Ryoji Matsushima

Inhibitory effect of porphyran, prepared from dried "Nori", on contact hypersensitivity in mice.

Porphyran is a major component of the red algae, Porphyra tenera and P. yezoensis, which are processed into a sheet type of dried food, “Nori”. Porphyran has been reported to activate murine macrophages by in vitro and i.p. injection studies. The contact hypersensitivity (CHS) reaction in mice is commonly used as a model to evaluate the anti-allergic activity of food and food components. We therefore studied the effect of porphyran on the CHS reaction in Balb/c mice to evaluate anti-allergic activity of porphyran. We found that an oral administration of porphyran (2% in drinking water) suppressed the CHS reaction (ear edema) induced by 2,4,6-trinitrochlorobenzene. We also found that porphyran suppressed the serum level of IgE and the production of interferon-γ (IFN-γ) in the challenged ear lobe. We conclude from these results that the CHS reaction was suppressed by oral porphyran due to the decreased serum level of IgE and the production of IFN-γ in the challenged ear lobe.

Quantitative determination of paralytic shellfish toxins in cultured toxic algae by LC-MS/MS

We developed a sample preparation and LC-MS/MS method for the determination of saxitoxins in toxic algae. Paralytic shellfish toxins (PSTs) were successfully separated by gradient elution on an amide column with the hydrophilic interaction mode and quantified with multiple reaction monitoring (MRM) detection in the positive ion mode. This method showed good performance in the summed LODs and LOQs for all 12 toxins, 25 and 84 nM, respectively. Next, extracts of cultured strains of a toxic dinoflagellate Alexandrium tamarense and a freshwater cyanobacteria Anabaena circinalis were treated in a short column of basic alumina and the toxic fractions were analysed by our LC-MS/MS method and by HPLC with fluorescence detection. Comparison of the results obtained by the two methods demonstrated that approximately equivalent results were obtained for both the dinoflagellate and the cyanobacteria. In addition, the retention time of the toxins showed acceptable shifts. Therefore, the clean-up of the toxic algal extracts by using the basic alumina column controlled unwanted chromatographic behaviour and variable ionisation efficiency during MS detection. LC-MS/MS for saxitoxins has great potential as a rapid analytical method for determining all primary saxitoxins in cultured algae.

Analysis of extracellular alginate lyase and its gene from a marine bacterial strain, Pseudoalteromonas atlantica AR06

Pseudoalteromonas atlantica AR06 is a marine bacterial strain that can utilize alginate as a sole source of carbon and energy. The extracellular protein fraction prepared from the AR06 cultivation media exhibited alginate lyase activity to depolymerize the alginate molecules having homopolymeric and heteropolymeric forms of mannuronate and guluronate so as to mainly convert into the dimer to tetramer. A DNA fragment encoding a portion of alginate lyase was amplified from AR06 genomic DNA by PCR using a set of degenerated primers, and then the whole alginate lyase gene, named alyA, and its flanking regions were obtained from a cosmid library of AR06 genomic DNA. The alyA mutant of AR06 showed (1) the loss of alginate depolymerization activity on alginate agar plate and (2) significant growth defects in alginate minimal medium; these defects were complemented by the introduction of the alyA gene. Furthermore, zymography and biochemical analyses revealed that three extracellular protein bands of AR06 had alginate lyase activities and that all three protein bands were derived from the nascent alyA gene product. These results clearly indicated that the alyA gene greatly contributes to the assimilation of alginate in AR06. The transcription of the alyA gene was induced by the presence of alginate in minimal medium, but its obvious induction was not observed in rich medium even in the presence of alginate.

