Identification of 24-O-β-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Dinophysis Blooms, Dinophysis and Prorocentrum Cultures, and Shellfish in Europe, North America and Australasia.

Alistair L Wilkins,Jane Kilcoyne,Morten Sandvik,Beatriz Reguera,Brent K Knudsen,Pilar Rial,Cheryl Rafuse,Frode Rise,Sabrina D Giddings,Michael J Boundy,Christopher O Miles,Elliott J Wright,Thomas Rundberget

doi:10.3390/toxins13080510

Abstract

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-β-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1–5, the 24-O-β-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4–6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

Highlights

Okadaic acid and dinophysistoxins (OA/DTXs) (e.g., 1–3, Figure 1) are marine polyether toxins associated with diarrhetic shellfish poisoning of human shellfish consumers worldwide
It should be noted that PP binding affinity is not the only factor that is important for determining the relative toxicity of OA analogues to human consumers, and toxic equivalency factors (TEFs) derived from animal models are typically used to estimate the toxicity of shellfish containing mixtures of OA/DTXs [17,20,21]
We report the isolation of 24-O-β-D-glucosides of OA and DTX2 (4 and 5) from a fraction obtained during the isolation of OA/DTXs and PTXs from Dinophysis blooms harvested in Spain and Norway, and their identification by liquid chromatography–highresolution tandem mass spectrometry (LC–HRMS/MS), nuclear magnetic resonance (NMR) spectroscopy, and enzymatic hydrolysis

Summary

Introduction

Okadaic acid and dinophysistoxins (OA/DTXs) (e.g., 1–3, Figure 1) are marine polyether toxins associated with diarrhetic shellfish poisoning of human shellfish consumers worldwide. The metabolism of OA/DTXs in shellfish leads to extensive conversion to 7-O-acyl fatty acid esters [15] which, appearing to be of somewhat reduced toxicity [16], are believed to be largely converted back to the free toxins during digestion [15] and are included in the regulatory framework by including a base-hydrolysis step during sample preparation [4,6]. It should be noted that PP binding affinity is not the only factor that is important for determining the relative toxicity of OA analogues to human consumers, and toxic equivalency factors (TEFs) derived from animal models are typically used to estimate the toxicity of shellfish containing mixtures of OA/DTXs [17,20,21]

Methods

Results

Conclusion