Historical Perspective on Paralytic Shellfish Poison - ACS Publications

9

Historical Perspective on Paralytic Shellfish

Poison

EDWARD J. SCHANTZ

Downloaded by FUDAN UNIV on March 23, 2017 | http://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch009

Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, WI 53706

S h e l l f i s h have become an important item i n the world's food supply. Rapid a i r transportation with good r e f r i g e r a t i o n has widened l o c a l seacoast markets of past years to many inland cities and areas throughout the world and commercial growing and harvesting of shellfish for t h i s market has become a growing industry. Shellfish such as mussels and clams that feed on microscopic organisms concentrate and r e t a i n b a c t e r i a , v i r u s e s , toxins and poisons associated with the organisms they feed on. One important hazard associated with shellfish consumption is p a r a l y t i c shellfish poisoning (PSP), caused by a highly l e t h a l neurotoxin which the s h e l l f i s h r e t a i n from feeding on c e r t a i n poisonous d i n o f l a g e l l a t e s . Out of about 1200 species of d i n o f l a g e l l a t e s only a few produce the p a r a l y t i c poison and occur s p o r a d i c a l l y along many sea coasts throughout the world. Shortly after eating shellfish that have fed on the poisonous d i n o f l a g e l l a t e s humans experience a t i n g l i n g sensation i n the l i p s and finger t i p s followed by progressive p a r a l y s i s of the limbs and finally death from respiratory p a r a l y s i s which may occur within 2 to 24 hours depending upon the dose. I f the dose i s small enough that one survives 24 hours, the prognosis i s good. No antidote i s known, but artificial r e s p i r a t i o n administered soon after symptoms appear i s the best treatment and no doubt has saved lives, at least i n borderline cases of poisoning. The purpose of t h i s report i s to review and present the important historical developments leading to the present understanding of the chemical nature of the poison including its chemical s t r u c ture and properties, the mechanism causing death and the present means of c o n t r o l to make s h e l l f i s h safer for human consumption. H i s t o r i c a l Background and Cause of PSP As long as medical records have been kept, poisoning of humans from eating clams and mussels has been recorded. The sporadic and unexplainable occurrence of poisonous s h e l l f i s h was indeed a very puzzling phenomenon. When mussels became poisonous they usually remained so for a few weeks and for no apparent reason soon became 0097-6156/ 84/0262-0099$06.00/0 © 1984 American Chemical Society

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


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p e r f e c t l y s a f e t o e a t a g a i n . A l t h o u g h many t h e o r i e s were proposed f o r t h e o c c u r r e n c e o f p o i s o n o u s s h e l l f i s h t h r o u g h o u t the e i g h t e e n t h and n i n e t e e n t h c e n t u r i e s , p a r t i c u l a r l y i n European c o u n t r i e s around t h e N o r t h Sea, t h e a c t u a l cause was not known u n t i l about 1927 when o u t b r e a k s o f PSP o c c u r r e d a l o n g the c o a s t o f C a l i f o r n i a . At t h a t time Sommer, Whedon, Meyer and t h e i r c o l l e a g u e s (1.2) a t t h e U n i v e r s i t y of C a l i f o r n i a observed a p a r t i c u l a r d i n o f l a g e l l a t e blooming around t h e C a l i f o r n i a sea m u s s e l beds a t the time many p e o p l e became s i c k and d i e d a f t e r e a t i n g the m u s s e l s . These i n v e s t i g a t o r s s u s p e c t e d t h a t the d i n o f l a g e l l a t e upon which t h e mussels were f e e d i n g may be p o i s o n o u s and found t h a t a c i d i c w a t e r e x t r a c t s o f t h e d i n o f l a g e l l a t e s and the mussels k i l l e d mice i n t h e same manner. The d i n o f l a g e l l a t e was i d e n t i f i e d as Gonyaulax c a t e n e l l a . To t e s t t h i s o b s e r v a t i o n Dr. Sommer and h i s a s s i s t a n t L u c i l e F o s t e r p l a c e d nonpoisonous mussels i n l a b o r a t o r y j a r s c o n t a i n i n g seawater c u l t u r e s o f G. c a t e n e l l a and found t h a t t h e mussels soon became p o i s o n o u s . When t h e s e mussels were p l a c e d i n j a r s c o n t a i n i n g nonpoisonous o r g a n i s m s , t h e mussels e x c r e t e d o r d e s t r o y e d t h e p o i s o n w i t h i n a week o r two, thus e s t a b l i s h i n g c o n c l u s i v e l y t h e r e l a t i o n s h i p o f G. c a t e n e l l a t o t h e p o i s o n o u s m u s s e l s . T h i s o r g a n i s m , l i k e most d i n o f l a g e l l a t e s , blooms o v e r a p e r i o d o f 2 t o 3 weeks and g r a d u a l l y d i e s o f f as o t h e r organisms bloom. I n t h e n a t u r a l s t a t e mussels become poisonous soon a f t e r a p o i s o n o u s d i n o f l a g e l l a t e blooms and t h e y e x c r e t e o r d e s t r o y t h e p o i s o n w i t h i n 2 t o 3 weeks a f t e r the bloom has d i s a p p e a r e d . About 9 5 % o f the p o i s o n i n a m u s s e l i s c o n t a i n e d i n the d a r k g l a n d o r hepatopancreas and i s q u i t e s t a b l e t o o r d i n a r y steaming o r c o o k i n g w h i c h , o f c o u r s e , makes the f o o d p o i s o n i n g problem more a c u t e . Poisonous s h e l l f i s h n e i t h e r l o o k nor t a s t e d i f f e r e n t from normal ones. A l t h o u g h heavy blooms o f d i n o f l a g e l l a t e s cause a d i s c o l o r a t i o n o f the water c a l l e d a r e d t i d e , t h e average p e r s o n c o l l e c t i n g s h e l l f i s h f o r human consumption cannot r e l y on a change i n t h e appearance o f the w a t e r . C o n c e n t r a t i o n s as low as 400 o r 500 c e l l s per ml are not v i s i b l e t o t h e naked eye but are s u f f i c i e n t t o make t h e mussels t o o p o i s o n o u s f o r human consumption. C e l l counts o f around 20,000 o r more per ml a r e n e c e s s a r y t o produce a r e d t i d e . I t would be a d v i s a b l e not t o e a t s h e l l f i s h i f a r e d t i d e bloom i s apparent. The o r g a n i s m may o r may not be p o i s o n o u s . The e a r l y work o f Sommer and a s s o c i a t e s e s t a b l i s h e d a good mouse assay f o r t h e p o i s o n i n s h e l l f i s h p r o d u c t s w h i c h made q u a n t i t a t i v e work w i t h t h e p o i s o n p r a c t i c a l . A mouse u n i t (MU) was d e f i n e d as the minimum amount o f p o i s o n t h a t would k i l l a 20-gram w h i t e mouse i n 15 m i n u t e s when one ml o f an e x t r a c t o f s h e l l f i s h was i n j e c t e d i n t r a p e r i t o n e a l l y . H i g h e r amounts t h a n the minimum k i l l i n s h o r t e r t i m e , i . e . , d e a t h t i m e s o f 3, 4, 6, and 8 m i n u t e s a r e e q u i v a l e n t t o 3.7, 2.5, 1.6, and 1.3 MU, r e s p e c t i v e l y , as i l l u s t r a t e d i n F i g u r e 1. The d i s c o v e r y o f the r e l a t i o n s h i p o f G. c a t e n e l l a t o p o i s o n o u s s h e l l f i s h by Sommer, Whedon and a s s o c i a t e s l e d Canadian i n v e s t i g a t o r s N e e d i e r (3) and P r a k a s h (4) t o the d i s c o v e r y t h a t Gonyaulax t a m a r e n s i s ( v a r . e x c a v a t a ) caused clams and s c a l l o p s a l o n g t h e n o r t h e a s t c o a s t o f N o r t h A m e r i c a and the n o r t h e a s t c o a s t o f England t o become p o i s o n o u s .



