19 Diarrhetic Shellfish Poisoning 1
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TAKESHI YASUMOTO , MICHIO MURATA , YASUKATSU OSHIMA , GAYLE K. MATSUMOTO , and JON CLARDY 2
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Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Tsutsumidori, Sendai 980, Japan Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY 14853
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General aspects of diarrhetic shellfish poisoning i n cluding epidemiology, geographical distribution, magnitude of impacts to public health and shellfish i n dustries are described. Succeeding to the previous identification of Dinophysis fortii as the origin of shellfish toxins and determination of dinophysistoxin -1 as 35(S)-methyl okadaic acid, isolation and structural determination of dinophysistoxin-3 and two novel polyether lactones named pectenotoxin-1 and -2, and identification of Dinophysis acuminata as the probable source of okadaic acid are newly reported. Diarrhetic shellfish poisoning (DSP) is a term proposed by the authors to a shellfish poisoning distinctly different from the paralytic shellfish poisoning (PSP) in both symptomatology and etiology. Unlike PSP, the predominant human symptoms of DSP are gastrointestinal disturbances and no fatal cases have been reported (1). Nevertheless, the high morbidity rate and worldwide distribution of DSP make i t a serious threat to both public health and shellfish industries. In a early stage of investigation we established that the origin of the shellfish toxins is the dinoglagellate Dinophysis fortii (2). Toxins named dinophysistoxins (DTX ) were found to be structurally related to okadaic acid, a C33 polyether fatty acid first isolated from sponges (3) and then from the dinoflagellate Prorocentrum lima (4). A polyether toxin isolated from mussels and coded dinophysistoxin-1 (DTX^) was identified as 35(5)-methyl okadaic acid in the previous study (5). Subsequent study revealed coexistence of numerous toxins with either similar or entirely different skeletons. The purpose of this paper is to present the general aspects of this relatively new type of shellfish poisoning and to report the isolation and structural determination of dinophysistoxin-3, and two novel polyether lactones named pectenotoxin-1 and -2. Detection of okadaic acid and isolation of pectenotoxin-3,-4, g
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Current address: Suntory Institute For Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka618,Japan
0097-6156/84/0262-0207$06.00/0 © 1984 American Chemical Society
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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and -5 as well as i d e n t i f i c a t i o n of Dinophysis acuminata probable source of okadaic acid are also described.
as the
Materials and Methods
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Materials. The following s h e l l f i s h specimens were collected for toxin analysis from the northeastern part of Honshu, Japan, during the i n f e s t a t i o n period: the mussel Mytilus edulis at Miyagi Prefecture, the scallop Patinopeoten yessoensis at Aomori Prefecture, the short-necked clam Tapes japonioa at Fukushima Prefecture, and Gomphina melanaegis at Ibaraki Prefecture. Mouse bioassay. The digestive glands of s h e l l f i s h were extracted thrice with acetone at room temperature. After removal of acetone by evaporation, the aqueous suspension was extracted thrice with d i ethyl ether, the combined ether solution was backwashed twice with small portions of water and evaporated. The residue was suspended i n 1% Tween 60 solution and s e r i a l l y diluted suspensions were i n j e c ted intraperitoneally into mice weighing 17-20 g each. The mice were observed for 24 hr and the minimum amount of toxin required to k i l l a mouse at 24 hr was defined as one mouse unit (1). Thin layer chromatography. Thin layer chromatography (TLC) was carr i e d out on precoated S i l i c a gel 60 plates (Merck) with the solvent system benzene-acetone-methanol-6N acetic acid (150:80:19:1). Toxins were detected by heating the plates after spraying 50% s u l f u r i c acid. Instruments. NMR spectra were measured with FX-100, FX-400 (JEOL) and NT-360 (Nicole) instruments, mass spectra with a Hitachi M-80 mass spectrometer, IR spectra with a JASCO A-202 spectrometer, and UV spectra with a Hitachi 124 spectrophotometer. Gas chromatography. Gas chromatographic analyses were conducted on a Hitachi 163 instrument equipped with hydrogen flame i o n i z a t i o n detectors. Okadaic acid and dinophysistoxin-1 were t r i m e t h y l s i l y lated with T r i S i l "Z" either intact or after d e r i v a t i z a t i o n with diazomethane into methyl esters. A glass column (3 x 800 mm) packed with 2% OV-101 on 60/80 mesh Uniport HP was used for analysis of okadaic acid and dinophysistoxin-1. The column temperature