Analysis of imidocarb in livestock and seafood products using LC-MS/MS

A simple method using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was investigated for the detection of the antiprotozoal drug imidocarb in 10 livestock and seafood products. Liquid chromatographic separation employed a TSK VMpak-25 column with ammonium acetate-acetonitrile as a mobile phase. Mass spectral acquisition was performed in the ESI positive-ion mode. Imidocarb was extracted from all samples using liquid extraction with acetonitrile under basic conditions. For samples other than honey, fat-soluble impurities were removed by acetonitrile-hexane partitioning. The salting-out technique was used for extraction from honey in order to improve the separation of the organic solvent and water added to the honey sample. The limit of quantitation was 0.005 μg/g (expressed as concentration in samples). The recoveries from all samples were 76-109%, and the repeatability and reproducibility were also satisfactory.     

LC–MS/MS with atmospheric pressure chemical ionisation to study the effect of UV treatment on the formation of vitamin D3 and sterols in plants

Some plant species are known to cause calcium intoxification in grazing animals. This has been attributed to the presence of vitamin D₃-like activity. However, research into the presence of vitamin D₃ in plants has been limited. One reason for this may be limitations in the analytical methods available for unambiguous detection and quantification of vitamin D₃. This paper presents a new method for determining vitamin D₃ and its sterol precursors. The method is based on saponification and extraction followed by solid phase clean-up of the compounds from plant leaves and detection by APCI-MS. Recoveries ranged from 101% to 114% and precision from 3% to 12%. Detection limits were 2–8 ng/g fresh weight for the substances tested. In a pilot study we found that Solanum glaucohyllum Desf. and Solanum lycopersicum L. produced vitamin D₃ after UV-treatment. The preliminary results presented suggest that vitamin D₃ formation in plants is dependent on light exposure.     

Antagonistic effect of the Ainu-selected traditional beneficial plants on the transformation of an aryl hydrocarbon receptor

Transformation of an aryl hydrocarbon receptor (AhR) is the initial step to express the multiple toxicity of halogenated and polycyclic aromatic hydrocarbons (HAHs and PAHs) including dioxins. Therefore, it has been suggested that suppression of the transformation induced by HAHs and PAHs leads to reduce their toxicological effects. In this study, the antagonistic effect of 110 indigenous plants (192 plant parts) used as medicine and/or food by the Ainu on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation was investigated. Of these, a stalk of Aralia elata (Miq.) Seemann and a bark of Fraxinus mandshurica Rupr. var. japonica Maxim. exhibited the strong antagonistic effect in a dose-dependent manner. An antioxidative activity and polyphenol content were also measured, and the strong correlation (r= 0.96) between these two parameters could be confirmed. However, correlation coefficients of the antagonistic effect of 192 extracts compared to their antioxidative activity and polyphenol content were 0.17 and 0.20, respectively. These results suggest that the Ainu-selected traditional beneficial plants are useful source for findings of novel AhR antagonists, and the antagonistic activity of these plants may be independent on their antioxidative activity and polyphenol content. PRACTICAL APPLICATION: Our findings lead to discovery of the valuable plants used by the Ainu and the novel active compounds useful for human's life, and furthermore, may contribute to the development of new medicines and functional foods.     

Isolation of methyl syringate as a specific aflatoxin production inhibitor from the essential oil of Betula alba and aflatoxin production inhibitory activities of its related compounds

Methyl syringate was isolated from the essential oil of Betula alba as an aflatoxin production inhibitor. It inhibited aflatoxin production of Aspergillus parasiticus and Aspergillus flavus with IC₅₀ values of 0.9 and 0.8 mM, respectively, without significantly inhibiting fungal growth. Methyl syringate reduced mRNA levels of genes (aflR, pksA, and omtA) encoding proteins required for aflatoxin biosynthesis. Methyl gallate, methyl 3,4,5-trimethoxybenzoate, and methyl 3-O-methylgallate inhibited both aflatoxin production and fungal growth of A. parasiticus and A. flavus. However, their acids and syringic acid did not inhibit aflatoxin production and growth of A. parasiticus significantly, although gallic acid inhibited aflatoxin production of A. flavus with selectivity. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of methyl syringate was much weaker than that of gallic acid. These results showed that methyl syringate has a unique inhibitory activity toward aflatoxin production with a different mode of action from that of gallic acid.     

