Tetrodotoxin Determination Methods - ACS Publications - American

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Tetrodotoxin Determination Methods

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YOSHIOONOUE ,TAMAO NOGUCHI , and KANEHISA HASHIMOTO 1

Laboratory of Marine Botany and Environmental Science, Faculty of Fisheries, Kagoshima University, Kagoshima, Japan Laboratory of Marine Biochemistry, Faculty of Agriculture, University of Tokyo, Tokyo, Japan

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch029

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High-performance l i q u i d chromatography-fluorometry, mass spectrometry and c a p i l l a r y isotachophoresis have been applied successfully to i d e n t i f y or quantitate tetrodotoxin i n small volumes of toxin extract from p u f f e r f i s h . A mouse bioassay method i s useful f o r screening of the t o x i c i t y of various organisms from affected areas, although t h i s method may not be s u f f i c i e n t for the i d e n t i f i c a t i o n of the toxin. Recently, d i s t r i b u t i o n of tetrodotoxin i n the marine ecosystem has expanded from pufferfishes to some other animals. Rapid and accurate determination of the toxin occurring i n those organisms i s becoming increasingly important from the public health standpoint. It i s common knowledge that many species of p u f f e r f i s h are toxic to man. In spite of such recognition, a great number of persons have been intoxicated from ingesting p u f f e r f i s h i n Japan. According to the 1982 food poisoning s t a t i s t i c s (Japan), 80% of the victims were associated with t h i s f i s h . Tetrodotoxin (abbreviated TTX below, Figure 1) i s named a f t e r the family Tetraodontidae into which most pufferfishes are c l a s s i f i e d . TTX has not been detected i n any other fishes except for the goby Gobius criniger. During 1935-1945 Professor Tani of Kyushu Imperial University, Japan, surveyed t o x i c i t y of various tissues from 19 species of puff e r f i s h inhabiting the surrounding waters of the northern Kyushu Island (Table I) (1, 2). He published a book "Toxicological Studies on Japanese Pufferfishes", which i s even recently c i t e d by many researchers dealing with TTX. As he describes, pufferfishes are mostly toxic, irrespective of the species, tissue and the season of catch. In addition, the toxic potency widely d i f f e r s even among specimens of the same species by one catch. The muscle of p u f f e r f i s h from temperate waters i s believed to be l i t t l e or nontoxic, but that from t r o p i c a l waters to be toxic. Several persons were k i l l e d by ingestion of the f l e s h of the p u f f e r f i s h caught o f f Vietnam i n 1959 (2). This p u f f e r f i s h was l a t e r i d e n t i f i e d as Lagocephalus lunaris lunaris. It has recently been 0097-6156/ 84/0262-0345$06.00/0 © 1984 American Chemical Society

In Seafood Toxins; Ragelis, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1984.


SEAFOOD TOXINS


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Table I. Toxicity of Japanese P u f f e r f i s h Species Ovary Testis Liver Skin Intestine Fugru niphobles B A A A C F. poecilonotum B B A A B B B F. vermicularis D A A vermicularis B B F. pardalis A A C B B D A F. vermicularis A porpnyreus B B F. ocellatus B D A obscurum F. chrysops B B C D Β D C F. rubripes D B Β rubripes F. xanthopterum B C B Β D F. stictonotum D D B C Β D B D Lagoçephalus laeviD D gatus mermis D D L. lunaris spadiceus D D D D D Liosaccus cutaneus D D D Canthigaster B C D C rivulaza D D D Diodon holacanthus D D D D Chilomycterus affinis D D Ostracion cmmaculatum D D D D D D D D Lactoria diaphana D D D D Aracana aculeata D D A: Strongly toxic, lethal at less than 10 g. B: Moderately toxic, not l e t h a l at less than 10 g. C: Weakly toxic, not l e t h a l at less than 100 g. D: Negative, not l e t h a l at less than 1000 g. -: No data available. Adapted with permission from Ref. 2. Copyright 1979, Japan S c i e n t i f i c Societies Press.

American Chemical Society Library 1155 16th St. n. w. Washington, 0.Ragelis, C. 20036 In Seafood Toxins; E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

Muscle C C C D D D D D D D D D D D D D D D D

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found that the muscle of pufferfishes inhabiting the Sanriku Coast of Japan i s often t o x i f i e d up to a level of several hundred mouse units (MU) per g (3). Since the lethal dose of TTX i n human i s about 10000 MU (4), the f l e s h of these pufferfishes may k i l l a man even when eaten i n a small amount less than 100 g. On the other hand, some pufferfishes which have not been eaten before, are sometimes marketed i n Japan. In our mouse bioassay test a high t o x i c i t y was detected i n the tissues from Tetraodon alboreticulatus, one of such pufferfishes; 2870 MU/g ovary and 31 MU/g l i v e r (5). Thus, re-examinâtion seems to be necessary f o r toxic potency of the pufferfishes landed from Japanese and adjacent waters.


