Agric. Biol. Chem.

, 50 (ll), 2853-2857, 1986 2853

Okadaic Acid as the Causative Toxin of Diarrhetic Shellfish

Poisoning in Europe
Masanori Kumagai, Toshihiko Yanagi, Michio Murata,
Takeshi Yasumoto,1 Marie Kat,* Patrick Lassus,**
and Jose A. Rodriguez-Vazquez***
Faculty of Agriculture, Tohoku University, Tsutsumidori,
Amemiyamachi, Sendai 980, Japan
* Netherlands Institute for Fishery Investigations, Haringkade 1,
P.O. Box 68, 1970 AB Ymuiden, The Netherlands
**IFREMER,Centre de Nantes, B.P. 1049,
44037 Nantes Cedex, France
***Department of Analytical Chemistry, Colegio Universitad de Vigo,
Apartado 874, Vigo, Spain
Received June 2, 1986

The toxic principle in mussels collected in France, Sweden, the Netherlands, and Spain that
caused diarrhetic shellfish poisoning was identified to be okadaic acid by its chromatographic
properties and spectral data.

Diarrhetic shellfish poisoning (DSP) was In this paper we report the isolation and
first described in Japan as a new kind of a identification of okadaic acid as the toxic
seafood disease,1~3) but a similar disease re-
principle in European mussels.
sulting from ingestion of mussels has become
knownto occur in Europe.4~6) Unlike para- MATERIALS AND METHODS
lytic shellfish poisoning, DSPhas not caused
deaths in humans. Yet the illness is a serious Materials. Frozen meat (2.7kg) of mussels {Mytilus
threat to both public health and the shellfish
industry because of its wide geographical
distribution and long infestation period. To
alleviate human suffering and economical
damage, shellfish toxicity is being monitored
with assays in animals. There is a need for a okadaicacid
dinophysistoxin-1
( OA
(DTX1)
) : R^H,
: R<|=H,
R2=H
R2=CH3
practical instrumental assay, however, to dinophysistoxin-3 (DTX3) : Ri=acyl, R2=CH3
avoid the inaccuracy and the inconvenience
J3
of assays in animals. To design a physico-
chemical assay requires identification of the
causative toxin or toxins. Recent findings that
Japanese scallops contain a numberof poly-
ether toxins with different skeletons (Fig. 1)7) pectenotoxin-1 (PTX1) : R= CH2OH
and toxicological properties8} urged us to pectenotoxin-2 (PTX2) : R= CH3

study the toxin composition of European Fig. 1. Structures of Diarrhetic Shellfish Toxins from
mussels in detail. Japanese Shellfish.

To whomcorrespondence should be addressed.