9. SCHANTZ

Historical Perspective on Paralytic Shellfish Poison

101

F i g u r e 1. Dose and time t o death r e l a t i o n s h i p s f o r s a x i t o x i n s . A p l o t o f the l o g o f the dose a g a i n s t the r e c i p r o c a l of the time r e s u l t s i n a s t r a i g h t l i n e r e l a t i o n s h i p . Reprinted with permission from Ref. 37. C o p y r i g h t 1973, Committee on Food P r o t e c t i o n NAS-NRC.


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In the e a r l y y e a r s t h e r e was much i n t e r e s t i n the amount o f p o i s o n n e c e s s a r y t o cause s i c k n e s s and death of humans. From a c c i d e n t a l cases of p o i s o n i n g a l o n g the c o a s t of C a l i f o r n i a where G. c a t e n e l l a commonly blooms, Sommer and c o l l e a g u e s e s t i m a t e d t h a t death of humans r e s u l t e d from the consumption of about 20,000 MU o f p o i s o n . However, a l o n g the S t . Lawrence e s t u a r y and Bay o f Fundy the Canadians observed death of persons w i t h a consumption as low as 5000 MU. I n t h e s e areas s h e l l f i s h become poisonous by f e e d i n g on G. t a m a r e n s i s .


P u r i f i c a t i o n - Chemical

and P h y s i c a l P r o p e r t i e s

Dr. Byron R i e g e l and Warren Stanger of N o r t h w e s t e r n U n i v e r s i t y and Dr. Hermann Sommer undertook i n 1944 a t the s u g g e s t i o n o f the N a t i o n a l Academy of S c i e n c e s the f i r s t o r g a n i z e d s t u d i e s t o i s o l a t e and c h a r a c t e r i z e the p o i s o n from the sea mussels ( M y t i l u s c a l i f o r n i a n i s ) and i n c o n n e c t i o n w i t h my work a t the U.S. Army B i o l o g i c a l L a b o r a t o r i e s I had the o p p o r t u n i t y t o t a k e p a r t i n the work a t t h i s t i m e . Our f i r s t o b j e c t i v e was t o o b t a i n s u f f i c i e n t poisonous mussels f o r our s t u d i e s , but the s p o r a d i c o c c u r r e n c e of the poisonous d i n o f l a g e l l a t e and poisonous sea mussels a l o n g the C a l i f o r n i a c o a s t made t h i s q u i t e d i f f i c u l t and meant t h a t o r g a n i z e d surveys f o r poisonous mussels had t o be c a r r i e d on throughout the summer months, the most l i k e l y p e r i o d of o c c u r r e n c e a l o n g the C a l i f o r n i a c o a s t . I f a poisonous d i n o f l a g e l l a t e bloom d i d o c c u r a t the time of low t i d e p e r i o d so t h a t above w a t e r c o l l e c t i o n s of mussels c o u l d be made, we had o n l y about one week t o make collections. S i g n i f i c a n t amounts of the p o i s o n were o b t a i n e d from the sea m u s s e l s , but a d d i t i o n a l and l a r g e r amounts were o b t a i n e d by t h e c o l l e c t i o n of poisonous A l a s k a b u t t e r clams (Saxidomas g i g a n t e u s ) . Through arrangements w i t h the U n i t e d S t a t e s Department o f t h e I n t e r i o r and the A l a s k a E x p e r i m e n t a l Commission i n 1948 I made c o l l e c t i o n s of poisonous b u t t e r clams throughout c e r t a i n areas o f s o u t h e a s t e r n A l a s k a w i t h the h e l p of H a r r i s Magnusson, John Dassow, J . S. Chambers and C l a r e n c e C a r l s o n of the K e t c h i k a n L a b o r a t o r y . Much of the d i g g i n g of b u t t e r clams was c a r r i e d out by l o c a l I n d i a n s who worked f o r l o c a l p a c k i n g p l a n t s . S m a l l amounts a l s o were o b t a i n e d by c u l t u r i n g the poisonous d i n o f l a g e l l a t e i n t h e l a b o r a t o r y a t Hooper F o u n d a t i o n , U n i v e r s i t y of C a l i f o r n i a and a t the B i o l o g i c a l L a b o r a t o r i e s , F o r t D e t r i c k , M a r y l a n d . The c u l t u r i n g at the B i o l o g i c a l L a b o r a t o r i e s was c a r r i e d out w i t h a x e n i c c u l t u r e s °f 9.' c a t e n e l l a and G. t a m a r e n s i s w i t h h e l p from L u i g i P r o v a s o l i , H a s k i n s L a b o r a t o r i e s , who p r o v i d e d the a x e n i c c u l t u r e s o f t h e s e organisms. The o r i g i n a l c u l t u r e o f G. c a t e n e l l a was c o l l e c t e d i n 1944 from a bloom i n Monterey Bay, C a l i f o r n i a by L u c i l e F o s t e r a t the Hooper F o u n d a t i o n , U n i v e r s i t y of C a l i f o r n i a and G. t a m a r e n s i s by P r o v a s o l i from the Bay o f Fundy. The p o i s o n s from the t h r e e sources appeared the same, but were kept s e p a r a t e throughout the work. They a l l produced the same s i g n s of p o i s o n i n g i n mice and a l l had s i m i l a r m o l e c u l a r w e i g h t s of 300 t o 400 as measured i n crude e x t r a c t s by the d i f f u s i o n r a t e of the p o i s o n i n a N o r t h r o p diffusion cell. T h e i r a c t i o n on c a t i o n exchange r e s i n s was a l s o s i m i l a r . The source from the A l a s k a b u t t e r clam siphons proved t o