Recent Findings on the D i s t r i b u t i o n of Tetrodotoxin i n Nature TTX had long been believed to d i s t r i b u t e exclusively i n pufferfishes. Twenty years ago, a p a r a l y t i c toxin was found i n the ovaries of the C a l i f o r n i a newt Taricha torosa and named tarichatoxin (6). Identity of t h i s toxin with TTX was demonstrated l a t e r . Tarichatoxin or TTX was also detected i n the skin, muscle, blood of the C a l i f o r n i a newt, as well as other species of the genus Taricha. The authors noticed the presence of t h i s p a r a l y t i c toxin i n a goby Gobius criniger inhabiting t r o p i c a l to subtropical seas (7). In the goby as well, TTX was recognized i n the tissues such as skin, viscera and muscle. Kim et a l . (8) isolated TTX from Costa Rican frogs of the genus Atelopus. Sheumack et a l . (9) found a p a r a l y t i c toxin i n secretions from the posterior s a l i v a r y gland of the blueringed octopus Octopus maculosus, and named maculotoxin. However, t h i s toxin was also i d e n t i f i e d as TTX. In 1979, a food poisoning case due to ingestion of the digestive gland of a trumpet s h e l l Charonia sauliae occurred i n Shimizu, Shizuoka, Japan. Rather unexpectedly, TTX was found to be responsible for t h i s incidence (10). Subsequent surveys showed that most of the digestive glands of trumpet s h e l l specimens collected from the adjacent waters were moderately to highly t o x i c . The highest t o x i c i t y score recorded was 1950 MU/g digestive gland. Since the average weight of digestive gland of t h i s s h e l l f i s h i s about 50 g, ingestion of the single digestive gland may have k i l l e d ten persons. TTX was also found i n some other gastropods such as the Japanese ivory s h e l l Babylonia japonica (11, 12), and the frog s h e l l Tutufa lissostoma (13). In connection with t h i s , some starfishes fed by those gastropods were found to contain TTX at s i g n i f i c a n t levels (14). In t r o p i c a l to subtropical waters, there l i v e toxic crabs. They contain p a r a l y t i c s h e l l f i s h poisons i n most cases. One of those toxic crabs, Atergatis floridus,inhabits also along the P a c i f i c Coast of the Japan Proper. Very recently, i t was excavated that the crabs collected from Miura Peninsula near Tokyo possess TTX as the major toxin, along with some p a r a l y t i c s h e l l f i s h poisons as the minor (15). The d i s t r i b u t i o n of TTX may be expanded further i n the future. Present Status of Determination of TTX TTX has so f a r been determined mainly by mouse assay (_4). This assay i s featured by s i m p l i c i t y , but has some demerits such as low sensit i v i t y , low accuracy, rather high cost of suitable mice, and local


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d i f f i c u l t y of i t s procurement. Meanwhile, the d i s t r i b u t i o n of TTX has been expanded from pufferfishes to some animals of various phyla, as described above. More sensitive and accurate assay methods may promote these lines of research. Under these circumstances, attempts have been made to develop chemical assay methods of TTX. In the near future, the mouse assay method may be replaced by chemical methods.


Chemical Assay Methods Two example-determinations of TTX using fluorometry coupled with high-performance l i q u i d chromatography (HPLC) are introduced here, along with some other promising assay techniques such as mass spec trometry. HPLC with Fluorometry Using A l k a l i

(16)

Gonads (10 g) of the p u f f e r f i s h Fugu pardalis are extracted with 25 mL of 0.02 M acetic acid i n a b o i l i n g water bath for 10 min. Af ter being cooled to room temperature, the extract i s f i l t e r e d . The f i l t r a t e i s passed through an Amberlite CG-50 column (NH^, 1.2 χ 3 cm). The column i s developed with 50 mL each of water and 0.5 M acetic acid. A defatting step may be introduced p r i o r to the column treatment when f a t - r i c h tissues l i k e l i v e r s are employed. The toxic eluate i s concentrated i n vacuo and made up to 20 mL with water. A 50- pL portion of the solution i s analyzed by HPLC. The HPLC units include a s t a i n l e s s - s t e e l column (400 χ 5 mm I.D.) with a cationic exchanger (Hitachi 3011 C), a loop injector (Kyowa Seimitsu Co.), two constant flow pumps (Seishin Pharmaceutical Co.) for eluant and reagent, reaction c o i l (Teflon tubing, 30 m χ 0.5 mm I.D.) and a fluorometer (Japan Spectroscopic Co., FP-110). The column i s eluted with 0.06 M c i t r a t e buffer (pH 4.0) at a flow rate of 0.5 mL/min. The eluate i s mixed with an equal volume of 4 Ν NaOH. On alkaline treatment, TTX gives r i s e to a fluorogenic substance, 2amino-6-hydroxymethyl-8-hydroxyquinazoline (C base) (17, 18). The excitation and emission wavelengths are set at 357 and 510 nm, res pectively. The chromatogram i s recorded on a National Pen Recorder VP-6611A. TTX gives a peak with a retention time of 26 min. The t o x i c i t y fluorescence r e l a t i o n i s linear over the range of 0.1-20 MU. The maximum v a r i a t i o n of the peak heights i s 3%, as tested on varied toxin levels. HPLC with Fluorometry Using o-Phthalaldehyde

