Comparative concentrations of brevetoxins PbTx-2, PbTx-3, BTX-B1 and BTX-B5 in cockle, Austrovenus stutchburyi, greenshell mussel, Perna canaliculus, and Pacific oyster, Crassostrea gigas, involved neurotoxic shellfish poisoning in New Zealand.

Previously, we found brevetoxins PbTx-3, BTX-B5 and BTX-B1 in cockle, Austrovenus (A.) stutchburyi, PbTx-2, PbTx-3 and BTX-B1 in Pacific oyster, Crassostrea (C.) gigas and PbTx-3 and BTX-B1 in greenshell mussel, Perna (P.) canaliculus following outbreak of neurotoxic shellfish poisoning (NSP) in New Zealand by isolation and/or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this study, procedures for quantitative determination of PbTx-2 and BTX-B5 were developed and those for PbTx-3 and BTX-B1 were further examined by LC-MS/MS. In mass spectrometry with an electrospray ionization interface operating in the positive or negative ion mode, the protonated ions [M+H]+ of PbTx-2 (m/z 895), [M+H]+ of PbTx-3 (m/z 897), [M-H]- of BTX-B5 (m/z 909), and [M-Na]- of BTX-B1 (m/z 1016) were generated abundantly, when 0.1% formic acid-acetonitrile was used as the mobile phase for column chromatography. The product ions of m/z 877, 725, 111 and 80 from PbTx-2, PbTx-3, BTX-B5 and BTX-B1 were identified, respectively, allowing unambiguous confirmation of these toxins by selective reaction monitoring LC-MS/MS analysis. High levels of PbTx-3 and BTX-B5 were detected in C. gigas, of PbTx-3, BTX-B1 and BTX-B5 in A. stutchburyi, and of PbTx-2, PbTx-3 and BTX-B5 in P. canaliculus by this LC-MS/MS method.     

Implication of brevetoxin B1 and PbTx-3 in neurotoxic shellfish poisoning in New Zealand by isolation and quantitative determination with liquid chromatography-tandem mass spectrometry.

Brevetoxin B1 (BTX-B1) was isolated from Austrovenus stutchburyi following the 1992-1993 outbreak of neurotoxic shellfish poisoning (NSP) in New Zealand. We report here the first isolation of PbTx-3 from the same shellfish and the development of a procedure for quantitative determination of PbTx-3 and BTX-B1. PbTx-3 was isolated by chromatography on columns of SiO2, ODS, and LH-20, followed by reverse-phase HPLCs. In mass spectrometry (MS) with an electrospray ionization (ESI) interface operating in the positive or negative ion mode, the abundant protonated ion [M+H]+ of PbTx-3 (m/z 897) and the de-sodiated ion [M-Na]- of BTX-B1 (m/z 1016) were generated, respectively. These served as precursor ions for collision-induced dissociation, and the product ions of m/z 725 from PbTx-3 and m/z 80 from BTX-B1 were identified, allowing unambiguous confirmation of these toxins by selected reaction monitoring liquid chromatography-tandem mass spectrometry (SRM LC-MS/MS) analysis. The determination limits were 0.4 and 2 ng/g for BTX-B1 and PbTx-3 at a signal-to-noise ratio of five, respectively. This LC-MS/MS method was successfully applied to determine BTX-B1 and PbTx-3 in the NSP-associated toxic shellfish. BTX-B1 was found in both A. stutchburyi and Perna canaliculus, but not in Crassostrea gigas, while PbTx-3 was found in all three.     

Intrinsic potency of synthetically prepared brevetoxin cysteine metabolites BTX-B2 and desoxyBTX-B2.

In mammals and shellfish, brevetoxins produced by the dinoflagellate Karenia brevis are rapidly metabolized to cysteine conjugates. These metabolites identified by mass spectrometry are produced in abundance in mammals and are potentially major bioactive products for intoxication. They are also abundant metabolites in shellfish where they are, in contrast to mammals, retained for prolonged periods, posing a potential threat to shellfish consumers. In this work, we analyze the intrinsic potency of the semi-synthetic cysteine brevetoxin sulfoxide (BTX-B2) and the cysteine brevetoxin (desoxyBTX-B2), each confirmed for purity by LC-MS and NMR techniques, on receptor site 5 of the voltage-gated sodium channels (VGSCs) in brain, heart and skeletal muscle. We show that both brevetoxin conjugates compete with the tritiated reduced parent brevetoxin ([(3)H]PbTx-3) in rat brain membrane preparations and in HEK cells expressing skeletal muscle or cardiac VGSC, albeit, with 8-16-fold lower affinity than the PbTx-3. On neuroblastoma cell assays we show a 3-fold reduction in cytotoxic potency for BTX-B2 relative to PbTx-3, and an 8-fold reduction for desoxyBTX-B2. In conclusion, the major transformation product of brevetoxin observed in diverse species through cysteine adduction and oxidation leads to metabolites with reduced potency on brain, skeletal muscle and heart cells.     

Accumulation and depuration rates of paralytic shellfish poisoning toxins in the shore crab Telmessus acutidens by feeding toxic mussels under laboratory controlled conditions.

Accumulation and depuration rates of paralytic shellfish poisoning toxins (PSP) in the crab Telmessus acutidens were investigated by feeding toxic and non-toxic mussels under laboratory controlled conditions. The crab accumulated toxins in the hepatopancreas in proportion to the amount of toxic mussels they ingested, and the toxicity in the crab hepatopancreas became 3.2 fold of that in the prey mussels after 20 days of feeding. During depuration, a fast reduction of the total toxicity was observed in the crab, and the retention rate of the toxicity after 5 days depuration with feeding of non-toxic mussels was 45.8+/-18.7%. The reduction of the toxicity was moderated in the later period of depuration, and the retention rates of the total toxicity after 10 and 20 days were 54.1+/-29.8% and 14.5+/-9.0%, respectively. The toxin profiles in the crab and mussel were investigated by high performance liquid chromatography, and reductive conversions of the toxins were observed when the toxins were transferred from the mussel to the crab. Consequently, high concentrations of GTX2 and GTX3, and STX that were not detected in the prey mussels, were found in the crab.