male and female rats. Maximum radioactivity (5.56 ng Eq./mL) was reached in 10 min. [³H]TTX was below detection in plasma after 24 h. The area under the curve from 0 to 8 h was 5.89 h·ng Eq./mL; mean residence time was 1.62 h and t½ was 2.31 h. Bile secretion accounted for 0.43% and approximately 51% of the dose was recovered in the urine, the predominant route of elimination. Approximately 69% was recovered, suggesting that hydrogen tritium exchange in rats produced tritiated water excreted in breath and saliva. Average total radioactivity in the stomach, lungs, kidney and intestines was higher than plasma concentrations. Metabolite analysis of plasma, urine and feces samples demonstrated oxidized TTX, the only identified metabolite. In conclusion, TTX was rapidly absorbed and excreted in rats, a standard preclinical model used to guide the design of clinical trials.

[Tetrodotoxin (TTX) Monitoring of Biological Specimens and Toxin Profile in a Food Poisoning Case Caused by the Scavenging Gastropod Nassarius (Alectrion) glans "Kinshibai"].

In November 2015, a patient presented with symptoms of toxicity after eating whole boiled samples of the scavenging gastropod Nassarius (Alectrion) glans "Kinshibai" in Nagasaki. This food poisoning case was the third recorded in Japan. The case was investigated by evaluation of the toxin profile of the gastropod, and monitoring of tetrodotoxin (TTX) levels in serum and urine sampled from the affected individual. One gastropod contained a harmful dose of TTX (2.5 mg/ individual in food residue sample 2). In biological samples, maximum TTX concentrations were 42.8 ng/mL in serum on the day after onset of symptoms. TTX urinary excretion was calculated to be 2.4 mg. From the measured TTX concentrations, it was estimated that a lethal dose had been ingested in this case. Moreover, it was found by LC-QqQ-MS/MS analysis and mouse bioassay that the toxicity of "Kinshibai" was not solely due to TTX. The remaining toxicity was thought to be due to 11-oxoTTX. As in previous poisoning cases, it was concluded that ingestion of this gastropod poses a high risk of food poisoning.

Tetrodotoxin poisoning caused by Goby fish consumption in southeast China: a retrospective case series analysis.

OBJECTIVES: To investigate an unusual outbreak of tetrodotoxin poisoning in Leizhou, southeast China, a case series analysis was conducted to identify the source of illness. METHODS: A total of 22 individuals experienced symptoms of poisoning, including tongue numbness, dizziness, nausea and limb numbness and weakness. Two toxic species, Amoya caninus and Yongeichthys nebulosus, were morphologically identified from the batches of gobies consumed by the patients. Tetrodotoxin levels in the blood and Goby fish samples were detected using liquid chromatography-tandem mass spectrometry. RESULTS: The tetrodotoxin levels in the remaining cooked Goby fish were determined to be 2090.12 µg/kg. For Amoya caninus, the toxicity levels were 1858.29 µg/kg in the muscle and 1997.19 µg/kg in the viscera and for Yongeichthys nebulosus, they were 2783.00 µg/kg in the muscle and 2966.21 µg/kg in the viscera. CONCLUSION: This outbreak demonstrates an underestimation of the risk of Goby fish poisoning. Furthermore, the relationships among the toxic species, climates and marine algae present should be clarified in the future.

Tetrodotoxin poisoning caused by Goby fish consumption in southeast China: a retrospective case series analysis

OBJECTIVES: To investigate an unusual outbreak of tetrodotoxin poisoning in Leizhou, southeast China, a case series analysis was conducted to identify the source of illness. METHODS: A total of 22 individuals experienced symptoms of poisoning, including tongue numbness, dizziness, nausea and limb numbness and weakness. Two toxic species, Amoya caninus and Yongeichthys nebulosus, were morphologically identified from the batches of gobies consumed by the patients. Tetrodotoxin levels in the blood and Goby fish samples were detected using liquid chromatography-tandem mass spectrometry. RESULTS: The tetrodotoxin levels in the remaining cooked Goby fish were determined to be 2090.12 µg/kg. For Amoya caninus, the toxicity levels were 1858.29 µg/kg in the muscle and 1997.19 µg/kg in the viscera and for Yongeichthys nebulosus, they were 2783.00 µg/kg in the muscle and 2966.21 µg/kg in the viscera. CONCLUSION: This outbreak demonstrates an underestimation of the risk of Goby fish poisoning. Furthermore, the relationships among the toxic species, climates and marine algae present should be clarified in the future. .

Acute paralysis after seafood ingestion.

The first European case of tetrodotoxin intoxication is reported in a patient who ingested a trumpet shellfish from the Atlantic Ocean in Southern Europe. He suffered general acute paralysis with respiratory failure necessitating ventilation. Early neurophysiologic studies showed complete peripheral nerve inexcitability, with no recordable sensory or motor responses, and normal electroencephalography. Tetrodotoxin was detected in high quantities in the patient's blood and urine through high performance liquid chromatography-mass spectrometry analysis. Seventy-two hours after admission the patient recovered normal strength, reflexes and sensation.

[Postmortem distribution of tetrodotoxin in tissues and body fluids of guinea pigs].

OBJECTIVE: To investigate the postmortem distribution of tetrodotoxin in tissues and body fluids of guinea pig, and to provide method and evidence for forensic identification and clinical diagnosis and treatment. METHODS: Guinea pigs were intragastric administrated with 100, 50, 15 microg/kg tetrodotoxin, respectively. The poisoning symptoms were observed. The samples of heart, liver, spleen, lung, kidney, brain, stomach, intestines, bile, heart blood and urine were collected. The concentrations of tetrodotoxin in tissues and body fluids were measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: After administrated with tetrodotoxin, all guinea pigs came out poisoning signs including tachypnea, weary and dead finally. Tetrodotoxin concentrations in lung, stomach, intestines and urine were higher, followed by blood, heart and brain. The concentration in bile was the lowest. CONCLUSION: Postmortem distribution of tetrodotoxin in guinea pig is uneven. The concentration in the lung, stomach, intestines, urine and heart blood are higher, those tissues could be used for diagnosis of tetrodotoxin poisoning.