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be the b e s t s u p p l y because, u n l i k e the m u s s e l s , 60 t o 70% o f t h e p o i s o n i n t h e whole body o f t h e clam was c o n t a i n e d i n the s i p h o n and remained t h e r e f o r p e r i o d s o f a y e a r o r more which made c o l l e c t i o n p o s s i b l e a t low t i d e any time the weather p e r m i t t e d . Another source o f t h e p o i s o n was made a v a i l a b l e t o me s h o r t l y a f t e r World War I I t h r o u g h t h e c o u r t e s y o f the Canadian Defense Research Board i n 1947. T h i s s o u r c e was the e n t r a i l s o f commercial s c a l l o p s c o l l e c t e d i n t h e Bay o f Fundy. The e n t r a i l s were from deep sea s c a l l o p s , which a r e u s u a l l y poisonous a t a l l t i m e s . The p o i s o n i n t h e s c a l l o p s however had d i f f e r e n t p r o p e r t i e s and d i d not f u n c t i o n w e l l on c a t i o n exchange r e s i n s . As a r e s u l t I s t o r e d them i n a c i d s o l u t i o n about pH 2 f o r l a t e r s t u d i e s ( 5 ) . R i e g e l and Sommer's e a r l y work i n 1944 e s t a b l i s h e d t h a t t h e p o i s o n from C a l i f o r n i a mussels was a b a s i c substance e a s i l y e x t r a c t e d w i t h a c i d i c water pH 2 and some p u r i f i c a t i o n was accomp l i s h e d on D e c a l s o and a c t i v a t e d carbon ( 6 ) . These s u b s t a n c e s were not v e r y e f f i c i e n t f o r p u r i f i c a t i o n . However the new A m b e r l i t e c a t i o n exchange r e s i n s ( A m e r l i t e XE-64) d e v e l o p e d by Rohm and Haas Company a t about t h i s time proved t o be i d e a l f o r t h e q u a n t i t a t i v e removal o f t h e p o i s o n from crude a c i d e x t r a c t s o f poisonous s h e l l f i s h c o n t a i n i n g as l i t t l e as 1 MU per mg o f s o l i d s . E x t r a c t s o f t h e hepatopancreas o f poisonous mussels u s u a l l y c o n t a i n e d 2 t o 8 MU per mg o f s o l i d s whereas e x t r a c t s o f t h e s i p h o n s o f poisonous b u t t e r clams v a r i e d c o n s i d e r a b l y but u s u a l l y c o n t a i n e d around 2 MU per mg o f s o l i d s . Research on p u r i f i c a t i o n methods a t t h e B i o l o g i c a l L a b o r a t o r i e s and w i t h R i e g e l ' s group a t N o r t h w e s t e r n U n i v e r s i t y r e s u l t e d i n p a r t i a l p u r i f i c a t i o n o f t h e p o i s o n on t h e r e s i n t o about 3000 MU per mg o f s o l i d s . F u r t h e r chromatography on acid-washed and a c t i v a t e d a l u m i n a brought the s p e c i f i c t o x i c i t y t o 5500 MU per mg. Repeated chromatography i n v a r i o u s ways d i d not i n c r e a s e the t o x i c i t y and t h e p o i s o n was c o n s i d e r e d i n a h i g h l y p u r i f i e d s t a t e i n 1954, t e n y e a r s a f t e r the work began. Subseq u e n t l y o v e r 20 grams o f h i g h l y p u r i f i e d s a x i t o x i n were p r e p a r e d a t t h e B i o l o g i c a l L a b o r a t o r i e s m a i n l y from b u t t e r c l a m s i p h o n s . The p o i s o n was found t o be a w h i t e h y g r o s c o p i c s o l i d v e r y s o l u b l e i n w a t e r , p a r t l y s o l u b l e i n methanol and e t h a n o l , but i n s o l u b l e i n most n o n p o l a r s o l v e n t s such as e t h y l and p e t r o l e u m e t h e r s . I t showed no a b s o r p t i o n i n t h e u l t r a v i o l e t and t i t r a t i o n showed two p K v a l u e s a t 8.2 and 11.5. The o p t i c a l r o t a t i o n was about 130. The m o l e c u l a r f o r m u l a was found t o be C i o l 7 7 ° 4 as t h e f r e e base w i t h a m o l e c u l a r w e i g h t o f 299 and as the d i h y d r o c h l o r i d e s a l t 372. The p o i s o n can be reduced w i t h hydrogen a t room temperature and p r e s s u r e i n t h e presence o f p l a t i n u m b l a c k t o produce a n o n t o x i c d e r i v a t i v e . One mole o f hydrogen was t a k e n up p e r mole o f p o i s o n i n t h i s r e a c t i o n . I t a l s o r e a c t e d w i t h c e r t a i n a r o m a t i c n i t r o compounds t o form a c o l o r e d complex i n much the same way as c r e a t i n i n e r e a c t s w i t h d i n i t r o p h e n o l i n the J a f f e t e s t and w i t h d i n i t r o b e n z o i c a c i d i n t h e B e n e d i c t - B e h r e t e s t . The reduced p o i s o n ( n o n t o x i c ) d i d n o t r e a c t w i t h t h e s e r e a g e n t s . The good c o r r e l a t i o n o f t h e s e t e s t s w i t h the p o i s o n suggested t o us t h e i r p o s s i b l e use as a q u a n t i t a t i v e c h e m i c a l t e s t t o r e p l a c e the mouse t e s t where mice a r e h a r d t o get and keep f o r assay p u r p o s e s . However, c e r t a i n i m p u r i t i e s c o n t a i n e d i n clam and mussel e x t r a c t s i n t e r f e r e d s u f f i c i e n t l y w i t h t h e t e s t f o r i t s p r a c t i c a l use as an a

H

N



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a s s a y , but i t was v a l u a b l e f o r our r e s e a r c h work w i t h the p u r i f i e d poison. The s p e c i f i c t o x i c i t y d e t e r m i n e d i n s e v e r a l Food and Drug A d m i n i s t r a t i o n l a b o r a t o r i e s o f the U n i t e d S t a t e s and i n c o o p e r a t i o n w i t h Canadian P u b l i c H e a l t h S e r v i c e s c o n f i r m e d the s p e c i f i c t o x i c i t y t o be 5500 i 500 MU per mg and one mouse u n i t e q u i v a l e n t t o 0.18 microgram. Our work on the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f the p o i s o n was p u b l i s h e d i n the J o u r n a l of the American C h e m i c a l S o c i e t y i n 1957 (7.8) and i n the Canadian J o u r n a l o f C h e m i s t r y i n 1961 ( 9 ) . Much i n t e r e s t was shown i n the p u r i f i e d p o i s o n and s m a l l amounts were donated t o p h y s i o l o g i s t s , p h a r m a c o l o g i s t s and c h e m i s t s t h r o u g h o u t the w o r l d f o r v a r i o u s s t u d i e s . As a r e s u l t i n 1964 Dr. M. H. Evans (10) of the S h e r r i n g t o n S c h o o l o f P h y s i o l o g y , London and Dr. C. Y. Kao (11) o f the S t a t e U n i v e r s i t y o f New Y o r k d i s c o v e r e d t h a t the p o i s o n caused death by b l o c k i n g s p e c i f i c a l l y the inward sodium i o n c u r r e n t i n nerve and muscle c e l l membranes, an a c t i o n s i m i l a r t o t h a t o f t e t r o d o t o x i n from t h e p u f f e r f i s h . (