2854
edulis) collected at Le Harve, France, in 1984, at the coast
of the Wadden Sea, the Netherlands, in 1982 (1.5kg), and
at Halevic, Sweden, in 1984 (1.5kg), and canned mussels
(about 600g) produced in Vigo, Spain, in 1983 were used
in this study. Only the digestive glands were used for toxin
estraction. Authentic okadaic acid (OA) isolated from
cultures cells of the dinoflagellate Prorocentrum lima9) was
used as the reference.
Isolation. A procedure described in the previous report7]
for the isolation of diarrhetic shellfish toxins of Japanese
scallops was used with slight modifications. The digestive
glands of mussels were minced and extracted three times at
room temperature with an each equal volume of acetone
each time. The oily residue obtained after evaporation of
the solvent was partitioned between hexane and 75%
aqueous EtOH. The ethanol layer was condensed to an
aqueous suspension and extracted with diethyl ether. The
ether-soluble material was chromatographedon a silica
gel (Wakogel C-100) column with ether-MeOH (40 : 1 and
then 1 : 1) and MeOH.The toxic residue obtained in the
second eluate was purified by gel permeation through a
Sephadex LH-20 column (2.8 x 120cm) using benzene-
MeOH(1 : 1) as the solvent. Toxic fractions from 280 to
320ml were combined and evaporated, and the residue
was chromatographed on a basic alumina column
(Woelm, activity 3) with CH2Cl2-MeOH (1 : 1) and 1%
NH4OH-MeOH(1 : 1) to obtain the toxin in the last elu-
ate. Further purification was done by combinations of the
following reversed-phase columns and solvents: Develosil
ODS 10/20 (Nomura Kagaku) and MeOH-0.005%
NH4OH(6 :4); Lobar column (Li Chroprep RP-8, size B)
and MeOH-H2O(8:2); and Develosil C-8 10/20 (2.0x
25cm) and MeOH-H2O (85:15). Separation
toxins was monitored with a UVspectromonitor at the
wavelength of 220nm. TLCand mouse assays were also
used for the monitoring. Because of the small amounts of
the toxin in the Swedish and Spanish samples, gel per-
meation chromatography was skipped. Half of the Dutch
sample was processed without alumina column chroma-
tography to obtain a labile component.
Gas chromatography (GQ. Purified samples were es-
terified with CH2N2in ether solution and then trimethyl-
silylated with Trisil 'Z' reagent (Pierce). The derivatives
were chromatographed on an OV-101 column (2% on
60/80mesh Uniport HP, 3x800mm) at 320°C under
conditions described previously.7)
Fluorometric liquid-chromatographic analyses. Toxic
fractions obtained by purification on columns of silica gel,
alumina, and Develosil C-8 were fluorescence-labeled with
9-anthryldiazomethane (ADAM, Funakoshi
ceutical) with a standard method.10) The derivative was
chromatographed on a Develosil ODS-5
(Nomura Kagaku, 4.6 x 250mm) with MeCN-H2O (95 :
5) under conditions described previously.n)
M. Kumagai et al.
was monitored with a fluoromonitor (Japan Spectroscopic
Co., FP-1 10C). The excitation and emission wavelengths
were set at 365 and 412nm, respectively. The minor toxic
compotent of Dutch mussels was hydrolyzed in a 0.2n
methanolic NaOHsolution at 80°C for 30min. The hy-
drolysate was acidified with a 1.0n HOAcsolution and
extracted with diethyl ether. The ether extract was chro-
matographed on a silica gel column with CHC13 and
CHCl3-MeOH(1 : 1). The residue in the second eluate
was tested for the presence of OAafter derivatization
to a 9-anthrylmethyl ester.
Spectral measurements. 1H NMRspectra were mea-
sured with an NT-360 (Nicolet) spectrometer, and 13C
NMRspectra with an FX-100 (JEOL) spectrometer. All
data were obtained in CDC13solution. The secondary ion
mass spectra (SIMS) were taken on a Hitachi M-80 mass
spectrometer, and the fast atom bombardment mass
spectra (FABMS) on a JEOL JMX-DX300mass spec-
trometer with glycerol as the matrix.
RESULTS
In all specimens of European mussels the
major toxin was eluted from alumina columns
with 1% NH4OH-MeOH(1:1), as the ref-
erence OAwas. The high recovery (90%) of
toxicity in this fraction from French and
Swedish mussels suggested that labile com-
ponents such as dipophysistoxin-3 or its ana-
logs contributed only sightly to the toxicity, if
any. Toxicity was undetectable in CH2C12-
of the
MeOH(1 : 1), suggesting that pectenotoxins
(PTXs) were absent in the specimens tested.
The chromatographic properties of the major
toxin of each kind of specimen on other col-
umns also agreed with those of OA. Com-
parison between the purified specimens and
OAby high performance liquid chromatog-
raphy (HPLC) on a Develosil C-8 column is
shown in Fig. 2a.
The major toxin of the French mussels was
obtained as a colorless solid (7mg) judged
pure by TLC (Rf, 0.5). The lethality in mice
(1 80 /ig/kg, intraperitoneal injection) was com-
parable to that of OA (200/ig/kg). The XH
NMRspectrum (Fig. 3) and 13C NMRdata
Pharma- (Table I) agreed well with those of OA. The
SIMSshowed prominent ion peaks at m/z 843,
827, 805, 787, 769, and 751 (Fig. 3, inset),
column
which were assignable respectively to (M+
The eluate K)+, (M+Na)+, (M+H)+, (M+H-H2O)+,
 2855
Okadaic Acid from European Mussels
a) 1 b)

French /I V 1
i J\V__
Swedish LI 1

Dutch /I L&--^ ^

-.0 .
16 32 . min )) ( (_.96 .
104 .
112 .120 min

Fig. 2. Chromatograms of Toxins in European Mussels.

a) Okadaic acid fraction from French, Swedish, and Dutch mussels were chromatographed on a Develosil C-8
(2 x 25cm) column with MeOH-H2O(4: 1) at a flow rate of 0.7ml/min.
b) The minor toxin from Dutch mussels was chromatographed over a LiChroprep RP-8 (0.6 x 100cm) with
MeOH-H2Ofor 70min and then with MeOHat a flow rate of 3.0ml/min.
All chromatograms were monitored at 220 nm.

