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Objective@#To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with cation exchange-based solid phase extraction (SPE) for determination of tetrodotoxin (TTX) in salted pufferfish. @*Methods@#Evenly crushed salted pufferfish samples were subjected to ultrasound-assisted extraction with 0.5% acetic acid/50% methanol/water. The extract was cleaned with cation exchange-based SPE cartridge and eluted with 0.3% hydrochloric acid and 50% acetonitrile/water. The eluent was neutralized with ammonia and separated with a Waters XBridgeTM BEH Amide column (150 mm×3.0 mm, 1.7 μm), and determined using LC-MS/MS in a multiple reaction monitoring mode.@*Results@#The matrix effects of TTX were 85.7%-92.4%, and the matrix suppression effect was under effective control following clean-up procedures using the optimized SPE method. The TTX showed a good linear relationship at the range of 2.0 to 4 000 μg/kg, with a correlation coefficient (r2) of 0.999 2. The limits of detection and quantitation for TTX in sample matrix were 1.0 μg/kg and 2.0 μg/kg, respectively. The mean spiked-recovery rates were 81.2% to 96.5% at spiked amounts of 2.0, 200 μg/kg and 2 200 μg/kg, with relative standard deviations (RSDs) of 4.3% to 7.5%. The intraday accuracy and precision of TTX were 84.4% to 95.6% and 4.9% to 5.8% in quality control samples, and the interday accuracy and precision of TTX were 86.1% to 94.9% and 5.5% to 8.5% in quality control samples. The detection of TTX was 60.5% in 38 market-sold salted pufferfish products using the established LC-MS/MS method.@*Conclusion@#The established LC-MS/MS method is effective for accurate quantitative determination of TTX in salted pufferfish.
ABSTRACT
Tetrodotoxin (TTX) is a neurotoxin found in puffer fish and other marine organisms. It has been used as an inhibitor of voltage-gated sodium channels (VGSCs), which could selectively bind to the α-subunit on the outer vestibule of VGSCs, preventing sodium ions from entering the channel, resulting in pharmacological activities. As a typical sodium channel blocker, TTX shows a significant analgesic effect. TTX could selectively block Na+ channels without affecting other ion channels, therefore it could reduce the probability of adverse reactions caused by commonly used antiarrhythmic drugs. In addition, TTX has a significant role in detoxification and prevention of renal failure, so TTX has great potential as a medicine. The structure and physicochemical properties, mechanism of action, pharmacological activities and preparations of tetrodotoxin have been reviewed in this paper, so as to provide a general support for the evaluation of its druggability and application in the field of pharmacy.
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Objective To investigate the solubility and stability of Tetrodotoxin (TTX) in different solvents, and the effect of temperature and pH on its stability. Methods Solutions of TTX in different matrices were prepared. Their concentrations at different temperatures and pH buffers were determined by high performance liquid chromatography and their solubility and stability were analyzed and calculated. Results TTX was most soluble at pH 3.5 and its solubility decreased as the pH increased. TTX degraded most rapidly under strong alkali conditions, with complete degradation after 20 min of reaction at 0.1 mol/L sodium hydroxide and 70 ℃. The stability test results similarly demonstrated that TTX was least stable under alkaline conditions. In a PBS buffered solution at 37 ℃, pH 7.4, TTX concentration began to decrease consistently at 1~10h, with a degradation rate of 88.07±0.27% after 28 days. Conclusion TTX is readily soluble in acidic aqueous solutions at pH 3.5 and almost insoluble in alkaline aqueous solutions. Its stability is closely related to the temperature and pH of the medium. It is more stable in acidic aqueous solutions and easily degrades under alkaline conditions, and its degradation process could be accelerated by increasing temperature.
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Objective To establish a detection method for the determination of tetrodotoxin (TTX) in sustained-release microspheres. Methods The HPLC separation of tetrodotoxin was performed on an Agilent ZORBAX SB-C18 column (4.6mm×150mm,5 μm) with acetonitrile, 8mmol/L sodium heptane sulfonate containing 0.005% TFA (5:95) (pH 4.0) as the mobile phase. The flow rate was 1.0 ml/min. The UV detection wavelength was 200 nm and the column temperature was 30 °C. Results The method had good specificity and linearity of TTX in the concentration range of 1−20 μg/ml. The intra-day precision, inter-day precision, stability and repeatability of the method were good, and the average recoveries were found between 98.0% and 102.0%. Conclusion This study established an HPLC method which was suitable for the determination of tetrodotoxin sustained-release microspheres. The method is accurate and reliable within the applicable range, with strong specificity, which could lead to quantitative detection.