(ΟΡΑ) (19, 20)

Ovaries (100 g) of the p u f f e r f i s h Fugu vermicularis porphyreus are extracted with 1% acetic acid i n methanol. The extract i s concen trated i n vacuo and defatted by shaking with chloroform. The defat ted extract i s treated with activated charcoal. The toxin adsorbed i s eluted with 1% acetic acid i n 20% ethanol. The eluate i s evapo rated i n vacuo to dryness. The residue i s dissolved i n a small amount of water and adjusted to pH 6 with 1 N Na0H. The toxic solution i s applied to an Amberlite IRC-50 column (NH*, 2.5 χ 45 cm) and de veloped with 2 L of water, and then 1 L each of 1 and 10% acetic acid. The toxic fractions are freeze-dried, dissolved i n 1 mL of water and analyzed by HPLC. +


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The HPLC system comprises a Hitachi 638-50 analyzer with a 65010 spectrofluorometer, a 056 recorder operating at 10 mV f u l l scale, a s t a i n l e s s - s t e e l column (150 χ 4 mm I.D.) with a Hitachi 3013 C ionexchanger, and an ΟΡΑ unit with a reaction c o i l (10 m χ 0.3 mm I.D.). Ten m i c r o l i t r e s of toxic solution are placed on the column equilibrated with 0.005 M acetic acid. A three-step gradient elution with acetic acid i s then applied: 0.005-0.015 M, 0-15 min; 0.015-0.15 M, 15-30 min; 0.15-0.50 M, 30-80 min. The flow ra£e of the elution and the column pressure are 1 mL/min and 110 kg/cm , respectively. The ΟΡΑ reagent f o r HPLC i s prepared according to the method of Benson and Hare (21). The fluorescence reaction i s performed i n a 55 °C water bath. ΟΡΑ reacts with the guanidino group of TTX pre sumably to form a fluorescent product, l - a l k y l t h i o - 2 - a l k y l i s o i n d o l e (22, 23). TTX i s monitored at 453 nm with 332-nm excitation. Peak areas are calculated by a data processing system of the analyzer. The chromatogram of TTX from the column i s shown i n Figure 2. Although a multitude of peaks appear on analysis of a crude puffer toxin preparation, only one of them provides a retention time (20.0 min) compatible to that of TTX standard. The i d e n t i t y of TTX and other main contaminants has been confirmed by mouse bioassay, TLC, electrophoresis and amino acid analysis. The peak area i s proportional to the amount of toxin applied. The l i n e a r i t y of t h i s relationship i s maintained up to 100 nM TTX or more. The lower l i m i t of ΟΡΑ detection i s 1-2 nM f o r TTX. Five or more assays indicate that the r e l a t i v e standard deviation f o r TTX i s + 2.5%. Major i n t e r f e r i n g substances found i n the crude toxin prepara t i o n are c i t r u l l i n e , ethanolamine, ornithine, lysine and arginine. The presence of more than 10 nM of these amines makes d i f f i c u l t the detection of TTX because of t h e i r intense fluorescence. A similar effect i s also noted with 100 uM ammonium ions. Mass Spectrometry (24) The mass spectrum of TTX can d i r e c t l y be measured by fast atom bom bardment- or secondary ion-mass spectrometry. In the former a JEOL JMS DX-300 mass spectrometer equipped with a JEOL JMA-3100 data system i s used; xenon provides the primary beam of atoms. Accelera t i o n voltage of the primary ion i s 3 kV. Scanning i s repeated within a mass range of m/z 100 to 1000. TTX i s dissolved i n 0.05 M acetic acid at a concentration of approximately 10 pg/pL. One m i c r o l i t r e each of t h i s TTX solution and glycerol as matrix are placed on the sample stage of the mass spectrometer, mixed well, and introduced into the ion chamber of the spectrometer. Both p o s i t i v e and negative mass spectra of TTX are then measured. As shown i n Figure 3, TTX exhibits (M+H) and (M+H-H 0) ion peaks at m/z 320 and 302, respectively, i n the p o s i t i v e mass spec trum, and an (M-H)~ peak at m/z 318 i n the negative. In the secondary ion-mass spectrometry, a Hitachi M-80B mass spectrometer equipped with a Hitachi M-0101 data system i s applied as above. +

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Tetrodotoxin Determination Methods - ACS Publications - American

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