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Enzymatic hydrolysis of esterified diarrhetic shellfish poisoning toxins and pectenotoxins

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Abstract

Okadaic acid (OA) and dinophysistoxins-1 and -2 (DTX1, DTX2), the toxins responsible for incidents of diarrhetic shellfish poisoning (DSP), can occur as complex mixtures of ester derivatives in both plankton and shellfish. Alkaline hydrolysis is usually employed to release parent OA/DTX toxins, and analyses are conducted before and after hydrolysis to determine the concentrations of nonesterified and esterified toxins. Recent research has shown that other toxins, including pectenotoxins and spirolides, can also exist as esters in shellfish, but these toxins cannot survive alkaline hydrolysis. A promising alternative approach is enzymatic hydrolysis. In this study, two enzymatic methods were developed for the hydrolysis of 7-O-acyl esters, “DTX3,” and the carboxylate esters of OA, “diol-esters.” Porcine pancreatic lipase induced complete conversion of DTX3 to OA and DTXs within one hour for reference solutions. The presence of mussel tissue matrix reduced the rate of hydrolysis, but an optimized lipase concentration resulted in greater than 95% conversion within four hours. OA-diol-ester was hydrolyzed by porcine liver esterase and was completely converted to OA in less than 30 min, even in the presence of mussel tissue matrix. Esters and OA/DTX toxins were all monitored by LC–MS. Further experiments with pectenotoxin esters indicated that enzymatic hydrolysis could also be applied to esters of other toxins. Enzymatic hydrolysis has excellent potential as an alternative to the conventional alkaline hydrolysis procedure used in the preparation of shellfish samples for the analysis of toxins.

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References

  1. Botana LM (2000) Seafood and freshwater toxins: pharmacology, physiology and detection. Marcel Dekker, New York

    Google Scholar 

  2. Yasumoto T, Murata M (1993) Marine toxins. Chem Rev 93:1897–1909

    Article  CAS  Google Scholar 

  3. Yasumoto T, Murata M, Oshima Y, Sano M, Matsumoto GK, Clardy J (1985) Diarrhetic shellfish toxins. Tetrahedron 41:1019–1025

    Article  CAS  Google Scholar 

  4. Hu T, Doyle J, Jackson D, Marr J, Nixon E, Pleasance S, Quilliam MA, Walter JA, Wright JLC (1992) Isolation of a new diarrhetic shellfish poison from Irish mussels. J Chem Soc Chem Commun 39–41

  5. Bailojan C, Takai A (1988) Inhibitory effects of a marine sponge toxin, OA, on protein phosphatases. Biochem J 256:283–290

    Google Scholar 

  6. Cohen P, Holmes CFB, Tsukitani Y (1990) Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci 15:98–102

    Article  CAS  Google Scholar 

  7. Mestrovic V, Pavela Vrancic A (2003) Inhibition of alkaline phosphatase activity by okadaic acid, a protein phosphatase inhibitor. Biochimie 85:647–650

    Article  CAS  Google Scholar 

  8. Haystead TAJ, Sim ATR, Carling D, Honnor RC, Tsukitani Y, Cohen P, Hardie DG (1989) Effects of the tumor promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature 337:78–81

    Article  CAS  Google Scholar 

  9. Fujiki H, Suganuma M (1993) Tumor promotion by inhibitors of protein phosphatases 1 and 2A: the okadaic acid class of compounds. Adv Cancer Res 61:143–194

    Article  CAS  Google Scholar 

  10. Dahl E, Johannessen T (2001) Relationship between occurrence of Dinophysis species (Dinophyceae) and shellfish toxicity. Phycologia 40:223–227

    Article  Google Scholar 

  11. Hu T, Marr J, de Freitas ASW, Quilliam MA, Walter JA, Wright JLC, Pleasance S (1992) New diol esters of okadaic acid isolated from cultures of the dinoflagellates Prorocentrum lima and Prorocentrum concavum. J Nat Prod 55:1631–1637

    Article  CAS  Google Scholar 

  12. Pan Y, Cembella AD, Quilliam MA (1999) Cell cycle and toxin production in the benthic dinoflagellate Prorocentrum lima. Mar Biol 134:541–549

    Article  CAS  Google Scholar 

  13. Quilliam MA, Hardstaff WR, Ishida N, McLachlan JL, Reeves AR, Ross NW, Windust AJ (1996) In: Yasumoto T, Oshima Y, Fukuyo Y (eds) Harmful and toxic algal blooms. Production of diarrhetic shellfish poisoning (DSP) toxins by Prorocentrum lima in culture and development of analytical methods. IOC/UNESCO, Paris, pp 289–292