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Objective To evaluate the analgesic effect of tetrodotoxin (TTX) in four types of acute pain models and provide experimental support for its rational application. Methods Mice or rats were intramuscularly pretreated with morphine (1 mg/kg) or TTX (0, 0.5, 1, 2, 4 and 8 μg/kg) 40 min before acetic acid writhing test, formalin stimulation test, hot plate test or tail flick test. Pain response or pain threshold were recorded, and inhibition rate was calculated during the tests. The arachidonic acid of serum was determined by Elisa. Results Significant analgesic effects were observed with morphine in all four acute pain models. TTX dose-dependently reduced the number of writhing induced by acetic acid and inhibited the pain response induced by formalin during phase I and phase II, with the highest inhibition rate of more than 80.00% in two pain models. TTX showed analgesic effect in tail flick test and hot plate test, with the highest inhibition rate of 25.00% and 19.79%, respectively. Both acetic acid and formalin increased arachidonic acid in animal serum, but TTX had no significant inhibitory effect on the releasing of arachidonic acid. Conclusion TTX showed significant analgesic effect in the chemical stimulation pain models induced by acetic acid and formalin, but limited analgesic effect was observed on the physical stimulation pain model induced by heat (hot plate and hot water). TTX may produce analgesic effect by blocking the inflammatory mediators mediating pain response.
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Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.
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Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.
Subject(s)
Animals , Rats , Analgesia , Ganglia, Spinal , Growth Differentiation Factor 15 , Sensory Receptor Cells , Sodium Channels , Tetrodotoxin/pharmacologyABSTRACT
Objective: To provide electrophysiological evidence for RIN-14B cells as an useful model of enterochromaffin cells (EC) and to study the role of Nav1. 3 channel in the control of its excitability. Methods: Resting membrane potential was recorded and the effects of TTX and ICA-121431 were examined by current-clamp in cultured RIN-14B cells. The effects of TTX and ICA-121431 on Na+ current of RIN-14B cells were examined by voltage-clamp. Results: RIN-14B cells had a resting potential around -60 mV and fired action potentials when stimulated with depolarizing current pulses. The action potential was completely blocked by TTX and inhibited by ICA-121431 in a dose-dependent manner. TTX blocked activation and inactivation of sodium current. In addition ICA-121431 dose-dependently inhibited activation of Na+ current. Conclusion: The action potential of RIN-14B cells is induced by TTX-sensitive sodium channel and the excitability is controlled by Nav1.3. These results suggest RIN-14B cells are similar to EC and it may be a good model of EC.
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Migraine is a neurological disorder characterized by recurrent and disabling severe headaches. Although several anticonvulsant drugs that block voltage-dependent Na⁺ channels are widely used for migraine, far less is known about the therapeutic actions of carbamazepine on migraine. In the present study, therefore, we characterized the effects of carbamazepine on tetrodotoxin-resistant (TTX-R) Na⁺ channels in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na⁺ currents were measured in medium-sized DiIpositive neurons using the whole-cell patch clamp technique in the voltage-clamp mode. While carbamazepine had little effect on the peak amplitude of transient Na⁺ currents, it strongly inhibited steady-state currents of transient as well as persistent Na⁺ currents in a concentration-dependent manner. Carbamazepine had only minor effects on the voltage-activation relationship, the voltage-inactivation relationship, and the use-dependent inhibition of TTX-R Na⁺ channels. However, carbamazepine changed the inactivation kinetics of TTX-R Na⁺ channels, significantly accelerating the development of inactivation and delaying the recovery from inactivation. In the current-clamp mode, carbamazepine decreased the number of action potentials without changing the action potential threshold. Given that the sensitization of dural afferent neurons by inflammatory mediators triggers acute migraine headaches and that inflammatory mediators potentiate TTX-R Na⁺ currents, the present results suggest that carbamazepine may be useful for the treatment of migraine headaches.
