Se-enriched mycelia of Pleurotus ostreatus: distribution of selenium in cell walls and cell membranes/cytosol.

The incorporation of Se to fungi has been studied, focusing on element distribution among different cellular compartments and, in particular, polysaccharide structures contained in cell walls. Se-enriched mycelia of Pleurotus ostreatus were obtained in submerged cultures. The incorporation of selenium from the growth medium to mycelia was observed with the relative distribution between cytosol plus cell membranes fraction (CCM) and cell walls fraction (CW) of about 44 and 56%, respectively. CCM fractions were analyzed by size exclusion chromatography with on-line UV (280 nm) and ICP-MS detection (80Se). The results obtained showed selenium binding to components of different molecular masses (about 24% of total selenium coeluted with the compounds of molecular mass > 10 kDa). A polysaccharide-containing fraction of mycelia was treated alternatively with Tris-HCl at pH 7.5 or with chitinase. Better solubility and increased contribution of low molecular mass compounds were observed in chitinase extracts (UV detection), confirming the degradation of polysacharides by the enzyme. The total area under the ICP-MS chromatogram of chitinase extract was 2 times higher with respect to the area for Tris-HCl extract. Furthermore, the relative contribution of selenium in the low molecular mass fraction (molecular mass < 1 kDa) in chitinase extract was 72% as compared to 45% in Tris-HCl extract (based on peak area measurements with respect to total area under the chromatogram). The results obtained suggest selenium binding to chitin-containing polysaccharide structures in fungi cell walls.

Selenium speciation in dill (Anethum graveolens L.) by ion pairing reversed phase and cation exchange HPLC with ICP-MS detection.

In the present work, speciation of selenium in dill (Anethum graveolens L.), supplemented with sodium selenite during its growth, was performed using ion pairing reversed phase and cation exchange chromatography. Heptafluorobutyric acid (HFBA) was used as the ion-pairing agent in reversed phase chromatography. In cation exchange chromatography, two different gradient programs were employed for the identification of selenospecies using pyridinium formate as the mobile phase. Low molecular weight selenocompounds were extracted from root, stem and dill leaf with 0.1M HCl. Enzymatic digestion was used for the extraction of selenospecies related to high molecular weight compounds. The chromatograms obtained from different parts of the plant revealed major differences in the type of selenospecies as well as their concentrations. The major selenospecies found in different parts of the plant is Se-methyl-selenocysteine (MeSeCys). Another major Se species identified is Se-methyl-selenomethionine (MeSeMet), which has the highest relative concentration in the root indicating possible Se volatilization from that part of the plant. Selenomethionine (SeMet) is present in minor quantities in all parts of the plant.

Metallomics approach to trace element analysis in ustilago maydis using cellular fractionation, atomic absorption spectrometry, and size exclusion chromatography with ICP-MS detection.

Huitlacoche is the ethnic name of the young fruiting bodies of Ustilago maydis, a common parasite of maize. In Mexico and other Latin American countries, this fungus has been traditionally appreciated as a local delicacy. In this work a metallomics approach was used with the determination of eight elements in huitlacoche by electrothermal atomic absorption spectrometry as one facet of this approach. The results obtained indicated relatively lower concentrations of commonly analyzed metals, as referred to the data reported for other mushroom types. This effect was ascribed to different accessibilities of elements, depending on fungus substrate (lower from plant than from soil). Subcellular fractionation was accomplished by centrifugation of cell homogenates suspended in Tris-HCl buffer. Recoveries of the fractionation procedure were in the range of 71-103%. For six elements (Cr, Cu, Fe, Mn, Ni, and Pb), the mean relative contributions in cytosol, cell walls, and mixed membrane fraction were 50.7, 48.2, and 1.1% respectively. To attain the molecular weight distribution of compounds containing target elements as an additional aspect of the metallomics approach, the fungus extract (1% sodium dodecyl sulfate in Tris-HCl, 30 mmol L(-)(1), pH 7.0) was analyzed by size exclusion chromatography with UV and ICP-MS detection. With spectrophotometric detection (280 nm), the elution of high molecular weight compounds was observed in the form of one peak (MW > 10 kDa), and several lower peaks appeared at higher retention times (MW < 10 kDa). On ICP-MS chromatograms, a coelution of (59)Co, (63)Cu, (57)Fe, (202)Hg, (60)Ni, and (80)Se with the first peak on the UV chromatogram was clearly observed, indicating that a fraction of each element incorporated with high molecular weight compounds (12.7, 19.8, 33.7, 100, 19.4, and 45.8%, respectively, based on the peak area measurements). From a comparison of (80)Se and (33)S chromatograms (for sulfur analysis, the extract was obtained in the absence of SDS), both elements coeluted with the first UV peak, but their lower molecular weight compounds were apparently different. These findings may contribute to a better understanding of the accumulation of elements in mushrooms.