Organosoluble Fraction Research Articles

Complementary ESI and APPI high resolution mass spectrometry unravel the molecular complexity of a soil humeome

High resolution mass spectrometry (HRMS) can resolve thousands of compounds in complex mixtures such as natural organic matter. However, HRMS is seldom sufficient to fully resolve the molecular heterogeneity of Humus in the soil matrix, especially if no preliminary simplification of Humus complexity is applied and if a single ionization technique is used. Here we show that HRMS, when applied with both photoionization (APPI) and electrospray ionization (ESI) and combined with the extensive molecular simplification provided by a humeomic fractionation, significantly increases identification of the molecular composition of soil Humus. Different sequential extractions separate the soil Humeome in three organosoluble fractions (ORG1-3) and two hydrosoluble fractions (AQU2 and RESOM), which showed distinct molecular characteristics. The ORG fractions were particularly homogeneous and rich in alkyl compounds including unsaturated hydrocarbons and lipid compounds found mainly in ORG 1 and ORG 3, but also aromatic compounds comprising lignin-like molecules and condensed structures mainly detected in ORG2. The AQU2 fractions revealed greater complexity and heterogeneity due to the simultaneous detection of sugars, amino sugars, tannins and N-containing compounds not detectable in appreciable concentrations in other fractions. The most recalcitrant RESOM fraction contained highly reduced molecules and condensed structures. The combined use of APPI and ESI showed a marked selectivity in the detection of chemically different molecules separated in each fraction, thereby enhancing their molecular characterization. Specifically, APPI tended to ionize less oxidized and N-containing molecules, and compounds with high concentrations of unsaturation or aromatics, while ESI was more prone to detect highly oxidized compounds consisting of large O/C ratios. Our findings indicate that the combination of a soil Humeome fractionation with a detailed high resolution characterization of differently ionized molecules in the separated fractions enables a far deeper understanding of the molecular composition of soil Humus and the comprehension of its environmental reactivity.

Molecular properties of the Humeome of two calcareous grassland soils as revealed by GC/qTOF-MS and NMR spectroscopy

A Humeomic fractionation revealed the humus molecular composition of two uncropped calcareous soils of Northern France and differentiated the soils Humeome by extracting humic components first unbound to the organo-mineral matrix and then liberated from their progressively stronger intermolecular and intramolecular ester and ether linkages. We separated organo- (ORG1-3) and water-soluble (AQU2 and AQU4) fractions, a final extractable fraction (RESOM) and soil residues. Organo-soluble fractions were studied by GC coupled with high-resolution mass spectrometry (GC/qTOF-MS), all fractions underwent mono- and two-dimensional liquid-state NMR (except for the iron-rich AQU4 fraction), while solid-state 13C-CPMAS-NMR spectroscopy analyzed soil residues. The Calcaric Leptosol (A) showed a larger mass extraction than the Calcaric Cambisol (B), and a greater cumulative C and N content in its Humeome. Both soils showed the greatest weight yield for AQU4 fraction, followed by ORG2, RESOM, ORG1, AQU2, and ORG3. ORG2 was the most differentiating fraction between the two soils for both compound concentration and diversity, showing a larger C content for soil A than for soil B and a different distribution in aromatic compounds, fatty acids, and dicarboxylic acids. No significant differences between soils were found for ORG 3, suggesting similar processes of OM stabilization for its recalcitrant components, mostly hydrophobic esters of alkanoic, hydroxy, and aromatic acids with linear alkanols. We confirmed that Humeomic fractionation coupled to advanced analytical instrumentations enabled a detailed molecular characterization of the soil Humeome and differentiated between the two calcareous grassland soils and the other soils previously subjected to Humeomics.

Molecular dynamics of organic matter in a tilled soil under short term wheat cultivation

Soil organic matter (SOM) or humus is essential for the agricultural and environmental functionality of soils. Humus comprises small-size heterogeneous organic molecules arranged in complex meta-stable suprastructures, whose composition can be greatly affected by land management. Here, we report the molecular dynamics of the fractions extracted from an agricultural soil cropped with wheat after one and three years of tillage. The total molecular components, named Humeome, of soil under wheat found in both hydrosoluble and organosoluble fractions isolated by the Humeomic procedure, as determined by GC–MS and high-resolution Orbitrap LC–MS, were compared to the Humeome characterized in the same soil when cropped with maize. While the three-years tillage did not vary the total soil organic carbon under both wheat and maize, the carbon recovered for the sum of Humeomic fractions isolated from soil was significantly larger for maize than for wheat, thus suggesting a general destabilization of SOM under wheat cropping. Moreover, the soil Humeome under wheat resulted more hydrophilic than under maize. While fatty acids and carbohydrates were periodically replenished by crop residues, nitrogen-containing molecules, such as amides and heterocyclic nitrogen, and iron-bound molecular systems were the SOM components mostly reduced under wheat. The losses of these compound classes from the soil Humeome was possibly attributed to the exudation differences between wheat and maize cropping. These results reveal that the molecular dynamics and stability of SOM molecular components can be controlled by crops even in a short term.