S t u d i e s w i t h the P u b l i c H e a l t h S e r v i c e and Food and Administration

Drug

Through a c o o p e r a t i v e arrangement between the Department of Defense and the P u b l i c H e a l t h S e r v i c e i n 1957 I c a r r i e d out s t u d i e s w i t h Ε. T. J e n s e n , Κ. Η. L e w i s , J . Ε. Campbell and E. F. Mc F a r r e n on the use of the p u r i f i e d p o i s o n f o r the e s t a b l i s h m e n t of a s t a n d a r d i z e d mouse assay p r o c e d u r e f o r the p a r a l y t i c p o i s o n i n s h e l l f i s h p r o d u c t s ( 1 2 ) . Many of t h e s e s t u d i e s were c a r r i e d out c o o p e r a t i v e l y w i t h Canadian P u b l i c H e a l t h A g e n c i e s i n v o l v e d w i t h PSP. To e s t a b l i s h a more n e a r l y a c c u r a t e and u n i f o r m mouse assay between l a b o r a t o r i e s , we p r e p a r e d a s o l u t i o n c o n t a i n i n g 100 ug o f the h i g h l y p u r i f i e d p o i s o n ( s a x i t o x i n ) i n 20% e t h a n o l - w a t e r s o l u t i o n a t pH 3 w i t h HC1 as a r e f e r e n c e s t a n d a r d . T h i s s o l u t i o n was d i s p e n s e d i n 5 ml ampoules and f u r n i s h e d f r e e o f charge by the Food and Drug A d m i n i s t r a t i o n t o l a b o r a t o r i e s engaged i n the q u a n t i t a t i v e assay o f s h e l l f i s h products. The use of the r e f e r e n c e s t a n d a r d i n c r e a s e d the c o n f i d e n c e and use o f the mouse assay and h e l p e d p r o t e c t a g a i n s t poisonous s h e l l f i s h g e t t i n g on the commercial m a r k e t s . A l s o , the r e f e r e n c e s t a n d a r d made p o s s i b l e the r e p o r t i n g o f t h e p o i s o n c o n t e n t o f s h e l l f i s h as ug per 100 grams of meats and e s t a b l i s h e d a b a s i s f o r an o f f i c i a l AOAC mouse assay f o r the p o i s o n (12.13). The Food and Drug A d m i n i s t r a t i o n s e t a top l i m i t o f 80 micrograms of p o i s o n per 100 grams of s h e l l f i s h meats as m a r k e t a b l e f o r human consumption. T h i s l i m i t i s w e l l below the 1 t o 4 mg deemed n e c e s s a r y i n foods t o cause s i c k n e s s and d e a t h o f humans. C h e m i c a l S t r u c t u r e o f the P a r a l y t i c P o i s o n s Our e a r l y s t u d i e s on the c h e m i c a l s t r u c t u r e of s a x i t o x i n were hampered by the f a c t t h a t the h i g h l y p o l a r n a t u r e o f the m o l e c u l e p r e v e n t e d good c o n d i t i o n s f o r c r y s t a l l i z a t i o n . A l t h o u g h we had c r y s t a l l i z e d s a x i t o x i n as the h e l i a n t h a t e i n 1957 ( 7 ) , the m o l e c u l e was too l a r g e t o be u s a b l e f o r s t r u c t u r e d e t e r m i n a t i o n by c r y s t a l l o g r a p h e r s i n X - r a y d i f f r a c t i o n s t u d i e s . I n J u l y 1972 I moved t o the U n i v e r s i t y of W i s c o n s i n , Department of Food M i c r o b i o l o g y and T o x i c o l o g y and c o n t i n u e d the r e s e a r c h on s a x i t o x i n w i t h P r o f e s s o r s