was maintained at 315°C and nitrogen flow rate at 30 ml/min. For fatty acid analyses a glass column (3 x 2000 mm) packed with 10% DEGS on Chromosorb WAW DMCS 60/80 mesh was used. Column temperature was kept at 165°C and the nitrogen flow rate at 30 ml/min. The r e f e r ence fatty acids were purchased from Wako Pure Chemicals. Isolation of toxins. The digestive glands of s h e l l f i s h were extracted with acetone at room temperature. After removal of the acetone by evaporation, the aqueous suspension was extracted with d i e t h y l ether. The ether soluble residue was successively chromatographed twice over s i l i c i c acid columns with following solvents: benzene to benzene-methanol (9:1), and d i e t h y l ether to d i e t h y l ether-methanol (1:1). To avoid degradation of dinophysistoxin-3 by contaminant acid, the s i l i c i c acid was washed with d i l u t e sodium hydroxide solution and then with water p r i o r to a c t i v a t i o n at 110°C. Toxic r e s i due obtained i n the second eluates was separated into two fractions
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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19.
YASUMOTOETAL.
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by passing through Sephadex LH-20 column (28 x 100 mm) using benzenemethanol (1:1) solution. Fractions containing dinophysistoxin-3 (# 24-26, 10 ml each) and those containing other toxins (# 28-31) were combined respectively. Dinophysistoxin-3 was further p u r i f i e d on LiChroprep RP-8 column with methanol-water (20:1) and then on uBondapak C^g column with methanol-water (93:7). The toxic f r a c t i o n containing dinophysistoxin-1 and pectenotoxins was chromatographed f i r s t over a Lobar column (LiChroprep RP-8, sizeB) using methanolwater (17:3) as solvent and next on an alumina column (Woelm, basic, a c t i v i t y III) with following solvents: chloroform, chloroform-methan— o l (9:1), chloroform-methanol (1:1), methanol, and methanol-1% ammonium hydroxide (1:1). Pectenotoxins recovered from the f i r s t two eluates were treated on LiChroprep RP-8 column with a c e t o n i t r i l methanol-water (2:2:3). F i n a l separation of pectenotoxins was achieved on a Develosil column (4 x 250 mm, Nomura Kagaku) using dichloromethan-methanol (98:2) as solvent. Dinophysistoxin-1 was eluted from the alumina column with aqueous methanol containing ammonium hydroxide. Further p u r i f i c a t i o n was achieved by repeating chromatography on LiChroprep RP-8 column (5 x 1000 mm) using acetonitrile-methanolwater (3:2:2) as solvent. Separation and p u r i f i c a t i o n of toxins were monitored by mouse bioassay, TLC, and a UV-spectromonitor at wave length 220 nm for dinophysistoxin-1 and -3, and at 235 nm for pectenotoxins. P u r i f i c a t i o n of okadaic acid from mussel specimen was carried out i n e s s e n t i a l l y the same manner as employed for p u r i f i c a t i o n of dinophysistoxin-1. Results General outline of DSP. During the period of 1976-1982, more than 1,300 people were o f f i c i a l l y diagnosed as DSP cases i n Japan. Frequency of signes and symptoms i n patients were as follows: diarrhea (92%), nausea (80%), vomiting (79%), abdominal pain (53%), and c h i l l (10%). Incubation period ranged from 30 min to several hr but seldom exceeded 12 hr. Around 70% of the patients developed symptoms within 4 hr. Suffering may l a s t for 3 days, i n severe cases, but leaves no a f t e r - e f f e c t . Causative s h e l l f i s h e s were the mussels Mytilus edulis and M, ooruscum, the scallops Patinopeoten yessoensis and Chlamys nipponensis akazara, and the short-necked clams Tapes japoniaa and Gomphina melanaegis. The method of cooking did not a l t e r the t o x i c i t y of the causative s h e l l f i s h e s but i n t o x i c a t i o n could be avoided i f the digestive glands were eliminated beforehand. The minimum amount of toxin to induce symptoms i n an adult was estimated to be 12 mouse units from the analyses of the l e f t overs of the patient meals. The maximum allowance l e v e l of toxins i n s h e l l f i s h meat was set by the government regulation at 5 mouse units/100 g meat. Infestation period ranges from A p r i l to September and the highest t o x i c i t y of s h e l l f i s h i s overved during May to August, though i t may vary l o c a l l y . Heavy i n f e s t a t i o n of s h e l l f i s h normally occurs i n northeastern part of Japan. In southwestern areas s h e l l f i s h t o x i c i t y i s low and decreases rapidly. The causative organism which transfer toxins to s h e l l f i s h was i d e n t i f i e d as Dinophysis f o r t i i (2). Mussels and scallops may become toxic beyond the regulation l e v e l i n the presence of this dinoflagellate at c e l l density of 200/1, or even lower (2). Gas chromatographic analysis confirmed, for the f i r s t
Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.