Subject(s)
Animals , Rats , Action Potentials , Anticonvulsants , Carbamazepine , Headache , Kinetics , Migraine Disorders , Nervous System Diseases , Neurons , Neurons, Afferent , Sodium Channels , Trigeminal GanglionABSTRACT
Objective In order to analyze of poisoning causes,a new method was established utilizing hydrophilic liquid chromatography-tandem triple quadrupole mass spectrometer (HILIC-MS/MS) coupled with dispersive solid phase extraction for rapid qualitative and quantitative analysis of tetrodotoxin in nassarius and shellfish.Methods Sample (1.0 g) was extracted with 0.1％ acetic acid in boiling water bath,purified by dispersive solid phase extraction with 50 mg hydrophilic-lipophilic balance (HLB),5 mg graphitized carbon black (GCB) and protein precipitation with acetonitrile,and then filtered through a polytetrafluoroethylene (PTFE) membrane.The analytes were separated on a HILIC column,and detected in selected reaction monitoring (SRM) mode via positive electrospray ionization.The matrix matching and external standard method was used for quantification.Results Tetrodotoxin showed good linearity in the concentration range between 2.0 and 40.0 ng/ml,the correlation coefficient was higher than 0.999.The detection limit of tetrodotoxin in seafood was 10.0 pg/kg.The rates of recovery varied between 74.2％ and 87.9％ with relative standard deviations from 2.3％ to 9.1％ at spiked concentrations of 25,100 and 200 pg/kg.The proposed method was applied in the detection of tetrodotoxin in shellfish and nassarius from coastal cities of Zhejiang Province.Conclusion The method was accurate,fast,easy to operate,which could meet the requirements of public health emergency testing or routine testing.
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Objective To assess the toxicity of the pufferfish Takifugu oblongus, from Chennai coast, Tamil Nadu, India and to detect the presence of Tetrodotoxin (TTX). Methods The toxicity was evaluated by mouse bioassay using Swiss Albino mice which were expressed in mouse units (MU). Gross anatomical features were observed which is followed by histopathology of the dead mice tissues to establish the toxicity. Instrumental analysis for the presence of tetrodotoxin was also performed through GC–MS and HPLC. Results The toxicity of ovary was the maximum with 163 MU/g and lowest toxicity was observed in skin with 75.88 MU/g. Histopathological analyses of the dead mice showed various cellular degenerations and inflammations. The amount of Tetrodotoxin detected through GC–MS and HPLC was more reliable and sensitive than the customary mouse bioassay as instrumental analyses were able to detect even nanograms of the toxin. Conclusions The present study evidently proved that Takifugu oblongus is highly toxic and consumption of the same can pose serious threat for health and possible lethality to humans.
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@#To elucidate possible mechanisms and targets of honokiol(Hnk)for hte treatment of pain, the effects of Hnk on tetrodoxin-senstive(TTX-S)sodium current(INa)were studied in acutely dissociated mouse dorsal root ganglion(DRG)neurons with whole-cell patch clamp technique. Hnk inhibited TTX-S INa currents in a concentration-dependent manner. At 30 μmol/L, Hnk obviously shifted the steady state activation of TTX-S INa toward more positive potentials by 10. 2 mV and prolonged the time course of recovery of sodium current, yet with no significant difference on recovery from inactivation of TTX-S sodium channel.
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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. .
Subject(s)
Adult , Aged , Animals , Female , Humans , Male , Middle Aged , Fishes, Poisonous , Foodborne Diseases/etiology , Perciformes , Tetrodotoxin/poisoning , Chromatography, Liquid , China/epidemiology , Disease Outbreaks , Foodborne Diseases/epidemiology , Retrospective Studies , Tandem Mass Spectrometry , Tetrodotoxin/bloodABSTRACT
A method was developed for the determination of tetrodotoxin in marine organisms by high perfor-mance liquid chromatography-mass spectrometry with immunoaffinity column. The samples were extracted with 1% acetic acid methanol solution and diluted with phosphate buffer at pH 7-8. After cleaned up by immuno-affinity column, the samples were analyzed by LC-MS/MS and quantitatively determined by external standard method. The chromatographic separation was performed on an ACQUITY UPLC BEH Amide column with gradient elution by using acetonitrile and 5 mol/L ammonium acetate solution containing 0. 1% formic acid as mobile phase. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode. Linear ranges of TTX was in the range of 0. 3 -20. 0 μg/L with correlation coeffi-cient more than 0. 997. The quantification limit of the method was 0. 3 μg/kg. The recoveries of standard addition for tetrodotoxin were 88. 7%-102. 3%, and the relative standard deviation was 2. 0%-6. 4%. The method could be used to identify and quantify tetrodotoxin in marine organisms with satisfactory reproducibility and sensitivity.
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In this brief communication the authors report eleven cases of human poisoning caused by ingestion of pufferfish meat. Three patients (two children and one adult) were seriously affected. The circumstances that precipitated the poisoning are discussed as well as the clinical aspects observed. No deaths were registered and the patients did not present sequelae after the episode.