9. SCHANTZ


105

F r a n k S t r o n g and H e i n r i c h Schnoes i n the Department o f B i o c h e m i s t r y . I n 1971 Wong e t a l . ( 1 4 ) , w o r k i n g w i t h Rapoport on the s t r u c t u r e by d e g r a d a t i o n of the m o l e c u l e at the U n i v e r s i t y o f C a l i f o r n i a , r e p o r t e d i n the J o u r n a l o f the American C h e m i c a l S o c i e t y a proposed s t r u c t u r e f o r s a x i t o x i n . A l t h o u g h l a t e r shown t o be i n c o r r e c t , i t d i d e s t a b l i s h t h a t s a x i t o x i n e s s e n t i a l l y i s a t e t r a h y d r o p u r i n e d e r i v a t i v e . D u r i n g the y e a r s t h a t f o l l o w e d quest i o n s were r a i s e d by some i n v e s t i g a t o r s r e g a r d i n g the p r o p e r t i e s o f the r e p o r t e d s t r u c t u r e t h a t d i d not seem i n a c c o r d w i t h the p r o p e r t i e s o f s a x i t o x i n . As a r e s u l t we f e l t t h a t more e f f o r t s h o u l d be p l a c e d on o b t a i n i n g a good c r y s t a l l i n e d e r i v a t i v e so t h a t c r y s t a l l o g r a p h i c s t u d i e s c o u l d be used f o r s t r u c t u r e d e t e r m i n a t i o n . At the l a b o r a t o r y a t the U n i v e r s i t y of W i s c o n s i n w i t h S t r o n g , Schnoes and V a r t a n G h a z a r o s s i a n we produced a c r y s t a l l i n e d e r i v a t i v e o f s a x i t o x i n w i t h p-bromobenzene s u l f o n i c a c i d which was v e r y s u i t a b l e f o r c r y s t a l l o g r a p h i c s t u d i e s . Jon C l a r d y and colleagues, then a t Iowa S t a t e U n i v e r s i t y , c a r r i e d out the c r y s t a l l o g r a p h i c s t u d i e s and d e t e r m i n e d the s t r u c t u r e as p r e s e n t e d i n F i g u r e 2A. T h i s s t r u c t u r e f o r s a x i t o x i n (15.) was p r e s e n t e d at the f i r s t C o n f e r e n c e on P o i s o n o u s D i n o f l a g e l l a t e s a t B o s t o n , Mass. i n November 1974 and p u b l i s h e d i n the J o u r n a l o f the American C h e m i c a l S o c i e t y , March, 1975 ( 1 6 ) . On the b a s i s o f t h i s s t r u c t u r e , T a n i n o e t a l . (17.) w i t h K i s h i a t H a r v a r d U n i v e r s i t y s y n t h e s i z e d d , l - s a x i t o x i n and i t was found a c t i v e i n b l o c k i n g sodium c h a n n e l s i n the same manner as n a t u r a l s a x i t o x i n . L a t e r Bordner e t a l . (18) w o r k i n g w i t h Rapoport c o n f i r m e d our s t r u c t u r e f o r s a x i t o x i n w i t h an X-ray a n a l y s i s o f s a x i t o x i n e t h y l hemiketal. A l t h o u g h t h e s e s t u d i e s s o l v e d the s t r u c t u r e f o r s a x i t o x i n , the n e u t r a l or weakly b a s i c p o i s o n from s c a l l o p s , and t h a t produced i n c u l t u r e s o f G. t a m a r e n s i s . was s t i l l a m y s t e r y . I t s poisonous a c t i o n appeared i d e n t i c a l t o s a x i t o x i n ( 1 9 ) , but because o f i t s weakly b a s i c a c t i o n on c a t i o n exchange r e s i n s ( A m b e r l i t e CG-50) and e l e c t r o p h o r e s i s i t was assumed t o be o f d i f f e r e n t s t r u c t u r e . A p p r o x i m a t e l y 85% of the p o i s o n from e a s t c o a s t s c a l l o p s and G. t a m a r e n s i s c u l t u r e s was e l u t e d from the c a t i o n exchange r e s i n above pH 4 and the remainder o f 15% was removed below pH 3. On the o t h e r hand 90 t o 95% of the p o i s o n from the west c o a s t m u s s e l s , b u t t e r clams and a x e n i c G. c a t e n e l l a c u l t u r e was e l u t e d o n l y below pH 3. The e x t r a c t s o f poisonous s c a l l o p v i s c e r a I o b t a i n e d from the Canadians back i n 1947 were s t o r e d i n a c i d s o l u t i o n and moved t o the U n i v e r s i t y of W i s c o n s i n i n 1972 a l o n g w i t h e x t r a c t s o f c u l t u r e d G. t a m a r e n s i s c e l l s . A f t e r s t a n d i n g i n a c i d s o l u t i o n f o r o v e r 20 y e a r s we found t h a t the p o i s o n now chromatographed on the c a t i o n exchange r e s i n s s i m i l a r t o s a x i t o x i n ( 2 0 ) . However the major poison i n extracts of f r e s h l y c o l l e c t e d s c a l l o p e n t r a i l s obtained from the Canadian Department o f Human S e r v i c e s i n 1978 was e l u t e d from the c a t i o n exchange r e s i n s above pH 4 i n l i n e w i t h the o r i g i n a l observations. I t was t h e r e f o r e assumed t h a t some c o n v e r s i o n took p l a c e i n the m o l e c u l e t h a t l e d t o a more b a s i c s t r u c t u r e . S h o r t l y a f t e r we were s u c c e s s f u l i n o b t a i n i n g the s t r u c t u r e o f s a x i t o x i n , S h i m i z u and c o l l e a g u e s a t the U n i v e r s i t y of Rhode I s l a n d p u r i f i e d the p o i s o n from e a s t c o a s t s c a l l o p s and r e p o r t e d t h a t the s t r u c t u r e o f the major p o i s o n from s c a l l o p s and G. t a m a r e n s i s . based on NMR s t u d i e s , was 11-hydroxy s a x i t o x i n which he c a l l e d



106

S E A F O O D TOXINS

g o n y a u t o x i n I I and i t s epimer g o n y a u t o x i n I I I ( 2 1 ) . T h i s work e s t a b l i s h e d t h e s i m i l a r i t y o f G. t a m a r e n s i s p o i s o n t o s a x i t o x i n but i n our minds i t was q u e s t i o n a b l e i f the proposed s t r u c t u r e would be weakly b a s i c i n c h a r a c t e r and i n l i n e w i t h the p r o p e r t i e s o f G. t a m a r e n s i s p o i s o n . Gregory Boyer i n our l a b o r a t o r y a t t h e U n i v e r s i t y o f W i s c o n s i n had a l s o p u r i f i e d the p o i s o n from f r e s h l y c o l l e c t e d s c a l l o p s on B i o - g e l P-2 and a c i d i c AI2O3 columns and upon e l e m e n t a l a n a l y s e s found, i n a d d i t i o n t o t h e elements i n s a x i t o x i n , one mole of s u l f u r per mole o f p o i s o n i n the form o f s u l f a t e . NMR s t u d i e s showed t h i s group i n the 11 p o s i t i o n making t h e c o r r e c t s t r u c t u r e f o r the major p o i s o n from s c a l l o p s , and a l s o f o r the p o i s o n from G. t a m a r e n s i s . as t h e s u l f o n i c a c i d e s t e r o f 11-hydroxy s a x i t o x i n as shown i n F i g u r e 2B. T h i s s t r u c t u r e was i n l i n e w i t h the weakly b a s i c p r o p e r t i e s o f G. t a m a r e n s i s p o i s o n and was p u b l i s h e d by Boyer e t a l . i n 1978 i n the J o u r n a l o f t h e C h e m i c a l S o c i e t y (London) C h e m i c a l Communications ( 2 2 ) . S h i m i z u i n 1978 p u b l i s h e d the s t r u c t u r e o f an i n t e r e s t i n g s a x i t o x i n d e r i v a t i v e , 1 - N - h y d r o x y s a x i t o x i n , which he i s o l a t e d from s c a l l o p s and c a l l e d n e o s a x i t o x i n (23) as shown i n F i g u r e 2C. S u b s e q u e n t l y Boyer i n 1980 p u b l i s h e d the s t r u c t u r e o f neo-11h y d r o x y s a x i t o x i n s u l f a t e and i t s epimer ( 2 4 ) . Some v e r y i n t e r e s t i n g d e r i v a t i v e s o f s a x i t o x i n r e c e n t l y have been i s o l a t e d by H a l l and c o l l e a g u e s o f the U n i v e r s i t y o f A l a s k a from d i n o f l a g e l l a t e s o f the genus P r o t o K o n y a u l a x found a l o n g t h e c o a s t o f A l a s k a (25). The s p e c t r o s c o p i c s t u d i e s performed i n c o l l a b o r a t i o n w i t h H. K. Schnoes and h i s a s s o c i a t e s a t the U n i v e r s i t y of W i s c o n s i n suggested t h a t two o f t h e s e were c a r b a m o y l - N - s u l f o l l a - h y d r o x y s a x i t o x i n s u l f a t e and i t s 11B epimer, as shown i n F i g u r e 2D. X-ray a n a l y s i s by S. D. D a r l i n g a t the U n i v e r s i t y o f Akron c o n f i r m e d t h e l a t t e r s t r u c t u r e ( 2 6 ) . These compounds were n e u t r a l i n c h a r a c t e r . I t s p r o p e r t i e s w i l l be d e s c r i b e d i n more d e t a i l by o t h e r speakers a t t h i s symposium. The r e p o r t o f t h e N - s u l f o c a r b a m o y l group i s t h e f i r s t r e p o r t e d i n a n a t u r a l p r o d u c t and the a t t e n u a t i o n o f t o x i c i t y a s s o c i a t e d w i t h s u l f o n a t i o n o f t h e carbamoyl group i n t r o d u c e s a new a s p e c t o f s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s i n t h i s group o f n e u r o t o x i n s . S a x i t o x i n has t h e h i g h e s t s p e c i f i c t o x i c i t y o f any o f t h e s e p o i s o n s i s o l a t e d thus f a r . G h a z a r o s s i a n (27.) and G h a z a r o s s i a n e t a l . (28) had found t h a t removal o f the carbamoyl group by t r e a t m e n t o f s a x i t o x i n w i t h 3 Ν HC1 a t 100*C reduced t h e t o x i c i t y t o about 60%. As s t a t e d p r e v i o u s l y i n our e a r l i e r work (8.27) and t h a t o f Koehn e t a l . ( 2 9 ) , r e d u c t i o n of the h y d r a t e d ketone a t p o s i t i o n 12 t o a monohydroxy group reduces the t o x i c i t y t o around 1% o f the o r i g i n a l . As mentioned above we o b t a i n e d most o f the p o i s o n f o r our c h e m i c a l and b i o l o g i c a l work from the A l a s k a b u t t e r clam. However our s e a r c h f o r a d i n o f l a g e l l a t e o r any o t h e r poisonous organism i n t h e water as a s o u r c e o f t h e p o i s o n i n the clams was never d e f i n i t e l y a c c o m p l i s h e d . I n 1948, and i n some y e a r s f o l l o w i n g , a l o n g w i t h a s u r v e y f o r t o x i c b u t t e r clams i n S o u t h e a s t e r n A l a s k a by t h e s t a f f a t t h e F i s h e r y P r o d u c t s Research L a b o r a t o r y a t K e t c h i k a n , I c o l l e c t e d p l a n k t o n i n areas where t h e clams were found to be t o x i c . Mouse assays o f the p l a n k t o n showed no t o x i c i t y . M i c r o s c o p i c e x a m i n a t i o n o f the water i n t h e s e areas showed heavy growth o f m i c r o s c o p i c p l a n k t o n but l i t t l e o r no e v i d e n c e o f