Table I. 13C NMRSpectral Data of Reference (M+H-2H2O)+ and (M+H-3H2O)+. The

OAand the Major Toxin of French Mussels
(CDCI3, 25 MHz) toxin was thus identified as OA.
F ren ch F re n c h The major toxin of the Swedish mussels was
R efe ren c e R efere n c e obtained as a colorless solid (1mg). It was
m u sse l m u ssel
O A O A
to x m to x m indistinguishable from OA by TLC (Rf, 0.5) or
by HPLC (Fig. 2a). The SIMS showed the
4 2.9 4 2.7
17 6.8 presence of ion peaks at m/z 827 and 805
4 2.2 4 2.2
37 .3 37.4
corresponding to (M-|-Na)+ and (M+H)+ of
144. 7 144. 7
13 9. 1 13 9.3 36. 0 3 5. 9 OA, respectively.
136. 5 13 6.3 3 5.3
33. 2
35.4
3 3.2
The Dutch mussels afforded about 1 mg of a
13 1. 1 13 1. 3
12 1.7 12 1. 5 32. 9 32. 9 colorless toxic solid, which was indistinguish-
112 .5 1 12.5 3 1. 8 3 1. 7 able from OA by TLC (Rf, 0.5), HPLC (Fig.
3 1. 1 3 1. 1 å 2a) or by GC (retention time 8'48"). The
10 5.7 10 5.7 30 .7 30 .5 FABMS had a prominent peak at m/z 805,
9 6.5 9 6.5 30 .3 30. 4
9 5.6 9 5.6 27. 4 27. 4 which was assignable to M+H)+of OA. In
84 .8 84 .9 27. 4 27. 4 the Dutch mussels, a minor toxin accounted
79 .2 79 .2 27. 4 2 7.4 for 10% of the mouse toxicity. It was eluted
7 6.5 7 6.5 27. 4 27. 4 from the LiChroprep RP-8 column with
7 6.5 26. 4 26. 4
7 6.5
7 5. 1 7 5. 1 25. 4 2 5.5
MeOH (Fig. 2b). Fluorometric HPLC analy-
7 1. 5 7 1.6 2 3. 1 2 3. 1 sis of ADAMderivatives of the hydrolysis
7 1.4 7 1.6 1 8.8 1 8.8 products indicated the presence of a com-
1 6.2
7 1. 1 7 1. 1 1 6.2 ponent identical in retention time with the ref-
6 9.8 69 .8 1 5.9 1 5.9
69 .1 6 9. 1 10.7 1 0.7 erence 9-anthrylmethyl okadaate.
64.8 6 4.8 The chromatographic properties of the ma-
60.3 6 0.4 jor toxin of the Spanish mussels agreed with
those of OAon columns ofalumina, silica gel,
2856 M. Kumagai et ah

I805(M+H) +

843

I I827I
LJIljI jl,.

I I 1 I i | i r-i i | 1 i i i | 1 1 1 r 1 1 1 1 1 1 1 1 1 , 1 1 r-t 1 1 1 1 , 1
8 7 6 5 4 3 2 1 0 PPM

Fig. 3. XHNMR(360MHz, CDC13) and Secondary Ion Mass Spectra of the Maior Toxin from French
Mussels.

and Develosil C-8. No spectral measurement The presence of OAin Japanese mussels coin-
was attempted because the sample was small. cided with the presence of D. acuminata, but
Fluorometric HPLC analysis of the ADAM not D. fortii,7) providing more evidence that
derivative of the toxin showed a prominent D. acuminata synthesizes OA. Both OAand
peak identical with 9-anthrylmethyl okadaate DTX1have strong diarrheagenicity.13)
in its retention time. Another difference between Japanese and
European shellfish is the absence of PTXsin
DISCUSSION the latter. The coexistence of toxins that differ
both in skeletons and in functional groups
All European mussels contained OAas their has been an obstacle to use of a specific in-
principal toxin, unlike Japanese mussels, the strumental assay for Japanese shellfish. The
major toxin of which is a 35-methyl derivative conventional bioassay, which measures
of OA (dinophysistoxin-1, DTX1).3) The
mouse lethality, is also difficult because dino-
Dutch mussels contained a polar component physistoxins differ from PTXs in their
biological activity. The simple toxin com-
corresponding
(DTX3),
in polarity to dinophysistoxin-3
the major toxin of Japanese scal- position of European mussels reported here
lops,^ in small amounts. However, the toxin will make instrumental assay, such as by
liberated OAupon hydrolysis whereas DTX3 fluorometric HPLC,a suitable monitor of
liberated DTX1. The difference in the toxin shellfish toxicity.
structures seems to be attributable to the dif-
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Okadaic Acid from European Mussels
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