Improvement of Versatility and Analytical Range of AOAC Official Method 2015.06 for Selenium.

AOAC Official Method 2015.06 is not applicable for infant formula without selenium addition because of lack of sensitivity. In addition, Method 2015.06 specifies hydrogen gas as the cell gas of inductively coupled plasma (ICP)-MS instruments. There are only a few manufacturers who have formally adopted hydrogen gas. To expand the applicability of Method 2015.06 for infant formulas with lower selenium content and for ICP-MS instruments that do not use hydrogen gas as the cell gas, we modified the conditions of Method 2015.06. The results exhibited a good linearity (coefficient of determination >0.999) when the range of standard concentration was set from 0.4 to 16.0 µg/L and the cell gas was replaced with helium gas. The measurement precision was improved to an intermediate precision RSD value of 3.49%, and the recovery factor was 103.1%. This study demonstrates that helium gas can be used as the cell gas (easing restrictions in selecting an ICP-MS instrument) and expands the applicability of this method to infant formula samples with lower selenium content by modifying the sample preparation method.

The relationship between ergosterol and mycotoxin contamination in maize from various countries.

Maize is a good substrate for fungal growth and production of toxic secondary metabolites or mycotoxins. The relationships between the fungal biomarker ergosterol (ERG) and mycotoxins such as aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) were investigated in maize collected from four different geographic locations. ERG and mycotoxins were measured by high-performance liquid chromatography with UV and fluorescence detection. ERG did not correlate with AFs in 139 analysed samples. OTA contamination was found in only one sample from the North American region. A significant correlation (r (2) = 0.82) was observed between ERG and ZEA. AFs and ZEA were found in 47% of all samples. Half of the samples contained more than two mycotoxins. Levels of ERG and mycotoxin contamination differed by geographical region. North American and Asian samples had higher frequencies and levels of ERG and mycotoxin contamination. No AF contamination was observed in European samples (limit of detection 0.025 µg/kg for AFB1). We conclude that samples containing less than 3 mg/kg ERG in most cases do not exceed the EU maximum limits for AFs, OTA and ZEA.

Adsorption of zearalenone to Japanese acid clay and influencing factors.

Zearalenone (ZEA) mainly contaminates grains such as corn and wheat, causing damage to livestock through ingestion of contaminated feed. Recently, various clays have been added to the feed to adsorb mycotoxins and to prevent mycotoxicosis of animals fed contaminated feeds. However the adsorption mechanism of the mycotoxin to clay is not well understood. In this study, a method to analyze the level of adsorption of ZEA to clay was developed using Japanese acid clay. Changes to the amount of the clay, concentration of ZEA, shaking time, and other parameters were evaluated to determine their influence on adsorption. The adsorption isotherms were also developed. Under conditions that mimic the gastrointestinal tract of swine, 100 % of ZEA was adsorbed to clay at a pH equivalent to the stomach, while the level of desorption under intestinal basic conditions was 1.8 %. Thus Japanese acid clay has a high ability to absorb ZEA with very little desorption under gastrointestinal conditions of the swine. Isothermal analysis suggests that the Japanese acid clay is potentially highly efficacious as a ZEA adsorbent.

Modified use of a commercial ELISA kit for deoxynivalenol determination in rice and corn silage.

