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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/14590

Title: 台灣產有毒螺類之毒性、蓄毒機制及毒素結合蛋白之研究
Studies on Toxicity, Toxin Accumulating Mechanism and Toxin-Binding Protein of Toxic Gastropods in Taiwan
Authors: Pai-An Hwang
黃培安
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 河魨毒;吸引性試驗;毒素結合蛋白
Tetrodotoxin;Attracting test;Toxin-binding protein
Date: 2004
Issue Date: 2011-06-30T07:40:12Z
Abstract: 河魨毒(TTX)與麻痺性貝毒(PSP)為常見的兩種海洋生物毒, 其作用機制為阻塞神經細胞上之鈉腔體,導致神經訊息無法傳遞。自2001至2004間,在台灣共發生3起螺類中毒案件,經分析得知:榧螺科的橙口榧螺、台灣榧螺和平瀨榧螺、織紋螺科的球織紋螺、素面織紋螺和橡子織紋螺體內含有河魨毒,且並未檢出含有麻痺性貝毒,因此判定上述三起螺類中毒事件是由河魨毒所引起。截至目前,尚未開發出有效防治螺類蓄積毒素之方法,且對於中毒患者也無專一的治療藥劑可供使用,因此全面性的對台灣產螺類進行毒性市場調查,為防範螺類中毒事件的最佳方法之ㄧ。自民國91年至93年間,於台灣地區進行市場調查,於6個縣市中採集了11種螺類,樣品數共170粒。經毒性及毒成分分析得知,在嘉義及屏東地區所採集到的斑節楊桃螺及震旦海音螺含有河魨毒,毒素分佈於中腸腺及肌肉中。其餘的大玉螺、腰帶玉螺、條紋髻螺、花點鶉螺、鶴頭骨螺、花球琵琶螺、台灣鐘螺、千手玫瑰螺及赤蛙螺均未檢出含有毒性。 食物中毒事件中致毒成分之鑑定,大多根據殘存食物來進行檢驗。由於食物中成分複雜,純化毒素需經過繁複步驟,因此本實驗開發LC-MS檢測法,以中毒患者的尿液及血液為材料,對人類尿液及血液中微量河魨毒做定性及定量分析,以供作為鑑定致毒成分的另一種方法。此LC-MS檢測方法的極限為1 ng TTX/mL,檢量線範圍介在1-600 ng TTX/mL之間 (Y=11.88X+1098.27, R2=0.9947),回收率在95%左右。自橡子織紋螺中毒案件中取得4位中毒患者之尿液及血液,經 C18 Sep-Pak cartridge column及3,000分子量高速離心過濾膜純化河魨毒,而後以LC-MS進行河魨毒的定性及訂量分析。結果顯示4位中毒患者的尿液中均可檢出含有河魨毒,4件血液樣品中僅3件檢出含有河魨毒,且尿液中河魨毒的量高於血液。 根據有毒螺類毒性資料表得知,不同種螺類所含的毒素量差異甚大,此外少數螺類同時含有河魨毒及麻痺性貝毒。為了解不同種螺類毒性差異的原因,以及為何僅少數螺類可蓄積麻痺性貝毒,以利用河魨毒及麻痺性貝毒吸引性試驗,以瞭解螺類可能之蓄毒機制。在河魨毒吸引性試驗中發現,有毒螺類的河魨毒相對吸引百分比與螺類的毒性呈現正相關(Y=1.050X+3.443, R=0.8061),並且螺類的毒性亦與其河魨毒耐受性呈現正線性關係(Y=0.113X+294.602, R=0.8143)。在麻痺性貝毒吸引性試驗中亦有相似的結果,有毒螺類的麻痺性貝毒相對吸引百分比與螺類的毒性呈現正相關(Y=3.716X+0.363, R=0.8427),而螺類的毒性亦與其麻痺性貝毒耐受性呈現正線性關係(Y=0.091X+210.090, R=0.6803)。結果顯示毒性愈高的螺類愈受河魨毒所吸引,造成螺類毒性有高低差異,且麻痺性貝毒對有毒螺類也具有吸引力,使得部分螺類可同時蓄積兩種毒素。 河魨毒保有生物對河魨毒的耐受性較無毒生物高,而河魨毒保有生物是如何運送、累積毒素,且不被自身毒素毒害,為本研究探討的重點之ㄧ。經Sepharose CL-6B膠體過濾分析得知,已知有毒螺類大玉螺、細紋玉螺、橙口榧螺、台灣榧螺和平瀨榧螺肌肉中含有TTX-binding 高分子物質,此物質可包覆河魨毒,使河魨毒以無毒的型式存在。其個別結合河魨毒之能力為0.12, 0.62, 0.45, 0.28和0.35 MU/mg。這五種螺類之TTX-binding 高分子物質可被蛋白質酵素及酸水解,但不被RNase T2、 DNase、deoxyribonuclease I和α-amylase水解,此外額外添加河魨毒於TTX-binding高分子物質中發現,TTX-binding高分子物質不具有中和河魨毒的能力。另取細紋玉螺的TTX-binding高分子物質,以Q Fast-Flow陰離子交換樹脂及BioSep-SEC-S 2000高效能液相層析管柱依序進行純化,發現其蛋白結合河魨毒之能力分別上升為3.5 MU/mg和4.8 MU/mg。此高分子物質經純化後可得河魨毒結合蛋白,其分子量約為434K,再經SDS-PAGE分析得知河魨毒結合蛋白是由兩個次單元組成,分子量分別為272和47K。顯示部分河魨毒在細紋玉螺體內,是被河魨毒結合蛋白所包覆,而以無毒的型式存在。
Tetrodotoxin (TTX) and paralytic shell poisons (PSP) are widely known marine neurotoxins, which act by blocking the entry of sodium ion into sodium channels of neuronal membranes. During 2001 to 2004, there were 3 gastropod poisoning incidents in Taiwan. Based on food poisoning incidents demonstrated that gastropods Olividae Oliva miniacea, O. mustelina, and O. hirasei, Nassariidae Nassarius glans, Zeuxis sufflatus, and Niotha clathrata contained TTX. While PSP was not detected in those causative gastropods, so it was concluded that the causative agent of the above gastropod poisoning incidents was TTX. Until now, it is no known strategy to prevent gastropods from accumulating the toxin and without a specific remedy for poisoning patients either. Therefore, the overall screening test of Taiwanese gastropods is the best way to prevent the happening of gastropod poisoning incident. During 2002 to 2004, 170 specimens of 11 species gastropods were collected from 6 prefectures in eastern, western and southern Taiwan. Both Harpa