9. SCHANTZ


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Poison

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A. Saxitoxin

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B. 11-hydroxysaxitoxin sulfate

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C. Neosaxitoxin

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D. Carbamoyl-N-sulfo11-hydroxysaxitoxin sulfate

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3

References

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Boyer et a l 22 Shimizu et a l 23

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Including the u and 0 epimers of these poisons at position 11 there are a t o t a l of 12 p a r a l y t i c s h e l l f i s h poisons. Figure 2. Structure of p a r a l y t i c s h e l l f i s h poisons



108

SEAFOOD TOXINS

d i n o f l a g e l l a t e s . A l s o mussels ( M y t i l u s e d u l u s ) showed l i t t l e o r no t o x i c i t y . The p l a n k t o n were c o l l e c t e d i n No. 25 s i l k n e t s . The survey was made i n a 5 0 - f o o t r e t i r e d navy boat c a l l e d t h e ••Researcher** a s s i g n e d t o t h e K e t c h i k a n L a b o r a t o r y and had l i v i n g space f o r e i g h t p e r s o n s . We c a r r i e d on board l a b o r a t o r y equipment i n c l u d i n g mice f o r assays o f clams and a microscope f o r e x a m i n a t i o n o f t h e w a t e r from t h e K e t c h i k a n a r e a t o t h e a r e a around Juneau. D u r i n g some o f o u r work a t t h e K e t c h i k a n l a b o r a t o r y we t r a n s p l a n t e d t o x i c b u t t e r clams i n areas where t h e i n d i g e n o u s clams were n o t t o x i c and a l s o t r a n s p l a n t e d nonpoisonous clams i n areas where t h e i n d i g e n o u s clams were t o x i c . Only a f t e r one y e a r was t h e t o x i c i t y o f t h e t r a n s p l a n t e d t o x i c clams s i g n i f i c a n t l y l o w e r e d . The nonpoisonous clams a c q u i r e d t o x i c i t y e q u i v a l e n t t o t h e i n d i g e n o u s clams i n about two months. I n 1949 L u c i l e F o s t e r and Hermann Sommer, i n c o o p e r a t i o n w i t h t h e F i s h e r y P r o d u c t s Research L a b o r a t o r y c o l l e c t e d p l a n k t o n i n these waters throughout t h e summer and i d e n t i f i e d G. c a t e n e l l a but o n l y i n e x c e e d i n g l y low c o n c e n t r a t i o n s i n areas where they d i d o c c u r . They showed no p a r t i c u l a r p a t t e r n s o f o c c u r r e n c e t h a t c o i n c i d e d w i t h t o x i c clam beds. E x a m i n a t i o n o f d i n o f l a g e l l a t e blooms t h a t o c c u r r e d d u r i n g t h i s p e r i o d c o n s i s t e d m a i n l y o f s p e c i e s o f N o c t i l u c a and were n o n t o x i c t o mice. From t h e s e f i n d i n g s we b e l i e v e d t h a t t h e b u t t e r clams were t o x i c , i n c o n t r a s t t o t h e low t o x i c i t y o f t h e m u s s e l s , because o f t h e slow r a t e a t which t h e clams d e s t r o y e d o r e x c r e t e d t h e p o i s o n from t h e s i p h o n whereas t h e mussels e x c r e t e d o r d e s t r o y e d t h e p o i s o n from t h e hepatopancreas a t a much f a s t e r r a t e . Although our surveys and t h o s e o f L u c i l e F o s t e r i n p a r t i c u l a r showed o n l y a minute presence o f G. c a t e n e l l a a l o n g w i t h t h e f a c t t h a t t h e p r o p e r t i e s o f t h e p o i s o n from t h e clam siphons and from t h e C a l i f o r n i a sea mussels appeared v e r y s i m i l a r , i t seemed l o g i c a l t o p o s t u l a t e t h a t a poisonous d i n o f l a g e l l a t e was t h e source o f t h e p o i s o n . A r e p o r t o f t h i s work was p u b l i s h e d i n t h e J o u r n a l o f P r o t o z o o l o g y i n 1964 ( 3 0 ) . I t i s i n t e r e s t i n g t o o , i n l i g h t of the recent f i n d i n g s of p o i sonous d i n o f l a g e l l a t e s by H a l l , R e i c h a r d t and Neve a t t h e U n i v e r s i t y o f A l a s k a , t h a t b u t t e r clam siphons c o l l e c t e d i n s o u t h e a s t e r n A l a s k a from 1948 t o 1958 f o r our o r i g i n a l work c o n t a i n e d o n l y s a x i t o x i n . C o u l d i t be t h a t t h e b u t t e r clams b i n d o n l y t h e s t r o n g l y b a s i c p o i s o n s i n t h e s i p h o n and a l l o w t h e weakly b a s i c and n e u t r a l ones t o pass through? I f t h e r e i s a reason f o r such s e l e c t i o n , perhaps i t i s t o ward o f f p r e d a t o r s . However, t h e main f u n c t i o n , i n my e s t i m a t i o n , f o r t h e presence o f t h e p o i s o n i n s h e l l f i s h i s t h a t i t has been a f a c t o r i n warding o f f human predators. Work on t h e c h e m i c a l s t r u c t u r e and p r o p e r t i e s o f t h e p o i s o n s from A l a s k a b u t t e r clam s i p h o n s , t h e hepatopancreas o f C a l i f o r n i a m u s s e l s , and a x e n i c c u l t u r e s o f G. c a t e n e l l a c a r r i e d on i n t h e B i o l o g i c a l L a b o r a t o r i e s and w i t h Rapoport a t t h e U n i v e r s i t y o f C a l i f o r n i a d e f i n i t e l y e s t a b l i s h e d t h a t t h e sea m u s s e l d i d not a l t e r t h e p o i s o n o b t a i n e d from G. c a t e n e l l a . The p o i s o n from a l l t h r e e sources had i d e n t i c a l s t r u c t u r e s ( 3 1 ) . The s i t u a t i o n w i t h t h e A l a s k a b u t t e r clam may be d i f f e r e n t however i n l i g h t o f t h e d i f f e r ent s a x i t o x i n d e r i v a t i v e s r e c e n t l y found i n t h e d i n o f l a g e l l a t e s i n t h a t r e g i o n . Perhaps t h e clam c o n v e r t s t h e s u l f o and s u l f a t e d e r i v a t i v e s t o t h e more t o x i c s a x i t o x i n .