The analysis of deoxynivalenol (DON) in silage samples using enzyme-linked immunosorbent assay (ELISA) often leads to an overestimation. To better analyze DON in rice and corn silages using a commercially available ELISA kit, a cleanup method using a MultiSep #226 column was developed. As a result, overestimation of DON by the influence of specific cross-reaction with acetyldeoxynivalenol (AcDON) was confirmed. In samples where AcDON was not detected by liquid chromatography with mass spectrometry (LC-MS), no samples showed a significant difference (P < 0.05) in DON amounts between ELISA with cleanup and LC-MS analysis. For the recovery study, blank silage was spiked with 0.5 or 1.0 mg/kg DON. The mean recoveries of DON determined by ELISA with cleanup and LC-MS analysis were 112 and 96%, respectively, and the relative standard deviation for the repeatability (RSDr) were 8.2 and 9.8%, respectively. No samples showed a significant difference (P < 0.05) in DON concentration determined by either ELISA or LC-MS analysis. A collaborative study to validate this rapid method was carried out using four samples, two rice and two corn silage, by 10 participating laboratories. Each sample was analyzed using blind duplicates. The mean values of DON detected were 1.5-2.3 mg/kg, RSDr and the relative standard deviation for the reproducibility (RSDR) were 4.1-12.7 and 7.6-23.4%, respectively, and the HorRat values were 0.5-1.6. Therefore, the overestimation of DON by the influence of nonspecific cross-reaction with sample matrix was reduced by the cleanup method using a MultiSep #226 column, and analysis of DON in silage was improved. This use of this method for estimation of DON contamination in silage allows rapid detection at the place of use that is likely to result in improved animal health.

Improvement and validation of the method to determine neutral detergent fiber in feed.

To improve the performance of the analytical method for neutral detergent fiber in feed with heat-stable α-amylase treatment (aNDFom), the process of adding heat-stable α-amylase, as well as other analytical conditions, were examined. In this new process, the starch in the samples was removed by adding amylase to neutral detergent (ND) solution twice, just after the start of heating and immediately after refluxing. We also examined the effects of the use of sodium sulfite, and drying and ashing conditions for aNDFom analysis by this modified amylase addition method. A collaborative study to validate this new method was carried out with 15 laboratories. These laboratories analyzed two samples, alfalfa pellet and dairy mixed feed, with blind duplicates. Ten laboratories used a conventional apparatus and five used a Fibertec(®) type apparatus. There were no significant differences in aNDFom values between these two refluxing apparatuses. The aNDFom values in alfalfa pellet and dairy mixed feed were 388 g/kg and 145 g/kg, the coefficients of variation for the repeatability and reproducibility (CV(r) and CV(R) ) were 1.3% and 2.9%, and the HorRat values were 0.8 and 1.1, respectively. This new method was validated with 5.8% uncertainty (k = 2) from the collaborative study.

Synthesis of pyranicin and its inhibitory action with bovine heart mitochondrial complex I.

Total synthesis of pyranicin was achieved using Cl2Pd(CH3CN)2-catalyzed diastereoselective cyclization of the allylic ester as the key step. The inhibitory activity of this compound for mitochondrial NADH-ubiquinone oxidoreductase (complex I) was slightly poorer than that of ordinary mono-THF acetogenins such as cis-solamin.

Synthesis, determination of the absolute configuration of tonkinelin, and inhibitory action with bovine heart mitochondrial complex I.

The first synthesis of two possible diastereomers of tonkinelin was achieved. By comparison of the optical rotation of two candidates of tonkinelin and the natural compound, it is suggested that the absolute configuration of natural tonkinelin is likely to be (17S,18S). The inhibitory activity of these compounds was examined with bovine heart mitochondrial NADH-ubiquinone oxidoreductase. These compounds showed remarkably weak inhibitory activity compared to ordinary acetogenins such as bullatacin.

Induced secretion of insulin-like growth factor binding protein-1 (IGFBP-1) in human hepatoma cell HepG2 by rubratoxin B.

The induction of insulin-like growth factor binding protein-1 (IGFBP-1) secretion by rubratoxin B was investigated using human hepatoma cell line HepG2; we also documented the involvement of stress-activated MAP kinases [c-Jun-N-terminal kinases (JNKs) and p38s] in this process. Rubratoxin B dramatically enhanced IGFBP-1 secretion, which peaked at a concentration of 40 microg/ml. The amount of IGFBP-1 mRNA increased with time and plateaued at 6 h. Compared with the amounts of IGFBP-1 secreted, the induction ratios of transcription were much smaller, indicating that IGFBP-1 secretion is regulated chiefly post-transcriptionally. The result of concomitant treatment with rubratoxin B and JNK inhibitor indicated that JNKs do not affect rubratoxin B-induced IGFBP-1 secretion. Alternatively, rubratoxin B-associated induction of IGFBP-1 secretion was marked in the absence of p38 inhibitor but attenuated in its presence. Therefore, p38s appear to stimulate rubratoxin B-induced IGFBP-1 secretion. Treatment with p38 inhibitor slightly increased the amount of rubratoxin B-induced IGFBP-1 mRNA. However this induction ratio was smaller than that of rubratoxin B-induced secretion, suggesting that p38s regulate IGFBP-1 secretion both transcriptionally and post-transcriptionally. In this study, we showed that rubratoxin B induces IGFBP-1 levels in HepG2 cells and p38s contribute to this process.

