Evaluation of microcystin contamination in blue-green algal dietary supplements using a protein phosphatase inhibition-based test kit

Abstract

The cyanobacterium Aphanizomenon flos-aquae (AFA), from Upper-Klamath Lake, Oregon, are used to produce blue-green algal (BGA) dietary supplements. The periodic co-occurrence of hepatotoxin-producing contaminant species prompted the Oregon Health Division to establish a limit of 1 μg/g microcystin (MC) for products sold in Oregon in 1997. At the federal level, the current good manufacturing practice (CGMP) regulations for dietary supplements require manufacturers establish a specification, and test, for limits on contaminants that may adulterate finished products. Despite this, several previous international surveys reported MC in BGA supplements in excess of 1 μg/g. The objectives of this study were (1) identify a reliable, easy to use test kit for the detection of MC in dried BGA materials and (2) use this kit to assess the occurrence of MC contamination in AFA-BGA dietary supplements in the U.S. A commercial protein phosphatase inhibition assay (PPIA), based on the enzyme PP2A, was found to have acceptable relative enzyme inhibition and accuracy for the majority of MC variants tested, including those most commonly identified in commercial samples, making the kit fit for purpose. Using the PPIA kit, 51% (26 of 51) distinct AFA-BGA products had MC ≥0.25 μg/g (the detection limit of the kit), 10 products had MC concentrations between 0.5 and 1.0 μg/g, and 4 products exceeded the limit (1.1–2.8 μg/g). LC-MS/MS confirmed PPIA results ≥0.5 μg/g and determined that MC-LA and MC-LR were the main congeners present. PPIA is a reliable method for the detection of MC contamination in dried BGA dietary supplements produced in the U.S. While the majority of AFA-BGA products contained ≥0.25 μg/g MC, most were at or below 1.0 μg/g, suggesting that manufacturers have adopted this level as a specification in these products; however, variability in recommended serving sizes prevented further analysis of consumer exposure based on the concentrations of MC contamination found.

Are Cyanotoxins the Only Toxic Compound Potentially Present in Microalgae Supplements? Results from a Study of Ecological and Non-Ecological Products

“…The presence of microcystin in microalgae-based food supplements has been previously documented, especially when they contain Aphanizomenon [ 6 , 21 , 27 , 28 , 33 , 35 , 45 , 46 ]. The more commercialized Aphanizomenon comes from Klamath Lake (USA), and several reports about the toxicity of this product are found in literature [ 28 , 29 , 30 , 46 ].…”

“…Microalgae from other taxonomic groups are also frequently consumed, of which the most popular are probably Chlorella and Haematococcus (Trebouxiophyceae and Chlorophyceae, respectively). These products are available in diverse forms (e.g., pills, capsules, powder, flakes, tablets) [ 19 , 20 , 21 ] and have been progressively included in other products, like biscuits, pasta, drinks or yoghurt [ 22 , 23 ]. As it is compulsory to only indicate the composition, daily intake and a warning about them not substituting healthy diet in most countries [ 24 , 25 ], the labels of these products are often inaccurate and do not offer information about geographic origin, if they are collected from nature, or if they are produced in close bioreactors or open systems.…”

“…The presence of Microcystis aeruginosa (Kützing) Kützing (the most important microcystin producer) in supplements of Aphanizomenon flos-aquae Ralfs ex Bornet and Flahault has been documented [ 28 , 29 ], but sometimes those containing A. flos-aquae are used to treat children with hyperactivity or attention deficiency syndrome [ 30 ]. Thus no clear idea is available of what a healthy daily dose actually is [ 17 , 21 , 30 , 31 ]. These products are normally bought with no medical prescription and are perceived as being safe [ 6 , 25 , 32 , 33 ].…”

Micro-droplet Hydrodynamic Voltammetry for the Determination of Microcystin-LR Based on Protein Phosphatase

“…The protein phosphatase inhibition assays and immunoassays are suitable substitutions for rapid detection of MC-LR because of its potency to inhibit the activities of protein phosphatases 1 and 2A (PPl, PP2A) as a similar manner to okadic acid [18,19], where the former methods are sensitive than later due to the provision of insight on toxicology [2]. Like MC-LR, other MC variants are also involving the potent inhibition of protein phosphatases and, therefore, PPIA has been used for the determination of MC contamination [20]. These assays are considered as the ideal toxicity assays for MC determination in the environmental samples [21][22][23].…”

Cyanobacteria: Review of Current Potentials and Applications

“…It is important for DNA synthesis and takes part in hemoglobin production [23,27]. A recent study reported that in the United States, numerous dietary supplements containing cyanobacterial species such as Aphanizomenon flos-aquae, Spirulina, and chlorophyte Chlorella are available in the consumer markets [28]. Microcystis and Oscillatoria are also present at the source; both these genera are known to produce microcystins and nodularins (toxic cyclic peptides) that can contaminate the raw materials.…”