Analysis of fumonisin B1 in corn by capillary electrophoresis.

Intact fumonisins contain two tricarballylic acid groups and can therefore acquire a net negative charge. The anionic nature of the fumonisins is the basis behind the widely used method for cleanup of corn with strong anion exchange (SAX) columns. This property also enables the fumonisins to be separated by electrophoretic techniques which, until now, have not been applied to the analysis of fumonisins in corn. Fumonisin B1, extracted from corn with 80/20 (v/v) methanol/water and isolated with a commercially available affinity column, was derivatized with fluorescein isothiocyanate for analysis by capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). Recoveries from corn fortified with 0.25 to 5.0 ppm FB1 averaged 89% (range 71 to 102%). As little as 0.05 ppm FB1 could be detected in corn. For corn naturally contaminated with FB1, the CZE-LIF method compared favorably to established SAX/HPLC and C18/HPLC methods. Capillary electrophoresis can be used for quantitation of FB1 in corn, with minimal use of organic solvents and provides an additional tool for confirming fumonisin contamination.

Distribution of fumonisins in food and feed products prepared from contaminated corn.

The fate and distribution of the fumonisins B1 (FB1) and B2 (FB2) were determined in products obtained from naturally contaminated corn used for ethanol fermentation and wet milling operations. Fumonisins are stable to the conditions used in ethanol fermentations and tend to concentrate in the distillers dried grain, a fraction generally used for animal feed. No toxin was found in the ethanol. Starch from wet milling of corn, naturally contaminated at 13.9 micrograms fumonisin B1/g, was free of detectable toxin. The other fractions contained fumonisins at the following levels: gluten (5.1-5.8 micrograms FB1/g, 4.7-4.9 micrograms FB2/g); fiber (2.7-5.7 micrograms FB1/g, 2.1-3.1 micrograms FB2/g); and germ (1.3-3.1 micrograms FB1/g, 0.7-1.6 micrograms FB2/g). The steep water and process water contained 22% of the recoverable fumonisins. A combination of analytical methodologies was required to determine fumonisins in the different products from the wet milling process.

An optimized MTT bioassay for determination of cytotoxicity of fumonisins in turkey lymphocytes.

In vitro cytotoxicity assays have been performed for detection and quantitation of fumonisins, as possible alternatives for whole animal testing. This study was undertaken to establish optimal in vitro conditions using turkey lymphocytes. Turkey lymphocytes were isolated from peripheral blood by Percoll gradient centrifugation. Cytotoxicity of fumonisin B1 (FB1) and B2 (FB2) was determined by exposing lymphocytes to FB1 or FB2 at concentrations of 0.01-25 micrograms/mL for 24, 48, or 72 h at 39 degrees C. The MTT bioassay was used to measure cell viability and proliferation. In metabolically active cells, the tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], was reduced to MTT formazan. Turkey lymphocytes that had been exposed in vitro to FB1 and FB2 for 48 and 72 h showed inhibition of cell proliferation that was dose-dependent. The 50% inhibitory dose for FB1 and FB2 was 0.4-5 micrograms/mL. Cells exposed to FB1 or FB2 exhibited high levels of cytoplasmic vacuolization and were unable to proliferate, whereas proliferation of control lymphocytes was observed at 48 and 72 h. FB2 was 3- to 4-fold more cytotoxic than FB1.

Lymphocyte cytotoxicity and erythrocytic abnormalities induced in broiler chicks by fumonisins B1 and B2 and moniliformin from Fusarium proliferatum.

Peripheral blood lymphocytes were isolated from broiler chicks that had ingested feed amended with autoclaved Fusarium proliferatum culture material containing fumonisin B1 (FB1), fumonisin B2 (FB2) and moniliformin. Lymphocyte viability was determined for birds that were placed on amended rations at day 1 or day 7 of age at three different levels of mycotoxins, ranging from 61-546 ppm FB1, 14-94 ppm FB2 and 66-367 ppm moniliformin. Reduction of the tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], to yield MTT formazan, based on mitochondrial metabolic activity, was used to assess cell viability. Lymphocyte cytotoxic effects were observed in all treatment groups on day 21; chicks that started on amended feed at day 1 of age were affected more than those that started at day 7. Abnormal erythrocytes resembling early stages of erythroblasts were observed in peripheral blood from test chicks. Abnormally shaped red cells (poikilocytes) having a spindle-shape with one or both ends pointed were present. Some red cells appeared to be undergoing mitosis. Both reduced lymphocyte viability and abnormal erythrogenesis occurred in chicks given feed amended with F. proliferatum culture material containing FB1, FB2 and moniliformin.

Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture.

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.

Gas chromatographic determination of deoxynivalenol in wheat.

Modifications to a published method are described for the determination of deoxynivalenol (DON) in wheat by gas chromatography with electron capture quantitation of the heptafluorobutyrate derivative. In the modified method, DON is extracted by shaking the sample with methanol-water on a wrist-action shaker, followed by filtration through rapid flow paper. One concentration step is eliminated, and a hexane wash is incorporated to remove toluene from the silica gel column. Recoveries of DON from wheat samples spiked at 0.1, 0.5, and 1.0 ppm ranged from 77.3 to 86.3% and averaged 81.5%.