Synthesis of murisolin, (15R, 16R, 19R, 20S)-murisolin A, and (15R, 16R, 19S, 20S)-16,19-cis-murisolin and their inhibitory action with bovine heart mitochondrial complex I.

The asymmetric total synthesis of murisolin, (15R, 16R, 19R, 20S)-murisolin A, and (15R, 16R, 19S, 20S)-16,19-cis-murisolin was performed by using an epoxy alcohol as a versatile chiral building block for synthesizing the stereoisomers of mono-THF annonaceous acetogenins. The inhibitory activity of these murisolin compounds was examined with bovine heart mitochondrial complex I, and they showed almost the same activity.

Stress-activated MAP kinases regulate rubratoxin B-caused cytotoxicity and cytokine secretion in hepatocyte-derived HepG2 cells.

The effects of stress-activated MAP kinases (SAPKs) on biological phenomena in HepG2 cells caused by the hepatotoxin rubratoxin B were investigated. The amounts of phosphorylated (active) SAPKs (c-Jun N-terminal kinases (JNKs) and p38s) were significantly increased after treating cells with rubratoxin B, suggesting that rubratoxin B exerts its toxicity through SAPK signal transduction pathways. Compared with rubratoxin B-treatment alone, treatment with both rubratoxin B and the JNK inhibitor SP600125 decreased cell morphology changes and the activity of the apoptosis-related enzymes caspase-3 and caspase-7, indicating that JNKs are involved in rubratoxin B-induced apoptosis. The p38 inhibitor SB203580 had the same general effects as SP600125; however, its effects were rather weak. The percent inhibition of cell proliferation by SAPKs were nearly the same with or without rubratoxin B, suggesting that the regulation of SAPKs is independent of rubratoxin B effects. SAPK inhibitors decreased rubratoxin B-induced secretion of interleukin-8 and macrophage colony stimulating factor; SP600125 impaired rubratoxin B-induced granulocyte-macrophage colony stimulating factor secretion, but SB203580 enhanced this secretion. The effects of SAPK inhibitors on the levels of cytokine mRNAs showed basically the same pattern as their effects on cytokine secretion, except that their relative effects on mRNA levels was smaller. Thus, SAPKs play important roles in rubratoxin B-induced cytokine secretion, mainly post-transcriptionally.

Rubratoxin B induced the secretion of hepatic injury-related colony stimulating factors in human hepatoma cells.

The induction of cytokine secretion by rubratoxin B was investigated using human hepatoma cell lines HepG2 and HuH-7. Interleukin (IL)-8, macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage (GM)-CSF were detected in the media of rubratoxin B-treated both cell lines, and their levels peaked at about 40 microg/ml. Rubratoxin B-induced cytokine secretion was enhanced by tumor necrosis factor (TNF)-alpha in HepG2 cells. While emodin increased GM-CSF secretion, the secretion of the others was decreased, indicating that they are regulated differently in rubratoxin B-treated HepG2 cells. To our knowledge, this is the first report that an exogenous stimulus induced the secretion of M-CSF and GM-CSF in hepatocyte-derived hepatoma cells, suggesting that rubratoxin B is an excellent model compound to study the mechanisms of M-CSF and GM-CSF secretion. Our results showed that hepatotoxin rubratoxin B has a potential to induce the secretion of these two CSFs, implying that these factors play roles in rubratoxin B-caused hepatic injury.