Search for Biomarkers of Neurotoxic Shellfish Poisoning in Humans by LC-MS/MS

摘要

Neurotoxic shellfish poisoning (NSP) is caused by the consumption of molluscan shellfish (e.g., oysters, clams) contaminated with brevetoxins. Brevetoxins are cyclic polyether toxins produced by the marine dinoflagellate Karenia brevis. Several brevetoxins have been isolated from K. brevis, and grouped according to their backbone structure (A- or B-type). Principal A-type brevetoxins are BTX-1 and BTX-7, and B-types BTX-2 and BTX-3 (Fig. 1). Brevetoxins are accumulated and extensively metabolized in filter-feeding molluscan shellfish during K. brevis blooms; metabolites include cysteine conjugates and oxidation/reduction products of brevetoxins BTX-1 and BTX-2. Fate of these metabolites in the human consumer is unknown. NSP is currently diagnosed from clinical symptoms and recent dietary history. Confirmatory tests are unavailable. We examined clinical specimens from NSP patients to identify potential biomarkers of brevetoxin intoxication. Urine specimens from several individuals hospitalized with symptoms consistent with NSP were examined, along with shellfish meal remnants. Shellfish remnants were extracted with 80percent methanol, and extracts cleaned-up by solid-phase extraction (SPE). Urine samples were cleaned-up and concentrated by using SPE (C_(18)). ELISA (antibody directed against brevetoxin backbone structure) and LC-MS/MS were used for brevetoxin screening and confirmation, respectively, in sample extracts.