Determination and validation of tetrodotoxin in human whole blood using hydrophilic interaction liquid chromatography-tandem mass spectroscopy and its application.

A sensitive analytical method was developed for the quantitative determination of tetrodotoxin (TTX), a powerful sodium channel blocker, in human postmortem whole blood. The sample mixture was cleaned up using cation exchange SPE catridge after protein precipitation by methanol and then separated on a PC-HILIC (phosphorylcholine hydrophilic interaction liquid chromatography) column (150 mm × 2.0mm i.d., 5 µm) using a isocratic elution of 1% acetic acid and acetonitrile. The identification of TTX was performed on tandem mass spectrometry with electrospray ionization interface in positive ion mode. The retention time of voglibose (internal standard) and TTX was 5.1 and 6.0 min, respectively. TTX and internal standard (voglibose) were monitored and quantitated using the ion transitions: the respective precursor to product ion combinations, m/z 320/302 for TTX and m/z 268/92 for voglibose in the multiple reaction monitoring (MRM) mode. The recovery of TTX and voglibose was 61.4% and 62.8%, respectively and the good accuracy (97.7-103.9%), linearity (2-1200 ng/mL) and reproducibility were shown in this method. The limit of detection and limit of quantification were 0.32 ng/mL and 1.08 ng/mL, respectively. This method was applied in the case of three fishermen who were poisoned (including one death) by unknown fish on their boat in October 2010. In this case, the levels of TTX were 27.2, 30.0 and 29.7 ng/mL in heart blood, peripheral blood and serum of a victim, were 3.1 and 12.1 ng/mL in peripheral blood and 3.9 and 12.8 ng/mL in serum of two survivors, respectively.

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.

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma.

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL(-1) for urine and 20 ng mL(-1) for plasma. The limit of detection and limit of quantification were 0.13 ng mL(-1) and 2.5 ng mL(-1), respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL(-1), but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng µmol(-1) creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.

[Determination of TTX in biological samples by LC-MS/MS].

OBJECTIVE: To propose a method for determination of tetrodotoxin (TTX) in human blood, urine and liver by liquid chromatography with tandem mass spectrometry (LC-MS/MS). METHODS: Solid phase extraction is used after the samples are precipitated, then the samples will be analyzed by LC-MS/MS. RESULTS: The limits of detection were 2 ng/mL in blood and urine and 4 ng/g in liver for TIX respectively, the linear correlation coefficients were not less than 0.9973, both of the intra-day and inter-day precisions were less than 12.80%, the recoveries for all kinds of specimens were more than 47.2%. CONCLUSION: This method is efficient, sensitive and accurate and was successfully validated for implementation in forensic toxicological analysis.

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.

Plasma protein binding of tetrodotoxin in the marine puffer fish Takifugu rubripes.

To elucidate the involvement of plasma protein binding in the disposition of tetrodotoxin (TTX) in puffer fish, we used equilibrium dialysis to measure protein binding of TTX in the plasma of the marine puffer fish Takifugu rubripes and the non-toxic greenling Hexagrammos otakii, and in solutions of bovine serum albumin (BSA) and bovine alpha-1-acid glycoprotein (AGP). TTX (100-1000 microg/mL) bound to protein in T. rubripes plasma with low affinity in a non-saturable manner. The amount of bound TTX increased linearly with the TTX concentration, reaching 3.92+/-0.42 microg TTX/mg protein at 1000 microg TTX/mL. Approximately 80% of the TTX in the plasma of T. rubripes was unbound in the concentration range of TTX examined, indicating that TTX exists predominantly in the unbound form in the circulating blood of T. rubripes at a wide range of TTX concentrations. TTX also bound non-specifically to H. otakii plasma proteins, BSA, and bovine AGP. The amount of the bound TTX in the plasma of H. otakii and BSA, respectively, was 1.86+/-0.36 and 4.65+/-0.70 microg TTX/mg protein at 1000 microg TTX/mL, and that in the bovine AGP was 8.78+/-0.25 microg TTX/mg protein at 200 microg TTX/mL.

Maturation-associated changes in toxicity of the pufferfish Takifugu poecilonotus.

From October 2006 to December 2007, wild specimens of the pufferfish Takifugu poecilonotus (93 females, 45 males) were collected from the Ariake Sea. Tissue toxicity was examined by mouse bioassay, and tetrodotoxin (TTX) content in the blood plasma by enzyme-linked immunosorbent assay. The relationship between toxicity and maturation was investigated based on changes in the gonadosomatic index: December-March in females and November-March in males, the 'maturation period'; April, 'just after spawning'; and the other months, the 'ordinary period'. Toxicity of both sexes was high throughout the year, but sharply declined in April. In all tissues examined (skin, liver, and ovary) other than testis, toxicity exceeded 1000 MU/g or 10,000 MU/individual in many individuals. Seasonal profiles of tissue toxicity differed markedly between sexes. In females, liver toxicity was high during the ordinary period, and ovary toxicity was high during the maturation period. In males, little maturation-associated change in the toxin distribution was observed. Plasma TTX levels were similar between the sexes (1.59-15.1 MU/ml), and fluctuated largely throughout the year without corresponding changes in tissue toxicity. The percentage of TTX binding to high molecular-weight substances in the plasma varied in association with maturation; the binding ratio fluctuated at relatively low levels during the ordinary period, and stabilized at a high level during the maturation period.