Chapter 8 Polyether Toxins Involved in Seafood Poisoning
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Takeshi Yasumoto and Michio Murata Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Tsutsumidori Amamiya, Sendai 980, Japan Recent studies o n marine toxins point to the involvement o f an increasing number o f novel polyether compounds i n seafood p o i s o n ings. Causative toxins for ciguatera, ciguatoxin, scaritoxin, and maitotoxin seem to have polyether skeletons, although their structures remain u n k n o w n . Palytoxin was found to be responsible for the fatal poisonings caused by ingestion o f xanthid crabs and trigger fish. Three classes o f polyethers, okadaic acid derivatives, pectenotoxins, and yessotoxin were isolated from bivalves i n connection w i t h diarrhetic shellfish poisoning. T h e etiology o f the toxins, toxicological properties, and determination methods are described. Ciguatera Ciguatera is a term given to intoxication caused by eating a variety o f fish inhabiting o r feeding o n coral reefs. Its occurrence is most prevalent i n the Caribbean and the tropical Pacific, affecting probably over 10,000 people annually. A l t h o u g h ciguatera has been k n o w n to the scientific w o r l d since 17th century, chemical studies o f causative toxin(s) have been handicapped by the extreme difficulty i n obtaining toxic materials. Thus, the disease had been defined symptomatologically rather than chemically, u n t i l Scheuer's group characterized some properties o f the principle toxin named ciguatoxin. T h e variability o f ciguatera symptoms, which include digestive, neurological, and cardiovascular disorders, and the diversity o f fish species involved, however, led scientists to explore additional toxins, mostly i n herbivorous fish. Consequently, scaritoxin and maitotoxin were found i n parrotfish and surgeonfish, respectively. C i g u a t o x i n . T h e toxin was isolated from moray eels and purified to crystals by Scheuer's group (7). Structural determination o f the toxin by x-ray o r N M R analyses was unsuccessful due to the unsuitability o f the crystals and due to the extremely small amount o f the sample. T h e toxin was presumed to have a molecular formula o f C ^ H ^ N O ^ from H R F A B - M S data ( M H , 1111.5570) and to have six hydroxyls, five methyls, and five double bonds i n the molecule (2). T h e number o f unsaturations (18 including the five double bonds) and the abundance o f oxygen atoms i n the molecule point to a polyether nature o f the toxin. T h e toxin, o r a closely related toxin i f not identical, is believed to be the principal toxin i n ciguatera. Ciguatoxin was separable o n an alumina c o l u m n into two interconvertible entities presumably differing only i n polarity (J). +
Scaritoxin. D u r i n g a survey o f ciguatera intoxication i n the G a m b i e r Islands, Bagnis et a l . observed that patients poisoned by parrotfish (Scaridae) suffered a longer period than conventional ciguatera symptoms, and postulated the presence o f a
0097-6156/90/0418-0120$06.00/0 o 1990 American Chemical Society
In Marine Toxins; Hall, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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8 . YASUMOTO & MURATA
Polyether Toxins Involved in Seafood Poisoning 121
new toxin, scaritoxin (4). Later, two toxins code-named S G I a n d S G 2 were isolated from t h e flesh o f t h e parrotfish Scarus gibbus a n d were suggested to correspond t o scaritoxin a n d ciguatoxin, respectively (5). Scaritoxin was distinguished from ciguatoxin o n a D E A E cellulose c o l u m n , w h i c h d i d n o t adsorb scaritoxin from chloroform solution but d i d absorb ciguatoxin. T h e two toxins were also separable by t h i n layer chromatography, i n w h i c h scaritoxin showed a higher R f value than ciguatoxin. T h e two toxins h a d t h e same pharmacological properties (6). Scaritoxin was suggested to be the less polar entity o f t h e two interconvertible forms o f ciguatoxin (7). T h e hypothesis, however, remains to be verified. T h e toxin has been detected i n some snappers, but t h e amount was never as significant as i n parrotfish (8). Assays of ciguatoxin. Determination o f ciguatoxin levels i n fish was carried o u t i n many laboratories by mouse assays. E n z y m e immunoassay to screen inedible fish has been proposed by H o k a m a (9). N o specific chemical assay has been developed, as information o n functional groups suitable for fluorescence labeling is n o t available. Analyses conducted i n the authors' laboratory o n remnant fish retrieved from patients' meals indicated that ciguatoxin content as l o w level as 1 p p b c o u l d cause intoxication i n adults. A n extremely high sensitivity and a sophisticated pretreatment method w i l l be required for designing a fluorometric determination method for t h e toxin.
