不同配比微生物毒性传感器对河豚毒素毒性的测定及与HPLC法比较

河豚毒素传感器响应性能与固定化菌体的量和辅料的量都有一定关系.设计微生物量为10 μL,不加辅料及加入1％、2％辅料3种微生物膜,以夹层法固定微生物膜,以氧电极为换能器,分别组装微生物传感器,通过测定传感器的线性范围、线性相关系数以及加标回收率等获知固定化膜的通透性的好坏和响应信号的大小,最后确定3种组合中较优膜为10 μL菌悬液加入1％的辅料.以最优膜组装毒性传感器与高效液相色谱方法的对照发现两者的差异不显著,表明试验建立的毒性微生物传感器方法可以很好地测定河豚毒素的毒性.     

Neurotoxin tetrodotoxin as attractant for toxic snails

ABSTRACT:   The attracting effect of a small dose of tetrodotoxin (TTX) on eight toxic snail species ( Polinices didyma, Natica lineata, Natica vitellus, Zeuxis sufflatus, Niotha clathrata, Oliva miniacea, Oliva mustelina and Oliva hirasei ) and two non-toxic species ( Pomacea canaliculata and Satsuma bairdi ) was investigated. Each toxic snail species was determined to contain TTX. The minimum lethal dose of TTX for most toxic snails was estimated to be >44.5 µg TTX/20 g body weight, but for non-toxic snails it was <3.6 µg TTX/20 g body weight. After the attracting test, it was found that for all toxic snails there was a significantly positive relationship between the comparative attracting variation and the toxicity reported, and this relationship was linear ( y  = 5.895 x  + 3.443, r  = 0.806). The relationship between TTX resistance ability and toxicity also had a positive correlation ( y  = 0.113 x  + 52.447, r  = 0.814). However, non-toxic species showed a negative response. The more toxic snails appeared to prefer TTX, indicating that TTX is an attractant for toxic snails.     

The chemical synthesis of tetrodoxin: an ongoing quest

This contribution reviews all the synthetic work on tetrodotoxin that has appeared in the literature through June 2011.     

河鲀毒素固相萃取-气相色谱-质谱法研究

建立了固相萃取-气相色谱-质谱法测定河鲀鱼中河鲀毒素的分析方法。河鲀鱼肉用2%乙酸甲醇溶液提取出河鲀毒素,石油醚脱脂,浓缩蒸干,然后用强碱将河鲀毒素水解成2-氨基-8-羟基-6-羟甲基-喹唑啉 (C9生物碱),水解液经过MCX阳离子交换固相萃取柱净化、BSTFA衍生,采用气相色谱 质谱法全扫描方式定性分析,定性离子376,392,407,选择离子扫描方式定量分析,定量离子392。河鲀毒素在0.05～5.0μg/mL范围内具有良好的线性,相关系数R²=0.997 5,样品添加浓度为0.02、0.10、0.50 mg/kg,测定6次,方法回收率为 64.0%～92.8%,相对标准偏差RSD为5.41%～8.63%,方法检测限为5.0 μg/kg。实验结果表明,建立的固相萃取-气相色谱-质谱法方法灵敏度高,净化效果好,定性、定量准确。     

中国沿海两例食用织纹螺中毒事件中织纹螺体内毒素分析

近年来,因食用织纹螺(Nassariusspp.)导致的中毒事件在中国沿海屡有发生。由于中毒患者的症状与麻痹性贝毒中毒症状相似,因此,许多中毒事件被归咎于麻痹性贝毒,认为织纹螺中的毒素与邻近海域的有毒赤潮有关,但也有研究发现螺体内存在河豚毒素。对此,本研究应用亲水性相互作用色谱柱建立了河豚毒素的液相色谱-质谱联用分析方法,对造成2002年和2003年两次中毒事件的织纹螺样品进行分析。结果表明,两批织纹螺样品中均含有高浓度的河豚毒素及其衍生物,包括三脱氧河豚毒素、脱水河豚毒素和单加氧河豚毒素等,而且两个织纹螺样品中的毒素组成非常相似。因此,导致这两起中毒事件的致毒因子是河豚毒素及其衍生物。江苏和福建两地织纹螺中毒素组成的相似性显示两地织纹螺可能具有相同或相近的毒素来源。     

云斑裸颊虾虎鱼体内各组织河鲀毒素的含量

我国曾发生多起食用虾虎鱼的中毒事件。从发生中毒事件的广东湛江徐闻县西连镇采集了有毒虾虎鱼,经形态学鉴定为云斑裸颊虾虎鱼(Yongeichthys criniger),其体内含河鲀毒素。采用酶联免疫法测定了云斑裸颊虾虎鱼各组织河鲀毒素(tetrodotoxin,TTX)的含量,结果显示每千克组织的TTX含量由高到低依次为肝脏(69.954 mg)、卵巢(9.510 mg)、皮肤(8.937 mg)、肌肉(5.181 mg)、眼睛(3.321 mg)。比野生暗纹东方鲀(Takifugu fasciatus)相应组织TTX含量均要高,毒性强烈。     

