Evidence for a Covalently Bound Form of Microcystin-LR in Salmon Liver and Dungeness Crab Larvae

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Abstract
The chemically unique nature of the C20 β-amino acid (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) portion of the microcystins has been exploited to develop a strategy to analyze for the total microcystin-LR (1; see Figure 1) burden in salmon liver and crab larvae tissues. Lemieux oxidation of microcystin-LR (1) gives 2-methyl-3-methoxy-4-phenylbutanoic acid (2), a unique marker for the presence of microcystins. The butanoic acid 2 can be isolated and detected by GC/MS from the livers of Atlantic salmon that received an ip injection of microcystin-LR (1) and from tissues of wild-caught crab larvae. The Lemieux oxidation−GC/MS results are compared with those from MeOH extraction−PPase analysis. Only ∼24% of the total microcystin-LR (1) burden in salmon liver tissue is found to be extractable with MeOH. Similarly, the Lemieux oxidation−GC/MS method detected 10 000-fold greater microcystin concentrations in Cypress Island Dungeness crab larvae than did the MeOH extraction−PPase method. The disparity in microcystin concentrations measured by the two methods is taken as direct evidence for the existence of covalently bound microcystins in vivo.