Maitotoxin ( M T X ) . T h e toxin was first detected i n the surgeonfish Ctenochaetus striatus and thus bears the Tahitian name o f the fish, maito (10). T h e toxin probably explains t h e epidemiological observation o f the different symptomatology i n patients intoxicated by surgeonfish (4). Subsequently t h e origin o f t h e toxin was identified as t h e dinoflagellate Gambierdiscus toxicus, a n d t h e toxin was isolate/! from cultures o f the organism as shown i n Figure 1. M a i t o t o x i n judged as pure by T L C and H P L C was obtained as a colorless solid (77); [a$ +16.8 (c 0.36, M e O H - H . O 1:1); U V ( M e O H - H 0 , 1:1) 230 n m (e 9600); mouse lethality, 0.13 /zg/kg (i.p.); soluble i n H 0 , M e O H , and dimethylsulfoxide, practically insoluble i n C H C 1 , acetone and M e C N . T h e toxin reacted positively to Dragendorffs reagent, but n o t to ninhydrin reagent. I n F A B mass spectra (Figure 2) a fragment i o n shows u p at 3299 ( M - S 0 N a + H ) ) " , suggesting that the toxin is disulfated c o m p o u n d . Determination o f S 0 ' liberated by solvolysis also supported t h e presence o f two sulfate ester groups i n t h e molecule. T h e molecular weight o f t h e toxin as disodium salt was thus estimated t o be 3,424.5±0.5. C N M R spectra o f t h e toxin (Figure 3) indicated t h e presence o f 1 6 0 ± 5 carbon signals. Twenty-one methyls, about 36 methylenes, and five methines were observed i n the aliphatic region. N o quaternary carbon appeared i n this range. I n t h e region for oxygenated carbons, o n e methylene, approximately 74 methines, a n d fifteen quaternary carbons were observed. E i g h t olefinic carbons were observed. Absence o f signals assignable to acetal/ketal o r to carbonyl suggested that M T X has n o repeating units, such as amino acids o r sugars. M a i t o t o x i n presumably has n o side chains other than methyls o r an exomethylene, n o r does it have any carbocyles, since a l l 22 trialkylated carbons are accounted for by 5 methyl doublets, 15 methyl singlets o n oxygenated carbons, o n e singlet methyl o n a n aliphatic carbon, and o n e exomethylene. T h e presence o f many hydroxyls a n d ether rings is reminiscent o f palytoxin (Figure 4). However, M T X exceeds palytoxin i n t h e number o f carbons, ether rings, and quaternary methyls. 2
2
3
3
2
2
4
1
3
M T X exceeds any other marine toxins k n o w n i n t h e mouse lethality (0.13 /ig/kg) and is 80 times more potent than commercial saponin ( M e r c k ) i n hemolytic activity. A c c o r d i n g t o Terao (72), M T X induced severe pathomorphological change in the stomach, heart and l y m p h o i d tissues i n mice and rats by i.p. injection o f 200
In Marine Toxins; Hall, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY
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Gambierdiscus toxicus Extracted with MeOH under reflux
1 Extract
Residue Extracted with 50% MeOH under reflux Extract i
Partitioned
80% MeOH
CH C1 2
2
Partitioned
BuOH
H0 2
| S i l i c a gel [CHCl -MeOH (7:3), 3
CHCl -MeOH 3
(1:1)]
(1:1)
| ODS (Fuji G e l , Q3) [50%,
70%, 100% MeOH]
70% MeOH j Develosil C8 15/30
[25%,
30%, 40%, 50% MeCN]
40% MeCN | Develosil C8 15/30 Toxic
[33.3% MeCN]
fraction
| Develosil TMS-5 [35% MeCN] MTX Figure 1. Procedure for extraction and purification o f maitotoxin.
In Marine Toxins; Hall, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
123 Polyether Toxins Involved in Seafood Poisoning
Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on November 1, 2015 | http://pubs.acs.org Publication Date: January 29, 1990 | doi: 10.1021/bk-1990-0418.ch008
8 . YASUMOTO & MURATA
Figure 2. Negative F A B mass spectra o f maitotoxin. T h e numbers denote the mass number at the centroid o f each peak. A A survey scan at a l o w resolution ( R = 3 0 0 ) . B . R e s o l u t i o n enhanced spectrum (R=3000) for i o n clusters at around mlz 3300. C . R e s o l u t i o n enhanced spectrum (R=3000) for i o n clusters at around m/z 3400.
In Marine Toxins; Hall, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
124
MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY
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