    Google Scholar 

  14. Morono A, Arevalo F, Fernandez ML, Maneiro J, Pazos Y, Salgado C, Blanco J (2003) Accumulation and transformation of DSP toxins in mussels Mytilus galloprovincialis during a toxic episode caused by Dinophysis acuminata. Aquat Toxicol 62:269–280

    Article  CAS  Google Scholar 

  15. Suzuki T, Mitsuya T (2001) Comparison of dinophysistoxin-1 and esterified dinophysistoxin-1 (dinophysistoxin-3) contents in the scallop Patinopecten yessoensis and the mussel Mytilus galloprovincialis. Toxicon 39:905–908

    Article  CAS  Google Scholar 

  16. Vale P, Sampayo MAD (2002) First confirmation of human diarrhoeic poisonings by okadaic acid esters after ingestion of razor clams (Solen marginatus) and green crabs (Carcinus maenas) in Aveiro Lagoon, Portugal and detection of okadaic acid esters in phytoplankton. Toxicon 40:989–996

    Article  CAS  Google Scholar 

  17. Castberg T, Torgersen T, Aasen J, Aune T, Naustvoll LJ (2004) Diarrhoetic shellfish poisoning toxins in Cancer pagurus Linnaeus 1758 (Brachyura, Cancridae) in Norwegian waters. Sarsia 89:311–317

    Article  Google Scholar 

  18. Marr JC, Hu T, Pleasance S, Quilliam MA, Wright JLC (1992) Detection of new 7-O-acyl derivatives of diarrhetic shellfish poisoning toxins by liquid chromatography–mass spectrometry. Toxicon 30:1621–1630

    Article  CAS  Google Scholar 

  19. Vale P, De Sampayo MA (2002) Esterification of DSP toxins by Portuguese bivalves from the northwest coast determined by LC–MS—a widespread phenomenon. Toxicon 40:33–42

    Article  CAS  Google Scholar 

  20. Suzuki T, Ota H, Yamasaki M (1999) Direct evidence of transformation of dinophysistoxin-1 to 7-O-acyl-dinophysistoxin-1 (dinophysistoxin-3) in the scallop Patinopecten yessoensis. Toxicon 37:187–198

    Article  CAS  Google Scholar 

  21. Hamano Y, Kinoshita Y, Yasumoto T (1986) Enteropathogenicity of diarrhetic shellfish toxins in intestinal models (studies on diarrhetic shellfish toxins I). J Food Hyg Soc Jpn 27:375–379

    Google Scholar 

  22. Yanagi T, Murata M, Torigoe K, Yasumoto T (1989) Biological activities of semisynthetic analogs of dinophysistoxin-3, the major diarrhetic shellfish toxin. Agric Biol Chem 53:525–529

    CAS  Google Scholar 

  23. Torgersen T, Aasen J, Aune T (2005) Diarrhetic shellfish poisoning by okadaic acid esters from brown crabs (Cancer pagurus) in Norway. Toxicon 46:572–578

    Article  CAS  Google Scholar 

  24. Hu T, Curtis JM, Walter JA, Wright JLC (1995) Identification of DTX-4, a new water-soluble phosphatase inhibitor from the toxic dinoflagellate Prorocentrum lima. J Chem Soc Chem Commun 597–599

  25. Hu T, Curtis JM, Walter JA, McLachlan JL, Wright JLC (1995) Two new water-soluble DSP toxin derivatives from Prorocentrum maculosum: possible storage and excretion products of the dinoflagellate. Tetrahedron Lett 36:9273–9276

    Article  CAS  Google Scholar 

  26. Suzuki T, Beuzenberg V, Mackenzie L, Quilliam MA (2004) Discovery of okadaic acid esters in the toxic dinoflagellate dinophysis acuta from New Zealand using liquid chromatography tandem mass spectrometry. Rapid Comm Mass Spectrom 18:1131–1138

    Article  CAS  Google Scholar 

  27. Needham J, Hu T, McLachlan JL, Walter JA, Wright JLC (1995) Biosynthetic studies of the DSP toxin DTX-4 and an okadaic acid diol ester. J Chem Soc Chem Commun 1623–1624

  28. Suzuki T, Yoshizawa R, Kawamura T, Yamasaki M (1996) Interference of free fatty acids from the hepatopancreas of mussels with the mouse bioassay for shellfish toxins. Lipids 31:641–645