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time, the presence of okadaic acid i n mussels collected i n A p r i l when D. f o r t i i was scarce (3 cells/1) but Dinophysis acuminata was r e l a t i v e l y abundant (500 c e l l s / 1 ) , indicating that D. acuminata was also implicated i n DSP. Properties of toxins. Dinophysistoxin-1 was isolated as a white amorphous s o l i d ; m.p. 134°C; +28 (c 0.046, chloroform); Rf i n TLC 0.42; minimum l e t h a l dose to mouse 160 jig/kg ( i . p . ) . EI mass spectrum gave a dehydrated ion peak at m/z 800, pointing to a composition C45H630i2- Overall features of both PMR and CMR spectra of dinophysistoxin-1 closely resembled those of okadaic acid (I,) except the presence of an a d d i t i o n a l methyl i n dinophysistoxin-1. Comparison of the spectra of okadaic acid and dinophysistoxin-1 and supplemental spin-spin decoupling measurements enabled us to assign dinophysistoxin-1 to 35(5)-methyl okadaic acid (II) (5). A component tentatively named dinophysistoxin-2 was s l i g h t l y more polar than dinophysistoxin-1 but i t s characterization was unsuccessful due to the extreme smallness of the sample s i z e . Pure dinophysistoxin-3 was obtained from the scallop digestive gland as a colorless s o l i d ; Rf i n TLC 0.57; minimum l e t h a l dose to mouse 500 ^g/kg ( i . p . ) ; no UV absorption maximum above 220 nm. The PMR spectrum of dinophysistoxin-3 resembled that of dinophysistoxin -1 but contained a d d i t i o n a l signals assignable to a f a t t y acid moiety;6 0.88 (3H, t, terminal methyl), £ 1.25 {ca. 25H, br s., methylenes), S 1.98 {ca. 2H, m, methylene neighboring a double bond), 6 2.20 (2H, dd, methylene adjacent to an ester), and S 5.35 {ca, 5H, o l e f i n i c protons) ppm. Gas chromatographic analyses of hydrolysis products confirmed the presence of dinophysistoxin-1 and following fatty acids: C i 4 (13%), C (29%), C i 3 (3%), C 4u> (9%), 20:5*>3 (23%), and C22:6*»>3 (23%). Integration of proton signals suggested that dinophysistoxin-3 i s a mixture of dinophysistoxin-1 derivatives having one of the above f a t t y acids i n an ester linkage. Another d i s t i n c t i o n between PMR spectra of dinophysistoxin-1 and -3 was that one oxymethine proton of the former {£ 3.40) was deshielded by 1.36 ppm {S 4.76) i n the l a t t e r . This proton was assigned to 7-H because i t s signal shape and coupling constants (dd, 11.8, 4.2 Hz) was compatible with the a x i a l - a x i a l and a x i a l - e q u a t o r i a l couplings of 7-H to 6-2H. Signals at S 4.10 (br d, 10 Hz) and S 4.07 (t, 10 Hz) assignable to 24-H and 27-H respectively (3) remained unchanged. Thus, attachment of a fatty acid at C24-OH or C27-0H was ruled out. Acylation of C2-0H was also denied because the resonance of 39-CH3 (
