Sensitive Detection of Ciguatoxins Using a Neuroblastoma Cell-Based Assay with Voltage-Gated Potassium Channel Inhibitors.

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 µg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.

Investigation of Foreign Amylase Adulteration in Honey Distributed in Japan by Rapid and Improved Native PAGE Activity Staining Method.

Foreign amylase addition to honey in an effort to disguise diastase activity has become a widespread form of food fraud. However, since there is no report on the investigation in Japan, we investigated foreign amylases in 67 commercial honeys in Japan. First, the α-glucosidase and diastase activities of honeys were measured, which revealed that only α-glucosidase activity was significantly low in several samples. As both enzymes are secreted from honeybee glands, it is unlikely that only one enzyme was inactivated during processing. Therefore, we suspected the presence of foreign amylase. α-Amylase in honey were assigned using protein analysis software based on LC-QTOF-MS. As a result, α-amylases from Aspergillus and Geobacillus were detected in 13 and 6 out of 67 honeys, respectively. To detect foreign amylases easily, we developed a cost-effective method using native PAGE. Conventional native PAGE failed to separate the α-amylase derived from honeybee and Geobacillus. However, when native PAGE was performed using a gel containing 1 % maltodextrin, the α-amylase from honeybee did not migrated in the gel and the α-amylase could be separated from the other two α-amylases. The results from this method were consistent with those of LC-QTOF-MS method, suggesting that the novel native PAGE method can be used to detect foreign amylases.

Evaluation of relative potency of calibrated ciguatoxin congeners by near-infrared fluorescent receptor binding and neuroblastoma cell-based assays.

Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays.

[Development of LC-MS and LC-MS/MS Methods for Free Asparagine in Grains].

New analytical methods for the determination of free asparagine （Asn）, which is a precursor of acrylamide, in grains were developed using LC-MS and LC-MS/MS. Asn was extracted from a sample with 5％ （w/v） aqueous trichloroacetic acid solution, appropriately diluted with 0.1％ （v/v） formic acid solution, and then analyzed by LC-MS or LC-MS/MS. HPLC separation was performed by isocratic elution on a Penta Fluoro Phenyl （PFP） column using 0.1％ （v/v） formic acid and acetonitrile mixture as the mobile phase. The calibration curve was linear in the range of 0.005-0.1 µg/mL. The mean recoveries from potato starch, non-glutinous rice flour and whole wheat flour ranged from 95.4 to 100.9％, repeatability （RSD） ranged from 0.9 to 6.0％, and within-laboratory reproducibility （RSDwr） ranged from 2.8 to 7.1％. Limits of quantitation （LOQs） were 7 mg/kg for potato starch, and 5 mg/kg for non-glutinous rice flour. In addition, an inter-laboratory study was performed in 10 laboratories using 5 kinds of grains （non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour）, which naturally contained free asparagine. The HORRATR values ranged from 0.4 to 1.0. These results are within the range of the procedural manual of the Codex Alimentarius Commission, confirming the effectiveness of the developed procedures.

[Inter-laboratory Study of an HPLC-UV Method for Free Asparagine in Grains].

In order to validate an HPLC-UV method for the determination of free asparagine, which is a precursor of acrylamide, in grains using dansyl derivatization, an inter-laboratory study was performed in 9 laboratories using 5 kinds of grains (non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour), which naturally contain free asparagine. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDr) were in the ranges of 0.5-2.2 and 2.3-5.9%, respectively. The HorRat values ranged from 0.4 to 0.6. These results were within the range of the procedural manual of the Codex Alimentarius Commission, and therefore the method is effective.

[Development of an HPLC-UV Method for Free Asparagine in Grains].

A novel analytical method was developed for the determination of free asparagine (Asn), which is a precursor of acrylamide, in grains. Asn was extracted from a sample with 5% (w/v) aqueous trichloroacetic acid solution, and the crude extract was cleaned up using a reversed-phase solid-phase cartridge. The cleaned extract was derivatized with dansyl chloride and analyzed by HPLC-UV. HPLC separation was performed by gradient elution on a ODS column using 0.01 mol/L ammonium acetate and acetonitrile mixture as the mobile phase. The calibration curve was linear in the range of 0.5-100 µg/mL. The mean recoveries from potato starch, non-glutinous rice flour and whole wheat flour ranged from 97.7 to 102.6%, the repeatability (RSDs) ranged from 0.8 to 2.0%, and the within-laboratory reproducibility (RSDwr) ranged from 1.4 to 6.2%. Limits of quantitation (LOQs) were 13 mg/kg for potato starch and 4 mg/kg for non-glutinous rice flour. The quantitative values obtained for about 15 kinds of grains using this developed method were approximately equal to those obtained with an automatic amino acid analyzer.