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

Abstract

The chemically unique nature of the C20 beta-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 approximately 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.

Rapid quantification of total microcystins in cyanobacterial samples by periodate-permanganate oxidation and reversed-phase liquid chromatography

“…These results suggest that lower amounts of permanganate are insufficient to generate MMPB from cyanobacterial samples; therefore, oxidization of cyanobacterial samples with a high dose of permanganate in the reaction solution should be considered. These findings differ from those of previous studies, which have usually been conducted using lower concentrations of permanganate [38][39][40][41][42][43][44][45][46][47].…”

“…The results revealed that the yields of MMPB under neutral or alkaline conditions were higher than those under acidic conditions, with the highest concentration being produced under weak alkaline conditions (pH ∼9). These results are consistent with those of previous studies in which the pH of the solution was adjusted to approximately 9 using potassium bicarbonate to produce MMPB in oxidation reactions [38][39][40][41][42][43][44][45][46][47]. Among the five pH values evaluated, the resulting MMPB concentrations were shown to have only minor differences after 1 h, indicating that MMPB was stable under different pH conditions.…”

“…This method is believed to be an accurate method for screening for total MCs because it can measure MMPB derived from the unusual C 20 amino acid, ␤-amino acid (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4, 6-dienoic acid (Adda), which is a common constituent moiety of MCs [7]. Oxidizing toxins using the Lemieux oxidation method [33][34][35][36][37] have been widely used as a highly sensitive method for the detection of total MCs in a wide range of sample matrices, including cyanobacteria [38,39], animal tissues [40][41][42][43][44] and serum samples [45][46][47]. However, no studies have been conducted to evaluate the influence of reaction conditions on the formation of MMPB during the oxidation of MCs using permanganate-periodate.…”

Optimal strategies for determination of free/extractable and total microcystins in lake sediment

“…To date, only Tsuji et al [15] have applied this MMPB method for total MCs detection in lake sediments; however, there still exists a problem in that the necessary very-low reaction temperature is difficult to achieve as it involves oxidation of the MCs to form MMPB by ozonolysis. Several studies have reported that oxidizing the toxins by Lemieux oxidation is a more sensitive analytical method for total MC detection, and this technique has been widely applied in a range of sample matrices, including cyanobacteria [19,20], or animal tissues [21][22][23][24][25][26]. Unfortunately, there is still little information available regarding the effects of the primary factors such as the reaction conditions and sediment characteristics (i.e., organic matter) on the oxidation efficiency, and application of the MMPB method using Lemieux oxidation to the analysis of lake sediment has still not been reported.…”

Effect of dietary cyanobacteria on growth and accumulation of microcystins in Nile tilapia (Oreochromis niloticus)

“…Microcystins can be accumulated in the tissues of fish (Tencalla et al, 1994;Soares et al, 2004), mussels (Amorim and Vasconcelos, 1999;Vasconcelos, 1995;Williams et al, 1997b;Dionisio Pires et al, 2004) and aquatic macrophytes (Pflugmacher et al, 1998) and also transferred from crab larva to salmons through food chain (Williams et al, 1997a). Many fishes are not able to avoid the ingestion of these toxic organisms through food in the waters such as small eutrophicated lakes and aquaculture ponds (Tencalla and Dietrich, 1997;Magalhães et al, 2001).…”