Stable isotope dilution analysis of the fusarium mycotoxin zearalenone

Zearalenone is a secondary metabolite produced by molds of the Fusarium genus. Beside its nonsteroidal molecular structure, zearalenone has estrogenic activity and can disrupt the function of the endogenous hormone 17beta-estradiol in animals and possibly in humans. It can frequently be found in all major cereal grains as well as in processed food. Because of the estrogenic properties of zearalenone and its metabolites, legal regulations are installed in the European Union setting maximum levels in cereals and cereal products. Routine analysis of zearalenone in various commodities is carried out by HPLC with fluorescence detection, but due to the development of multi-mycotoxin methods and the reduced sample cleanup, HPLC-MS/MS has become a fast and efficient alternative. However, to achieve a reliable quantitation with this technique suitable internal standards are required. This paper reports the synthesis of stable isotope labeled 3,5- d 2-zearalenone (ZON) as internal standard for stable isotope dilution analysis. Furthermore, a method for the analysis of zearalenone by HPLC-MS/MS using 3,5- d 2-zearalenone as IS has been developed. Fifteen cereal products from the German retail markets were analyzed, of which seven contained ZON in levels from 4.9 to 45.0 microg/kg.     

Thermal degradation of the Fusarium mycotoxin deoxynivalenol

Deoxynivalenol (DON) is a toxic secondary metabolite produced by molds of the Fusarium genus, which are able to infect cereal crops in the field. Concerning its rate of occurrence and mean concentration, DON is one of the most important mycotoxins in cereal commodities. Its toxic effects range from causing diarrhea, vomiting, and gastro-intestinal inflammation to noncompetitive inhibition of the biosynthesis of proteins in eukaryotic cells. To study the stability of DON under food-processing conditions such as cooking or baking, we performed model heating experiments and screened the residue for degradation products. Heating of DON and 3-acetyldeoxynivalenol (3-AcDON), especially under alkaline conditions, gave a mixture of compounds, which were isolated and structurally elucidated by NMR and MS experiments. Three of these compounds were already known (norDON A, norDON B, and norDON C), while four were new and named 9-hydroxymethyl DON lactone, norDON D, norDON E, and norDON F. The significance of the DON degradation products was checked by analyzing commercially available food samples. norDON A, B, and C were detected in 29-66% of the samples in mean concentrations ranging from 3 to 15 microg/kg. Furthermore, cell culture experiments using IHKE cells showed that the compounds that were detected in food samples are less cytotoxic in the formazan dye cytotoxicity assay compared to DON. Whereas DON revealed a median effective concentration (EC50) at 1.1 micromol/L, all other compounds did not show any significant effect up to 100 micromol/L. These findings indicate that the degradation of DON under thermal treatment might reduce the toxicity of DON contaminated food.     

Soil–atmosphere greenhouse gas exchange in a cool,temperate Eucalyptus delegatensis forest in south-eastern Australia

Forests are the largest C sink (vegetation and soil) in the terrestrial biosphere and may additionally provide an important soil methane (CH₄) sink, whilst producing little nitrous oxide (N₂O) when nutrients are tightly cycled. In this study, we determine the magnitude and spatial variation of soil–atmosphere N₂O, CH₄ and CO₂ exchange in a Eucalyptus delegatensis forest in New South Wales, Australia, and investigate how the magnitude of the fluxes depends on the presence of N₂-fixing tree species (Acacia dealbata), the proximity of creeks, and changing environmental conditions. Soil trace gas exchange was measured along replicated transects and in forest plots with and without presence of A. dealbata using static manual chambers and an automated trace gas measurement system for 2 weeks next to an eddy covariance tower measuring net ecosystem CO₂ exchange. CH₄ was taken up by the forest soil (?51.8 μg CH₄-C m^?2 h^?1) and was significantly correlated with relative saturation (S_r) of the soil. The soil within creek lines was a net CH₄ source (up to 33.5 μg CH₄-C m^?2 h^?1), whereas the wider forest soil was a CH₄ sink regardless of distance from the creek line. Soil N₂O emissions were small (<3.3 μg N₂O-N m^?2 h^?1) throughout the 2-week period, despite major rain and snowfall. Soil N₂O emissions only correlated with soil and air temperature. The presence of A. dealbata in the understorey had no influence on the magnitude of CH₄ uptake, N₂O emission or soil N parameters. N₂O production increased with increasing soil moisture (up to 50% S_r) in laboratory incubations and gross nitrification was negative or negligible as measured through ¹⁵N isotope pool dilution.The small N₂O emissions are probably due to the limited capacity for nitrification in this late successional forest soil with C:N ratios >20. Soil–atmosphere exchange of CO₂ was several orders of magnitude greater (88.8 mg CO₂-C m^?2 h^?1) than CH₄ and N₂O, and represented 43% of total ecosystem respiration. The forest was a net greenhouse gas sink (126.22 kg CO₂-equivalents ha^?1 d^?1) during the 2-week measurement period, of which soil CH₄ uptake contributed only 0.3% and N₂O emissions offset only 0.3%.     

Analysis of the Fusarium mycotoxin moniliformin in cereal samples using 13C2-moniliformin and high-resolution mass spectrometry

Moniliformin is a mycotoxin produced by fungi of the Fusarium genus and occurs as a contaminant of different cereals worldwide. This study describes the first application of isotopically labeled (13)C(2)-moniliformin for the analysis of moniliformin in cereals. Moniliformin is a small and ionic molecule that forms only a single sensitive fragment ion in the collision cell of a tandem mass spectrometer. Therefore, the methods described in the literature for this kind of instrument observe only a single mass transition and show a relatively poor sensitivity. The use of high-resolution mass spectrometry was described to be a suitable alternative technique for the detection of this compound and was therefore applied in this study. The developed method is based on the use of strong anion exchange columns for cleanup prior to HPLC analysis and has a recovery rate of 75.3%, a limit of detection (LOD) of 0.7 μg/kg, and a limit of quantitation (LOQ) of 2.5 μg/kg. Twenty-three different cereal samples were analyzed for their moniliformin content. Twenty of them showed positive results with levels up to 126 ± 12.2 μg/kg.     

Structure of a cofactor-deficient nitrogenase MoFe protein

One of the most complex biosynthetic processes in metallobiochemistry is the assembly of nitrogenase, the key enzyme in biological nitrogen fixation. We describe here the crystal structure of an iron-molybdenum cofactor-deficient form of the nitrogenase MoFe protein, into which the cofactor is inserted in the final step of MoFe protein assembly. The MoFe protein folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits. In this structure, one of the three alpha subunit domains exhibits a substantially changed conformation, whereas the rest of the protein remains essentially unchanged. A predominantly positively charged funnel is revealed; this funnel is of sufficient size to accommodate insertion of the negatively charged cofactor.