河豚毒素两种定量检测方法的比较研究

应用小鼠生物试验和间接竞争抑制酶联免疫吸附试验(ELISA)同步检测17份河豚鱼肝组织和20份河豚鱼肌肉组织中的河豚毒素含量,并对2种方法进行比较。结果表明：ELISA法与小鼠生物试验测得的结果相符合。ELISA法由于其测定程序简便易行,速度快,灵敏度高,在河豚毒素的定量检测以及在预防河豚鱼中毒方面具有广泛的应用前景。     

柱后衍生高效液相色谱法测定水产品中河豚毒素含量

采用柱后衍生高效液相色谱法检测水产品中的河豚毒素含量,选用河豚Takifugu）和织纹螺（Nassarius）为主要研究对象,样品先由0.1%乙酸提取,经过C18固相萃取柱净化。以庚烷磺酸钠为离子对试剂,应用反相离子对液相色谱分离河豚毒素,采用在线柱后衍生系统,在110 ℃高温和4 mol·L^-1 NaOH强碱条件下,将河豚毒素衍生化成具有荧光特性的C9碱,荧光检测器定量检测。河豚毒素在5～1 600 ng范围内呈良好的线性相关,相关系数r=0.999 7;1、2、8 μg·g^-1,3个浓度水平添加样,日内和日间的回收率为80.9%～91.2%、相对标准偏差为1.93%～5.57%;方法定量限为1 μg·g^-1;从添加样色谱图上看,河豚毒素目标峰附近没有干扰峰,定性准确性好。采用柱后衍生高效液相色谱法与小鼠生物法检测同一阳性河豚样,检测结果一致性好。实验结果表明,柱后衍生高效液相色谱法适用于水产品中河豚毒素的检测。     

Tetrodotoxin concentrations in Pleurobranchaea maculata: temporal, spatial and individual variability from New Zealand populations

Tetrodotoxin (TTX) is a potent neurotoxin that has been identified in a range of phylogenetically unrelated marine and terrestrial organisms. Tetrodotoxin was recently detected in New Zealand in Pleurobranchaea maculata (the grey side-gilled sea slug). From June 2010 to June 2011 wild specimens were collected from 10 locations around New Zealand. At one site (Narrow Neck Beach, Auckland) up to 10 individuals were collected monthly for 6 months. Attempts were also made to rear P. maculata in captivity. Tetrodotoxin was detected in samples from eight of the ten sites. The highest average (368.7 mg kg⁻¹) and maximum (1414.0 mg kg⁻¹) concentrations were measured in samples from Illiomama Rock (Auckland). Of the toxic populations tested there was significant variability in TTX concentrations among individuals, with the highest difference (62 fold) measured at Illiomama Rock. Tetrodotoxin concentrations in samples from Narrow Neck Beach varied temporally, ranging from an average of 184 mg kg⁻¹ in June 2010 to 17.5 mg kg⁻¹ by December 2010. There was no correlation between TTX levels and mass. The highest levels correspond with the egg laying season (June–August) and this, in concert with the detection of high levels of TTX in eggs and early larval stages, suggests that TTX may have a defensive function in P. maculata. Only one larva was successfully reared to full maturation and no TTX was detected.     

The Bacterial (Vibrio alginolyticus) Production of Tetrodotoxin in the Ribbon Worm Lineus longissimus—Just a False Positive?

We test previous claims that the bacteria Vibrio alginolyticus produces tetrodotoxin (TTX) when living in symbiosis with the nemertean Lineus longissimus by a setup with bacteria cultivation for TTX production. Toxicity experiments on the shore crab, Carcinus maenas, demonstrated the presence of a paralytic toxin, but evidence from LC-MS and electrophysiological measurements of voltage-gated sodium channel–dependent nerve conductance in male Wistar rat tissue showed conclusively that this effect did not originate from TTX. However, a compound of similar molecular weight was found, albeit apparently non-toxic, and with different LC retention time and MS/MS fragmentation pattern than those of TTX. We conclude that C. maenas paralysis and death likely emanate from a compound <5 kDa, and via a different mechanism of action than that of TTX. The similarity in mass between TTX and the Vibrio-produced low-molecular-weight, non-toxic compound invokes that thorough analysis is required when assessing TTX production. Based on our findings, we suggest that re-examination of some published claims of TTX production may be warranted.