Fish Protein Stimulated the Fibrinolysis in Rats

Objective: We hypothesized that fish protein affects blood coagulation and/or fibrinolysis, and compared the activity and amounts of factors involved in blood coagulation and fibrinolysis in rats fed the fish protein, which was treated to remove water-soluble and ethanol-soluble elements, from sardine (sardine protein). Methods: In the first experiment, rats were fed for 21 days an AIN-93G-based control diet, and diets in which the casein of the control diet was exchanged for sardine protein at 5, 10 and 20% levels. In the second experiment, rats were fed an AIN-93G control diet and diets containing 5% fish oil, 10% sardine protein or both (5% fish oil + 10% sardine protein) for 21 days. At the end of the experiments, blood coagulation time, hemostatic parameters and fibrinolysis parameters were measured. Results: The activated partial thromboplastin time (APTT), which is an assay for blood coagulation time in the intrinsic blood coagulation pathway, of rats fed the 20% sardine protein diet was significantly prolonged compared to that of rats fed the control diet. The prolonged APTT by dietary sardine protein was due to a significant decrease of the activities of plasma blood coagulation factors VIII, IX, XI and XII. On the other hand, dietary sardine protein significantly increased the activity of tissue-type plasminogen activator, and the amount of plasma plasmin–α2-plasmin inhibitor complex, which are markers of activated plasmin. Moreover, we observed that the 20% sardine protein diet increased the amount of plasma D-dimer, which is a degraded product of the fibrin polymer by plasmin. In the second experiment, the APTT and PT of rats fed the F diet were prolonged compared to those of rats fed the control diet, however the concentration and amount of fibrinolytic parameters in the plasma were almost the same as those of rats fed the control diet. In contrast, the F+S diet not only prolonged APTT and PT, but also increased the concentration and amount of fibrinolytic parameters in plasma. Conclusions: We consider that the beneficial effects to health and amelioration of cardiovascular and cerebrovascular diseases by fish consumption are caused by a combination of the suppressing effect on blood coagulation of n–3 polyunsaturated fatty acids and the promoting effect on fibrinolysis of fish protein.

LC-MS/MS analysis of palytoxin analogues in blue humphead parrotfish Scarus ovifrons causing human poisoning in Japan.

Since 1953, a total of 27 human poisoning cases caused by the consumption of blue humphead parrotfish, Scarus ovifrons, have been reported in Japan. Characteristic symptoms are severe muscle pain associated with rhabdomyolysis. Although it is believed that palytoxin, which is one of the most potent non-protein marine biotoxins, is the most likely causative toxin in blue humphead parrotfish poisoning, palytoxin has not been proven conclusively as the causative toxin because of lack of a reliable and sensitive analytical method for palytoxin. In 2011, human poisoning cases caused by the consumption of blue humphead parrotfish occurred in Miyazaki and Tokyo. In our present study, an LC-MS/MS method for palytoxin and its analogues in the blue humphead parrotfish samples causing the human poisoning cases in 2011 was developed and the samples were analysed by using the newly developed LC-MS/MS method. Palytoxin and its analogues were not detected in the samples from the food poisoning cases. The LC-MS/MS findings therefore do not support the recently accepted hypothesis that palytoxin is the causative agent in blue humphead parrotfish poisoning in Japan.

Assimilation, Accumulation, and Metabolism of Dinophysistoxins (DTXs) and Pectenotoxins (PTXs) in the Several Tissues of Japanese Scallop Patinopecten yessoensis

Japanese scallops, Patinopecten yessoensis, were fed with the toxic dinoflagellate Dinophysis fortii to elucidate the relative magnitude of assimilation, accumulation, and metabolism of diarrhetic shellfish toxins (DSTs) and pectenotoxins (PTXs). Three individual scallops were separately exposed to cultured D. fortii for four days. The average cell number of D. fortii assimilated by each individual scallop was 7.7 × 105. Dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2) and their metabolites were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) and the toxin content in individual tissues (digestive gland, adductor muscle, gill, gonad, mantle, and the others), feces and the seawater medium were quantified. Toxins were almost exclusively accumulated in the digestive gland with only low levels being detected in the gills, mantles, gonads, and adductor muscles. DTX1 and PTX2 were the dominant toxins in the D. fortii cells fed to the scallops, whereas the dominant toxins detected in the digestive gland of scallops were PTX6 and esterified acyl-O-DTX1 (DTX3). In other tissues PTX2 was the dominant toxin observed. The ratio of accumulated to assimilated toxins was 21%–39% and 7%–23% for PTXs and DTXs respectively. Approximately 54%–75% of PTX2 and 52%–70% of DTX1 assimilated by the scallops was directly excreted into the seawater mainly without metabolic transformation.

Quantitative Nuclear Magnetic Resonance Spectroscopy Based on PULCON Methodology: Application to Quantification of Invaluable Marine Toxin, Okadaic Acid.

