HPLC assay of zearalenone and reduced metabolites in S9 fractions of duck liver

HPLC analysis of zearalenone (ZEA), zearalenols (-ZOL and ß-ZOL) and zearalanols (-ZAL and ß-ZAL) was developed, in order to obtain a sensitive and reproducible method to quantify ZEA and its reduced metabolites in subcellular fractions of animal livers (S9 samples). Optimal in vitro metabolism was observed by incubating 5 mg S9 proteins with 0.016 μmol. ZEA. Acetonitrile and diethylether/chloroform mixture were compared for extraction, as well as different mobile phases and two detection modes in HPLC analysis. Extracted samples were eluted with water/acetonitrile (55:45, v/v) at a flow-rate of 1.0 ml/min-1, resulting in well separated peaks between ZEA and the metabolites. The limits of detection ranged from 0.5 to 2 ng/mg S9 proteins using UV, and from 0.04 to 4 ng/mg S9 proteins, using fluorescence detection. Fluorescence showed a ten-fold higher sensitivity than UV detection for ZEA and -ZOL. Repeatability (10 assays) was 2.7% to 6.99% for zearalenols. Day-by-day coefficients of variation for zearalenone and zeranols with UV detection were 3.3 to 8.5 %, and 2.5 to 4.3 %, respectively. This analysis applied to S9 samples from ducks after 30 min of ZEA incubation allowed to demonstrate that -ZOL is the main reduced metabolite in the duck. The present method is particularly adapted for studying in vitro metabolism of ZEA and inter-species variations

Variations in zearalenone activation in avian food species

Zearalenone (ZEA), a widely distributed oestrogenic fusariotoxin, constitutes a potential risk for human and animal health. ZEA is metabolised to the main metabolites identified in vitro and in vivo: alpha-zearalenol (α-ZOL) and beta-zearalenol (β-ZOL). The efficiency to produce alpha-reduced metabolites appears of particular interest in risk assessment as alpha-reduced metabolites constitute activated forms whereas beta-reduced metabolites are less oestrogenic than ZEA. In this study ZEA activation was compared in avian food species. ZEA and its reduced metabolites were quantified in subcellular fractions of six avian species and rat livers. The α-ZOL/β-ZOL ratio in rats was 19. The various avian food species cannot be considered to be equivalent in terms of ZEA reduction (P<0.001). Quails represented high “beta reducers”, with α-ZOL/β-ZOL ratio less than two. Weak “beta reducers” included on one part ducks and chickens showing α-ZOL/β-ZOL ratio greater than 3 and up to 5.6 and on a second part geese, showing a lower production of α-ZOL than other poultry. Comparisons of enzyme kinetics in ducks and in quails show that these variations can be explained by the action of various isoforms of dehydrogenases. These results are relevant to food safety, in the context of frequently inevitable contamination of animal feed

Comparison of two extraction methods for ergosterol determination in vegetal feeds

Ergosterol is the principal sterol of fungi in which it plays an essential role in cell membrane and other cellular constituents. This sterol is considered as a good marker of fungal contamination and of mycotoxin production. After validation of ergosterol quantification by HPLC-UV system (linearity range: 0.2 to 20.0 mg/ml, repeatability: 3.27%, between day precision: 4.75%), 2 extraction methods of ergosterol from 3 vegetal matrixes (maize, barley and wheat) were compared: the first one, normalized by the AFNOR, is based on solid phase extraction (SPE), while the other is based on liquid/liquid extraction (LLE). The LLE procedure allowed ergosterol extraction gains of around 20% for high initial sterol contents (3 to 5 mg/kg) in naturally contaminated matrixes or in spiked samples, and of 86% for low initial sterol contents (1-2 mg/kg) in maize. Moreover, the precision of ergosterol determination was comparable for the 2 methods even if it was slightly lower using LLE and was more affected by the initial ergosterol contents in vegetal matrix than by its nature. These results suggest that ergosterol contents in vegetal feeds would be underestimated with the official method (SPE) and emphasize the importance of the extraction step

Production and purification of fumonisins from a highly toxigenic Fusarium verticilloides strain