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109

The a b s o r p t i o n o f the p o i s o n by the hepatopancreas o f t h e m u s s e l and the s i p h o n o f the clam i s an i n t e r e s t i n g and u n e x p l a i n e d phenomenon. S e v e r a l y e a r s ago ( u n p u b l i s h e d ) I p l a c e d ground o r s l i c e d hepatopancreas o f mussels i n s o l u t i o n s o f s a x i t o x i n and found no s i g n i f i c a n t a b s o r p t i o n o f the p o i s o n and no more than was adsorbed by mouse l i v e r s l i c e s . Under normal c o n d i t i o n s i n t h e n a t u r a l s t a t e t h e hepatopancreas o f a s m a l l m u s s e l w e i g h i n g a few grams would accumulate up t o 10,000 MU o r more from the d i n o f l a g e l late. I t t h e r e f o r e seemed t o me t h a t the i n t a c t l i v e hepatopanc r e a s i n v o l v e d something o t h e r than j u s t a p h y s i c a l a d s o r p t i o n . P r i c e and Lee, however, a t Oregon S t a t e U n i v e r s i t y have p r e s e n t e d e v i d e n c e t h a t some o f t h e m e l a n i n pigments i n the s i p h o n o f t h e clam may be i n v o l v e d i n the a b s o r p t i o n o f the p o i s o n ( 3 2 ) . I n c o n c l u s i o n I must say p e r s o n a l l y t h a t my 40 y e a r s o f work on t h e p a r a l y t i c s h e l l f i s h p o i s o n s has been a r e w a r d i n g e x p e r i e n c e . The o p p o r t u n i t y o f w o r k i n g w i t h many c o l l e a g u e s throughout the y e a r s has l e d t o the accomplishment o f the p u r i f i c a t i o n o f t h e p a r a l y t i c p o i s o n and the d e t e r m i n a t i o n o f i t s s t r u c t u r e . The p u r i f i e d p o i s o n , s a x i t o x i n , has become a v a l u a b l e t o o l f o r nerve t r a n s m i s s i o n s t u d i e s f o r p h y s i o l o g i s t s , and p h a r m a c o l o g i s t s i n v a r i o u s l a b o r a t o r i e s throughout the w o r l d . A l s o i t has s e r v e d as a r e f e r e n c e s t a n d a r d i n the AOAC O f f i c i a l mouse assay used by t h e Food and Drug A d m i n i s t r a t i o n and o t h e r c o n t r o l a g e n c i e s i n t h e i r work on making s h e l l f i s h s a f e f o r human consumption. There a r e problems ahead t h a t we hope may be s o l v e d . One i s a s i m p l e t e s t procedure f o r the p o i s o n i n s h e l l f i s h t h a t c o u l d be c a r r i e d out r a p i d l y as a f i e l d t e s t by c o n t r o l a g e n c i e s and commercial f i s h e r m e n t o a v o i d c o l l e c t i n g poisonous s h e l l f i s h . Several l a b o r a t o r i e s are w o r k i n g on b e t t e r a n a l y t i c a l methods f o r t h e p o i s o n . Bates and Rapoport (33) a t the U n i v e r s i t y o f C a l i f o r n i a and Shoptaugh e t a l . (34) t the U n i v e r s i t y of New Hampshire have developed t e c h n i q u e s f o r the d e t e r m i n a t i o n o f s a x i t o x i n more s e n s i t i v e and s p e c i f i c than the mouse t e s t but the procedures and equipment r e q u i r e d make them i m p r a c t i c a l f o r f i e l d use. Other methods based on i m m u n o l o g i c a l t e c h n i q u e s f o r the development o f s i m p l e r methods a r e b e i n g worked on by Chu e t a l . (35) a t t h e U n i v e r s i t y o f W i s c o n s i n and G u i r e e t a l . ( r e p o r t a t t h i s symposium) a t the U n i v e r s i t y o f M i n n e s o t a . The s i m p l e mouse t e s t , a l t h o u g h not s p e c i f i c f o r the p o i s o n s , remains the method o f c h o i c e used by t h e Food and Drug A d m i n i s t r a t i o n . Another problem i s the d e v e l o p ment o f an a n t i d o t e f o r the p o i s o n t o be used i n c o n n e c t i o n w i t h a r t i f i c i a l r e s p i r a t i o n f o r the t r e a t m e n t o f p o i s o n e d p e r s o n s . I n t h i s r e s p e c t the Klamath I n d i a n s o f N o r t h e r n C a l i f o r n i a a r e r e p o r t e d (36) t o have used the sap from the sugar p i n e t r e e as t r e a t m e n t o f s i c k n e s s from e a t i n g mussels a t c e r t a i n times o f t h e y e a r . I n v e s t i g a t i o n s on the use o f sap from v a r i o u s p a r t s o f t h e sugar p i n e o b t a i n e d through the c o u r t e s y o f the F o r e s t r y Department o f the U n i v e r s i t y o f C a l i f o r n i a B e r k e l e y , d i d not r e l i e v e the s i g n s of s h e l l f i s h p o i s o n i n g i n mice. Funds f o r the s u p p o r t o f the work t h a t I was i n v o l v e d i n came from The Army B i o l o g i c a l L a b o r a t o r i e s ; the P u b l i c H e a l t h S e r v i c e ; the Food Research I n s t i t u t e , U n i v e r s i t y o f W i s c o n s i n ; and g r a n t s from the N a t i o n a l I n s t i t u t e s o f H e a l t h . a