Subject(s)
Humans , Animals , Foodborne Diseases , Tetraodontiformes , Tetrodotoxin/poisoning , Fish Venoms , BrazilABSTRACT
In this brief communication the authors report eleven cases of human poisoning caused by ingestion of pufferfish meat. Three patients (two children and one adult) were seriously affected. The circumstances that precipitated the poisoning are discussed as well as the clinical aspects observed. No deaths were registered and the patients did not present sequelae after the episode.(AU)
Subject(s)
Tetraodontiformes , Eating , Meat/toxicity , Research Report , Foodborne DiseasesABSTRACT
PURPOSE: We conducted this study in order to determine clinical features and prognostic factors in adults with acute tetrodotoxin (TTX) poisoning caused by ingestion of puffer fish. METHODS: In this retrospective study, 107 patients were diagnosed with TTX poisoning. The subjects were divided into two groups according to duration of treatment; Group I, patients were discharged within 48 hours (n=76, 71.0%), Group II patients were discharged after more than 48 hours (n=31, 29.0%). Group II was subsequently divided into two subgroups [IIa (n=12, 11.2%), IIb (n=19, 17.8%)] according to the need for mechanical ventilation support. RESULTS: In multivariable logistic regression analysis, the predictors of the need for treatment over 48 hours were dizziness (odds ratio [OR], 4.72; 95% confidence intervals [CI], 1.59-12.83), time interval between onset of symptom and ingestion (OR, 0.56; 95% CI, 0.16-0.97), PaCO2<35 mmHg (OR, 8.37; 95% CI, 2.37-23.59). In addition, predictors of the need for mechanical ventilation were a time interval between onset of symptoms and ingestion (OR, 0.54; 95% CI, 0.11-0.96) and PaCO2<35 mmHg (OR, 5.65; 95% CI, 1.96-18.66). CONCLUSION: Overall, dizziness, time interval between onset of symptoms and ingestion, DeltaDBP and PaCO2<35 mmHg predict the need for treatment over 48 hours, time interval between onset of symptoms and ingestion and PaCO2<35 mmHg predict the need for mechanical ventilation support after acute TTX poisoning.
Subject(s)
Adult , Humans , Dizziness , Eating , Logistic Models , Poisoning , Respiration, Artificial , Retrospective Studies , Tetraodontiformes , TetrodotoxinABSTRACT
Anultra-performancehydrophilicinteractionliquidchromatography-triplequadrupolemass spectrometric ( UPLC-MS/MS) method was developed for the determination of tetrodotoxin ( TTX) in human urine and plasma. After the sample was cleaned-up and concentrated by immunoaffinity column, the separation of the TTX was carried out on an Acquity UPLC BEH amide column (100 mm×2. 1 mm, 1. 7 μm) with gradient elution using mobile phases of 0. 1% ( V/V) formic acid in water and acetonitrile. The analyte was detected by positive electrospray ionization mass spectrometry in the multiple reaction monitoring ( MRM) mode, and quantified by external solvent standard calibration. The measuring ranges of TTX in urine and plasma were 0. 05-400 μg/L. The average recoveries were 92%-95% and 91%-96% for TTX respectively spiked in urine and plasma with relative standard deviations of 3 . 3%-7 . 2% and 3 . 9%-7 . 8% ( n=5 ) . The limits of detection (LOD, S/N=3) and limits of quantitation (LOQ, S/N=10) of TTX were 0. 02 μg/L and 0. 05μg/L for urine and plasma, respectively. This method is suitable for the detection of TTX in urine and plasma for both forensic and clinical purposes.
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Ingestion of puffer fish can result in severe and potentially lethal intoxication, referred to as tetrodotoxin intoxication. Tetrodotoxin is a potent neurotoxin well known for its ability to ability neuromuscular function. Tetrodotoxin is a specific and potent blocker of axonal sodium channel; it may block sodium channels in the axon of the neurons of the neurohypophysis, thereby inhibiting the release of vasopressin and causing diabetes insipidus neurotoxin. To our knowledge, previous report on diabetes insipidus causing tetrodotoxin is the only one case in Singapore. A married couple (69-year-old man and 57-year-old woman) ingested two green rough-backed puffer fish (Lagocephalus lunaris). They complained of paresthesia on perioral area and extremity and developed not only grade IV intoxication but also an increased urine output (4455 ml/day and 5035 ml/day), elevated serum sodium (157.4 mEq/L and 166.7 mEq/L) and elevated serum osmolality (324 mosmol/kg and 339 mosmol/kg), which suggested the development of diabetes insipidus. The administration of desmopressin nasal spray was successful in normalizing urine volume. Both were discharged on 20th and 18th hospital day, respectively, without any complications.
Subject(s)
Axons , Deamino Arginine Vasopressin , Diabetes Insipidus , Eating , Extremities , Neurons , Osmolar Concentration , Paresthesia , Pituitary Gland, Posterior , Sodium , Sodium Channels , Tetraodontiformes , Tetrodotoxin , VasopressinsABSTRACT
10.3969/j.issn.2095-4344.2013.23.001