Fumonisins are the major mycotoxins produced by Fusarium verticilloides and F. proliferatum fungi which are widely found as contaminants in corn and corn screenings. These molecules are hepatotoxic and nephrotoxic for several species and carcinogenic in rodents. Moreover their consumption was linked to high prevalence of human oesophageal cancer in certain geographic areas. The aim of this work was to improve FB1 production and purification procedures in laboratory conditions in order to produce large quantities of semi-purified toxin that may be used in experimental intoxications of farm animals. We used a highly toxigenic strain of Fusarium verticilloides (NRRL-3428) isolated from feeds. Influence of substrate, temperature, water content, culture recipient size and screen analysis of the substrate on fumonisin production was tested. Optimal production was obtained when strain was grown on coarsely cracked corn with 50% water content at 21°C during 5 weeks. This allowed the production of 3 to 4 g of fumonisin B1 per kg of culture material. The composition of the extracts was found to be as follow : 54% FB1, 8% FB2, 9% FB3 and 29% of pigments coming from corn. The ratio observed between FB1 and FB2 is comparable to the one reported in naturally contaminated corn. Further purification of these extracts on SAX columns led to the removal of pigments and to obtain of fumonisins extracts pure enough to be used for intra-venous or intra-peritoneal injection

An Examination of the Relationship Between Exclusionary Discipline and Academic Success

The purpose of this case study is to determine whether traditional methods of exclusionary punishment, such as detention, suspension, and expulsion affect the grades, attendance, and behavior of students who have been removed from the classroom. I will examine students from a middle school of 450 students to find out how removal from the regular classroom setting as a consequence of their behavior affects grades, attendance, and behavior as evidenced by school records and perceptions of the participants

Plant response to environmental conditions: assessing potential production, water demand, and negative effects of water deficit

This paper reviews methods for analyzing plant performance and its genetic variability under a range of environmental conditions. Biomass accumulation is linked every day to available light in the photosynthetically active radiation (PAR) domain, multiplied by the proportion of light intercepted by plants and by the radiation use efficiency. Total biomass is cumulated over the duration of the considered phase (e.g., plant cycle or vegetative phase). These durations are essentially constant for a given genotype provided that time is corrected for temperature (thermal time). Several ways of expressing thermal time are reviewed. Two alternative equations are presented, based either on the effect of transpiration, or on yield components. Their comparative interests and drawbacks are discussed. The genetic variability of each term of considered equations affects yield under water deficit, via mechanisms at different scales of plant organization and time. The effect of any physiological mechanism on yield of stressed plants acts via one of these terms, although the link is not always straightforward. Finally, I propose practical ways to compare the productivity of genotypes in field environments, and a “minimum dataset” of environmental data and traits that should be recorded for that

Toxicokinetics of fumonisin B1 in turkey poults and tissue persistence after exposure to a diet containing the maximum European tolerance for fumonisins in avian feeds

The kinetic of fumonisin B1 (FB1) after a single IV and oral dose, and FB1 persistence in tissue were investigated in turkey poults by HPLC after purification of samples on columns. After IV administration (single-dose: 10 mg FB1/kg bw), serum concentration–time curves were best described by a three-compartment open model. Elimination half-life and mean residence time of FB1 were 85 and 52 min, respectively. After oral administration (single-dose: 100 mg FB1/kg bw) bioavailability was 3.2%; elimination half-life and mean residence time were 214 and 408 min, respectively. Clearance of FB1 was 7.6 and 7.5 ml/min/kg for IV and oral administration respectively. Twenty four hours after the administration of FB1 by the intravenous route, liver and kidney contained the highest levels of FB1 in tissues, level in muscle was low or below the limit of detection (LD, 13 µg/kg). The persistence of FB1 in tissue was also studied after administration for nine weeks of a feed that contained 5, 10 and 20 mg FB1+FB2/kg diet. Eight hours after the last intake of 20 mg FB1+FB2/kg feed (maximum recommended concentration of fumonisins established by the EU for avian feed), hepatic and renal FB1 concentrations were 119 and 22 µg/kg, level in muscles was below the LD