    Article  CAS  Google Scholar 

  29. Fernández ML, Miguez A, Cacho E, Martinez A (1996) Detection of okadaic acid esters in the hexane extracts of Spanish mussels. Toxicon 34:381–387

    Article  Google Scholar 

  30. Mountfort DO, Suzuki T, Truman P (2001) Protein phosphatase inhibition assay adapted for determination of total DSP in contaminated mussels. Toxicon 39:383–390

    Article  CAS  Google Scholar 

  31. Gonzalez JC, Leira F, Fontal OI, Vieytes MR, Arevalo FF, Vieites JM, Bermudez Puente M, Muniz S, Salgado C, Yasumoto T, Botana LM (2002) Inter-laboratory validation of the fluorescent protein phosphatase inhibition assay to determine diarrhetic shellfish toxins: intercomparison with liquid chromatography and mouse bioassay. Anal Chim Acta 466:233–246

    Article  CAS  Google Scholar 

  32. Lee JS, Yanagi T, Kenma R, Yasumoto T (1987) Fluorometric determination of diarrhetic shellfish toxins by high-performance liquid chromatography. Agric Biol Chem 51:877–881

    CAS  Google Scholar 

  33. Quilliam MA (1995) Analysis of diarrhetic shellfish poisoning toxins in shellfish tissue by liquid chromatography with fluorometric and mass spectrometric detection. J AOAC Int 78:555–570

    CAS  Google Scholar 

  34. Quilliam MA, Hess P, Dell’Aversano C (2001) In: deKoe WJ, Samson RA, van Egmond HP, Gilbert J, Sabino M (eds) Mycotoxins and phycotoxins in perspective at the turn of the century. Recent developments in the analysis of phycotoxins by liquid chromatography–mass spectrometry. WJ deKoe, Wageningen, The Netherlands, pp 383–391

    Google Scholar 

  35. Aasen JAB, Hardstaff W, Aune T, Quilliam MA (2006) Discovery of fatty acid ester metabolites of spirolide toxins in mussels from Norway using liquid chromatography/tandem mass spectrometry. Rapid Comm Mass Spectrom 20:1531–1537

    Article  CAS  Google Scholar 

  36. Wilkins AL, Rehmann N, Torgersen T, Rundberget T, Keogh M, Petersen D, Hess P, Rise F, Miles CO (2006) Identification of fatty acid esters of pectenotoxin-2 seco acid in blue mussels (Mytilus edulis) from Ireland. J Agric Food Chem 54:5672–5678

    Article  CAS  Google Scholar 

  37. Quilliam MA, Ross NW (1995) Analysis of diarrhetic shellfish poisoning toxins and metabolites in plankton and shellfish by ion-spray liquid chromatography–mass spectrometry. In: Snyder AP (ed) Biochemical and biotechnological applications of electrospray ionization mass spectrometry. American Chemical Society, Washington, DC, pp 351–364

    Google Scholar 

  38. Suzuki T, Mackenzie L, Stirling D, Adamson J (2001) Pectenotoxin-2 seco acid: a toxin converted from pectenotoxin-2 by the New Zealand greenshell mussel, Perna canaliculus. Toxicon 39:507–514

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Drs. J. Aasen and T. Aune of the Norwegian School of Veterinary Science, Department of Food Safety and Infection Biology (Oslo, Norway) for contaminated shellfish tissue samples, Mr. W. Hardstaff and Ms. P. Blay for technical assistance, Ms. K. Boutilier for preliminary work with the esterase, and Drs. M. Laycock and T. Gill for valuable advice. Initial funding of ED’s graduate student support from Dr. J. Jellett (Jellett Rapid Testing Chester Basin NS) is gratefully acknowledged.

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Authors and Affiliations

  1. National Research Council Canada, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, NS, B3H 3Z1, Canada

    Erin Doucet, Neil N. Ross & Michael A. Quilliam

  2. Department of Process Engineering and Applied Science, Food Science Program, Dalhousie University, Halifax, NS, B3J 2X4, Canada

    Erin Doucet

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  1. Erin Doucet
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  2. Neil N. Ross
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  3. Michael A. Quilliam
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Correspondence to Michael A. Quilliam.

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Doucet, E., Ross, N.N. & Quilliam, M.A. Enzymatic hydrolysis of esterified diarrhetic shellfish poisoning toxins and pectenotoxins. Anal Bioanal Chem 389, 335–342 (2007). https://doi.org/10.1007/s00216-007-1489-3

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  • DOI: https://doi.org/10.1007/s00216-007-1489-3

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