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L i t e r a t u r e Cited 1. Sommer, H.; Whedon, W. F.; Kofoid, C. Α.; Stohler, R. ΑΜΑ Arch. P a t h o l . 1937, 24, 537. 2. Sommer, H.; Meyer, K. F. ΑΜΑ Arch. Pathol. 1938, 24, 560. 3. Needler, A. B. J . F i s h . Res. Board Canada 1949, 7, 490. 4. Prakash, A. J . Fish Res. Board Canada 1963, 20, 983. 5. Schantz, E . J. Ann. Ν. Y . Acad. Sci. 1960, 90, 843. 6. Sommer, H.; Monnier, R. P.; R i e g e l , B . ; Stanger, D. W.; Mold, J. D . ; Wikholm, D. M.; Kiralis, E. S. J . Am. Chem. Soc. 1948, 70, 1015. 7. Schantz, E . J.; Mold, J. D . ; Stanger, W. D . ; Shavel, J.; Riel, F. J.; Bowden, J. P . ; Lynch, J. M . ; Wyler, R. S.; R i e g e l , B.; Sommer, H. J . Am. Chem. Soc. 1957, 79, 5230. 8. Mold, J. D . ; Bowden, J. P . ; Stanger, D. W.; Maurer, J. E.; Lynch, J. M . ; Wyler, R. S.; Schantz, E. J.; R i e g e l , B. J . Am. Chem. Soc. 1957, 79, 5235. 9. Schantz, E. J.; Mold, J. D . ; Howard, W. L.; Bowden, J. P . ; Stanger, O. W.; Lynch, J. M . ; Wintersteiner, O. P, Dutcher, J . D.; Walters, D. R.; R i e g e l , B. Can. J. Chem. 1961, 39, 2117. 10. Evans, M. H. B r i t . Pharmacol. 1964, 22, 478. 11. Kao, C. Y.; Nishiyama, A. J . P h y s i o l . (London) 1965, 180, 50. 12. Schantz, E. J.; McFarren, E. F.; Schafer, M. L.; Lewis, Κ. H. J. Assoc. Off. Anal. Chem. 1958, 41, 160. 13. "Manual of Recommended Practice for Sanitary Control of the S h e l l f i s h Industry Part 1, U. S. Public Health Service, 1959. 14. Wong, J. L.; O e s t e r l i n , R . ; Rapoport, H. J . Am. Chem. Soc. 1971, 93, 7344. 15. Schantz, E . J.; Ghazarossian, V . E.; Schnoes, H. K.; Strong, F . M . ; Springer, J. P . ; Pezzanite, J. O.; Clardy, J. Proc. First International Conf. Toxic D i n o f l a g e l l a t e Blooms. 1974, p. 267. 16. Schantz, E. J.; Ghazarossian, V . E.; Schnoes, H. K.; Strong, F . M . ; Springer, J. P . ; Pezzanite, J . O.; Clardy, J. J . Am. Chem. Soc. 1975, 97, 1238. 17. Tanino, H.; Nakata, T . ; K i s h i , Y . J . Am. Chem. Soc. 1977, 99, 2818. 18. Bordner, J.; Thiessen, W. E.; Bates, Η. Α.; Rapoport, H. J . Am. Chem. Soc. 1975, 97, 6008. 19. Narahashi, T . ; Browdick, M. S.; Schantz, E. J. Environmental Letters 1975, 9, 239. 20. Ghazarossian, V . E.; Schantz, E. J.; Schnoes, H. K.; Strong, F . M. Biochem. Biophys. Res. Commun. 1975, 59, 1219. 21. Shimizu, T . ; Buckley, L . J.; Alam, M . ; Oshima, Y.; F a l l o n , W. E.; Kassai, H.; Miura, I.; G u l l o , V. P.; Nakanishi, K. J . Am. Chem. Soc. 1976, 98, 5414. 22. Boyer, G. L.; Schantz, E. J.; Schnoes, H. K. J . Chem. Soc. Chem. Commun. 1978, 889. 23. Shimizu, Y.; Hsu, C.; F a l l o n , W. E.; Oshima, Y.; Miura, I.; Nakanishi, K. J . Am. Chem. Soc. 1978, 100, 6791. 24. Boyer, G. L . Ph.D. Thesis, U n i v e r s i t y of Wisconsin, Madison, 1980. 25. Hall, S.; Reichardt, P. B.; Neve, R. A. Biochem. Biophys. Res. Commun. 1980, 97, 649. "



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26. Wichmann, C., Niemczura, W. P.; Schnoes, H. K.; Hall, S . ; Reichardt, P. Β . ; D a r l i n g , S. D. J . Am. Chem. Soc. 1981, 103, 6977. 27. Ghazarossian, V . E. Ph.D. Thesis, U n i v e r s i t y of Wisconsin, Madison, 1977. 28. Ghazarossian, V . E.; Schantz, E. J.; Schnoes, Η. K . ; Strong, F . M. Biochem. Biophys. Res. Commun. 1976, 68, 776. 29. Koehn, F. E.; Ghazarossian, V. E.; Schantz, E. J.; Schnoes, H. K . ; Strong, F. M. Bioorg. Chem. 1981, 10, 412. 30. Schantz, E. J.; Magnuson, H. W. J . Protozool. 1965, 11, 239. 31. Schantz, E. J.; Lynch, J. M . ; Vayvada, G . ; Matsumoto, K.; Rapoport, H. Biochemistry 1966, 5, 1191. 32. P r i c e , R. J.; Lee, J. S. J . F i s h . Res. Bd. Can. 1972, 29, 1659. 33. Bates, H. A.; Rapoport, H. J . A g r i . Food Chem. 1975, 23, 237. 34. Shoptaugh, N . H.; Ikawa, M . ; F o x a l l , T. L.; Sasner, J. J. "A Fluorometric Technique for the Detection and Determination of P a r a l y t i c S h e l l f i s h Poisons"; Carmichael, W. W., E d . ; The Water Environment; Plenum: New York, 1981; p. 427. 35. Chu, F. S.; Fan, T. S. L.; Li, S. W. 97th Ann. Meeting Assoc. Off. Anal. Chem. 1983, p. 77, Abst. 194. 36. Thompson, L . "To the American Indian" 1916 Cummins P r i n t Shop, Eurkea, Calif., p. 28. 37. Schantz, E. J. Seafood Toxicants. Toxicants Occurring Naturally i n Foods. National Academy of Sciences, Washington, D.C. 1983; p. 424. RECEIVED April 23, 1984


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