articularis and Hindsia sinensis collected from Chiay and Pingtung Prefecture were containing TTX. While, Polinices didyma, Natica vitellus, Phalium flammiferum, Tonna marginata, Murex haustellum, Ficus ficus, Chicoreus palmarosae, Calliostoma formosense and Bufonaria rana were not detected to contain TTX or PSP. Usually, the causative agents of TTX-food poisoning were identified from the toxin of food. However, the suspected food was sometimes discarded and no leftover was retained. Hence, the TTX diagnosis from patients is very important. The LC-MS method was developed to identify the trace amount TTX in human urine and blood. The detection limit of TTX was 1 ng/mL and the calibration curve was linear in the range of 1-600 ng/mL (Y=11.88X+1098.27, R2=0.9947). Recoveries were around 95% for TTX. The urine and blood samples from four patients who were intoxicated in N. glans food poisoning incident, were cleansed using a C18 Sep-Pak cartridge column and 3,000 MW cut-off Ultrafree microcentrifuge filters, and the eluate was filtered and determined by LC-MS. TTX was detected in all urine samples but in only three of four blood samples tested. TTX-bearing animals are generally known to be much more resistant to the lethal effect of TTX than TTX-lacking animals. In TTX attracting test, it was found that toxic gastropods showed significantly positive relationship between comparative attracting variation and the reported toxicity (Y=1.050X+3.443, R=0.8061), and the relationship between TTX resistance ability and the toxicity also has a positive correlation (Y=0.113X+294.602, R=0.8143). By the same way, in PSP attracting test, all toxic gastropods showed significantly positive linear relationship (Y=3.716X+0.363, R=0.8427) between comparative attracting variation and the toxicity reported. The relationship between PSP resistance ability and the toxicity also has a positive correlation (Y=0.091X+210.090, R=0.6803). It indicated that TTX and PSP are attractants for toxic gastropods. Toxic gastropods P. didamy, N. lineata, O. miniacea, O. mustelina and O. hirasei are TTX bearing animals, which accumulate TTX in their muscle and digestive gland. Analysis by gel filtration on Sepharose CL-6B revealed that the muscle extract of five kinds of toxic gastropods contained TTX-binding high molecular weight substance (HMWS) (1500~2000K). The TTX-binding capacity of those HMWS were 0.12, 0.62, 0.45, 0.28 and 0.35 MU/mg protein, respectively, but more TTX added into those HMWS showed no neutralizing ability. The HMWS of five toxic gastropods could be hydrolyzed with HCl and protease at 37℃, pH 7.4, but not with ribonuclease T2, deoxyribonuclease I and α-amylase. After the purification of N. lineata TTX-binding protein by Q Fast-Flow strong anion exchanger and BioSep-SEC-S 2000, the TTX-binding capacity raised to 3.5 MU/mg and 4.8 MU/mg protein, respectively. The molecular weight of TTX-binding protein of N. lineata was estimated to be about 434K, while it comprised at least two kinds of subunits with the molecular weight of about 272 and 47K under SDS-PAGE.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M90320016
http://ntour.ntou.edu.tw/ir/handle/987654321/14590
Appears in Collections:[食品科學系] 博碩士論文

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