ERETIC2 (Electronic Reference To access In vivo Concentrations 2) based on PULCON (Pulse Length–based Concentration determination) methodology is a quantitative NMR (qNMR) using an external standard. The performance of the PULCON method was assessed using maleic acid (MA). Quantification of the diarrhetic shellfish toxin and okadaic acid by PULCON was successfully consistent with that obtained by a conventional internal standard method, demonstrating that the PULCON method is useful for the quantification of invaluable marine toxins without any contaminations by an internal standard.

Influence of Temperature on Growth and Production of Pectenotoxin-2 by a Monoclonal Culture of Dinophysis caudata

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature affected the average specific growth rate (µ) during the exponential growth phase (EG), which increased from 15 °C to 30 °C, and then decreased at 32.5 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that this strain of D. caudata produced only pectenotoxin-2 (PTX-2) whose concentration increased significantly with incubation period, except at 32.5 °C. It was significantly different between temperatures ≤18 °C, ≥21 °C, and 32.5 °C. The cellular toxin production (CTP, pg·cell−1·day−1) showed variation with growth phase and temperature, except at 32.5 °C. The average net toxin production (Rtox) was not affected by temperature. During EG, the average specific toxin production rate (µtox) increased significantly with increase in temperature, reaching a peak of 0.66 ± 0.01 day−1 at 30 °C, and then decreased. Over the entire growth span, µtox was significantly correlated to µ, and this correlation was most significant at 27 and 30 °C. During EG, µtox was affected by both temperature and growth. This study shows that temperature affects growth and toxin production of this strain of D. caudata during EG. In addition, a positive correlation was found between toxin production and growth.

Anatomical Distribution of Diarrhetic Shellfish Toxins (DSTs) in the Japanese Scallop Patinopecten yessoensis and Individual Variability in Scallops and Mytilus edulis Mussels: Statistical Considerations

Diarrhetic shellfish toxins (DSTs) are a group of phycotoxins that include okadaic acid (OA)/dinophysistoxin (DTX) analogues. At present, detailed data on the distribution of DST is insufficient, and studies of the appropriate sample sizes are lacking. This study investigated the DST frequency distribution in scallops and mussels by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and a resampling analysis of existing data was carried out. The DST population-interval and the necessary sample size were also estimated. DSTs are localized in the scallop digestive-gland, and the DST concentrations in scallops were water-depth-dependent. DST concentrations in scallops and mussels showed normal distributions, but mussels tended to contain more DSTs than scallops. In the statistical resampling analysis of the acquired data on scallops and mussels, especially that using the bootstrap method, sample size was difficult to estimate when the DST variation was large. Although the DST population-interval could be statistically estimated from the sample standard deviation of three samples, the sample size corresponded to the risk management level, and the use of 13 or more samples was preferable. The statistical methods used here to analyze individual contents and estimate population content-intervals could be applied in various situations and for shellfish toxins other than DSTs.

Identification of Causative Ciguatoxins in Red Snappers Lutjanus bohar Implicated in Ciguatera Fish Poisonings in Vietnam

Ciguatera fish poisoning (CFP) is a type of food poisoning caused by the consumption of a variety of toxic ciguatera fish species in the tropical and subtropical waters. Although there have been a large number of suspected CFP cases in the Southeast Asian countries, few were confirmed with causative ciguatoxins (CTXs), and reliable information on the symptoms still remains rather limited. In the present study, CTXs in red snapper Lutjanus bohar, implicated in two suspected CFP cases in Vietnam in 2014 and 2016, were determined by use of the single-quadrupole selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS). Ciguatoxin-1B (CTX-1B), 54-deoxyCTX-1B, and 52-epi-54-deoxyCTX-1B were detected in the red snapper by our LC/MS method. Moreover, CTX-1B, 54-deoxyCTX-1B, and 52-epi-54-deoxyCTX-1B were further identified by the time of flight (TOF) LC/MS with the exact mass spectrum. The CTX profile of the red snapper in Vietnam is similar to those of ciguatera fish from Australia, Okinawa Islands in Japan, Kiribati, and Hong Kong. This is the first comprehensive report unambiguously identifying the causative toxins in fish implicated with reliable information on the poisoning symptoms in CFP in Vietnam and/or Southeast Asian countries.