Analysis of tetrodotoxin in puffer-fish by LC/MS

A simple and reliable method using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) for the analysis of tetrodotoxin in puffer-fish was developed. Tetrodotoxin in puffer-fish was extracted with 0.1% acetic acid by heating in a boiling water bath, and the extracts were cleaned up on a Bond Elut C18 (500 mg) cartridge. The LC separation was performed on a TSK-gel ODS 80Ts column (25 cm x 2 mm i.d.) using 5 mmol/L heptafluoro-n-butyric acid (HFBA)-methanol (99:1) as the mobile phase at a flow rate of 0.2 mL/min. Positive ionization produced a typical [M + H]+ molecular ion of tetrodotoxin (m/z 320.1). The calibration graph for tetrodotoxin was rectilinear from 0.1 to 1 microgram/mL with selected ion monitoring (SIM). The detection limit of tetrodotoxin in puffer-fish was 1 microgram/g (equivalent to ca. 5 MU/g).     

Detection of genetically modified organisms obtained from food samples

Genetially modified organisms (GMOs) were explored in food samples obtained from November 2000 to March 2003 in the Tokyo area by using PCR and real-time PCR techniques. The existence of Roundup Ready Soybean (RRS) was surveyed in processed foods derived from soybeans, such as tofu, boiled soybean, kinako, nama-age, abura-age, natto, miso, soymilk and yuba. RRS was detected in 3 of 37 tofu, 2 of 3 nama-age, 2 of 3 yuba and 3 of 3 abura-age samples. The CBH351 in 70 processed corn foods, NewLeaf Plus and NewLeaf Y in 50 processed potato foods, and 55-1 papaya in 16 papayas were surveyed. These GMOs were not detected among the samples. Qualitative and quantitative analyses of RRS and genetically modified (GM) corn were performed in soybean, corn and semi-processed corn products such as corn meal, corn flour and corn grits. RRS was detected in 42 of 178 soybean samples, and the amount of RRS in RRS-positive samples was determined. The content was in the range of 0.1-1.4% in identity-preserved soybeans (non-GMO), and 49.8-78.8% in non-segregated soybeans. On the other hand, GM corns were detected in 8 of 26 samples. The amount of GM corn in GM corn-positive samples was in the range of 0.1-2.0%.     

Levetiracetam Suppresses the Infiltration of Neutrophils and Monocytes and Downregulates Many Inflammatory Cytokines during Epileptogenesis in Pilocarpine-Induced Status Epilepticus Mice

Acute brain inflammation after status epilepticus (SE) is involved in blood–brain barrier (BBB) dysfunction and brain edema, which cause the development of post-SE symptomatic epilepsy. Using pilocarpine-induced SE mice, we previously reported that treatment with levetiracetam (LEV) after SE suppresses increased expression levels of proinflammatory mediators during epileptogenesis and prevents the development of spontaneous recurrent seizures. However, it remains unclear how LEV suppresses neuroinflammation after SE. In this study, we demonstrated that LEV suppressed the infiltration of CD11b⁺CD45^high cells into the brain after SE. CD11b⁺CD45^high cells appeared in the hippocampus between 1 and 4 days after SE and contained Ly6G⁺Ly6C⁺ and Ly6G⁻Ly6C⁺ cells. Ly6G⁺Ly6C⁺ cells expressed higher levels of proinflammatory cytokines such as IL-1β and TNFα suggesting that these cells were inflammatory neutrophils. Depletion of peripheral Ly6G⁺Ly6C⁺ cells prior to SE by anti-Ly6G antibody (NIMP-R14) treatment completely suppressed the infiltration of Ly6G⁺Ly6C⁺ cells into the brain. Proteome analysis revealed the downregulation of a variety of inflammatory cytokines, which exhibited increased expression in the post-SE hippocampus. These results suggest that Ly6G⁺Ly6C⁺ neutrophils are involved in the induction of acute brain inflammation after SE. The proteome expression profile of the hippocampus treated with LEV after SE was similar to that after NIMP-R14 treatment. Therefore, LEV may prevent acute brain inflammation after SE by suppressing inflammatory neutrophil infiltration.     

Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.