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Production of single or double 13C and 15N isotope enriched mycotoxins

The study of many mycotoxins does not raise difficulties when they are isolated, purified and in quantities sufficient for the usual detection methods used in biology (Chromatography, NMR, Mass Spectrometry, etc). When they are found in a biological fluid (blood, urine…), they are often in infinitesimal amount such as the effect of their biological matrices or the background noise that make their detection and their quantification very delicate.

The use of internal standards uniformly enriched with carbon 13 and/or nitrogen 15 makes it possible to obtain a signal more easily recognizable and identifiable thanks to the presence of the isotopes (peaks shifted in a mass spectrum for example). In collaboration with CEA Saclay (M. Delaforge) and CEA Cadarache (M. Pean), we have developed a strategy to produce in a single process, several fungi secondary metabolites uniformly enriched with 13C and 15N stables isotopes. This includes in first time, a plant culture in the presence of 10%, 50%, 76% or 100% 13CO2 as the only carbone source and in the presence of 10% or 100% 15N-enriched nitrogen salts and in second time, a subsequent solid culture of different filamentous fungi on plant biomass obtained from plant culture. As expected wheat, maize and fungal species uniformly incorporate enriched isotopes in their bio-products.
The fungal secondary metabolites obtained on this kind of subtract exhibit a characteristic isotopic cluster detectable by mass spectrometry. The figure 1 displays the comparison between 76% 13C versicolorin C experimental and theoretical mass spectra. The theoretical mass spectra was calculated with algorithm reported in Bravin et al., 2008.
We produced a large number of secondary metabolites from diverse fungal species, enriched at varied levels: 10% 13C zearalenone from Fusarium graminearum, 10% 13C deoxynivalenol from Fusarium graminearum, 76% 13C mycophenolic acid from Penicillium brevicompactum, 10% 13C and 76% 13C sterigmatocystine from Aspergillus nidulans.
This characteristic isotopic cluster is facilitating not only the structural elucidation of metabolites by mass spectrometry but also their detection in ppm or ppb allowing pharmacokinetic studies such as metabolism or transport across cell barriers.
For example, we produced in collaboration with Dr. S. Bretagne (Laboratoire de Parasitologie, Faculté de Medicine Creteil, France) such enriched compounds with high reliability in order to study sterigmatocystin metabolism in primary tracheal epithelial cells (Cabaret et al., 2010).

Chemical Characterization of fungal secondary metabolites

Since three years, the team focused on the chemical and toxicological characterization of secondary metabolites from the human pathogen fungi, Aspergillus fumigatus. We have developed a Liquid Chromatography coupled with Mass Spectrometry (LC-MS) method allowing detection of 37 metabolites from A. fumigatus extract. Several known compounds (tryptoquivaline F, fumiquinazoline C, questin, trypacidin, fumigaclavine C and monomethylsulochrin) have been purified in order to evaluate their toxicity on A 549 cells. Their identities have been checked by nuclear magnetic resonance (NMR) analysis and high resolution mass spectrometry (LC-HRMSn). Three new tryptoquivalines have been isolated and their complete characterizations are under way.