Structural characterization of carbon and nitrogen molecules in the Humeome of two different grassland soils

BackgroundThe surface layers of two German grassland soils, a silt loam soil from Dortmund (Soil A) and a sandy loam soil from Hannover (Soil B), were subjected to the Humeomics fractionation to identify the molecular composition of the soil Humeome.MethodsThe separated Humeomic fractions were analysed by high performance size exclusion chromatography hyphenated to a high-resolution electrospray-Orbitrap Mass Spectrometry (MS). Empirical formulae were obtained by mass data to describe the carbon and nitrogen molecular structures in the soil organic matter (SOM) of both soils.ResultsResults showed 175 masses for Soil A and 139 for Soil B distributed among the Humeomic fractions. Masses were classified according to molecular weight, unsaturation degree, oxygenation, and presence of nitrogen in the formula. The molecular information obtained with MS was consistent with the physical–chemical properties and environmental condition of the two grassland soils: (i) nitrogenated compounds were generally more numerous and more relatively abundant in the Humeome of Soil B, which was more anoxic and lower in C/N ratio than Soil A, (ii) highly oxygenated compounds were more numerous and abundant in the more oxic Soil A, (iii) most unsaturated formulae were comparatively more abundant in Soil A than Soil B, in line with differences in environmental conditions. The empirical formulae were then matched with their molecular structures, based on the application of ChemSpider and PubChem databases, and were found to be distributed into 16 specific chemical groups. The Van Krevelen plots built on the resulting carbon and nitrogen molecular structures provided a visual comparison of the Humeome of the two soils. The organosoluble fractions in both soils were dominated by aliphatic amides and saccharide ethers, while the hydrosoluble fractions comprised mainly aromatic amides, heterocyclic nitrogen compounds, and saccharide ethers.ConclusionsEven though the two soils contained different compounds, 66 molecules were found to be common. Based only on differences in soil texture and oxic conditions, these findings indicate the existence of similar molecular dynamics in the stabilization of organic matter in these two grassland soils. The main common group in the Humeome of both soils was the saccharide ethers, which were additionally bound to aromatic compounds in the hydrosoluble fractions. Our detailed molecular study of the Humeome of these grassland soils confirms the potential of the Humeomic procedure to assess not only the carbon and nitrogen molecular composition in SOM, but also the mechanism of their long-term persistence.

Scaling up a treatment to simultaneously remove persistent organic pollutants and heavy metals from contaminated soils

Soil washing is a treatment process that can be used to remediate both organic and inorganic pollutants from contaminated soils, sludges, and sediments. A soil washing procedure was evaluated utilizing about 100 g samples of soil that had been field-contaminated with arsenic, chromium, copper, pentachlorophenol (PCP), polychlorinated dibenzo- p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The highest level of mobilization/detoxification was achieved in three soil washes with a mixture of 0.1M [ S,S]-ethyelnediaminedisuccinate ([ S,S]-EDDS) and 2% Brij 98 at pH 9 with 20 min of ultrasonication treatment at room temperature. This combination mobilized 70% of arsenic, 75% of chromium, 80% of copper, 90% of PCP, and 79% of PCDDs and PCDFs, so that the decontaminated soil met the maximum acceptable concentrations of the generic C-level criteria regulated by the Ministère du Développement Durable, de l’Environnement et des Parcs for the Province of Québec, Canada. The organic pollutants were back-extracted from the aqueous suspension with hexane. Heavy metals were virtually completely precipitated from the aqueous washing suspension with Mg 0 particles at room temperature. The PCP was detoxified by catalytic hydrodechlorination with a stream of 5% (v/v) H 2-supercritical CO 2 that transported the organosoluble fraction through a reaction chamber containing 2% Pd/γ-Al 2O 3. In toto, this soil washing procedure demonstrates that persistent organic pollutants and selected heavy metals can be co-extracted efficiently from a field-contaminated soil with three successive washes with the same soil washing solution containing [ S,S]-EDDS and a non-ionic surfactant (Brij 98) in admixture. An industrial-scale ex situ soil washing procedure with a combination of a non-ionic surfactant and a complexing reagent seems to be a plausible remediation technique for this former wooden utility pole storage facility.

Characterization of by-products from organosolv pretreatments of yellow poplar wood ( Liriodendron tulipifera) in the presence of acid and alkali catalysts

Characterization of by-products generated during organosolv pretreatments of yellow poplar wood was performed in the presence of sulfuric acid and sodium hydroxide catalysts to elucidate the effects on further biological fermentation and provide the preliminary data for utilization of organosolv lignin. Monomeric sugars contents were 2.2–7.7% in organosoluble fraction with acid catalyst, while significantly lower amount of sugars (0.2–0.3%) were determined in that of with alkali catalyst. However, the concentrations of monomeric sugars were very low to ferment directly. Therefore, the oligomeric sugars in organosoluble fraction should be recovered to improve the economic feasibility of the organosolv pretreatment. Higher concentrations of inhibitory compounds such as acetic acid, 5-HMF and furfural were determined in the organosoluble fraction with acid catalyst. However, the concentrations were lower than the minimum inhibitory concentration to the microorganism for further biological process regardless of catalyst. Thus, the compounds could not inhibit the biological fermentation of organosoluble fraction. Precipitates, solid by-products, were also characterized by diverse analytical methods and the analysis results revealed that the precipitates were mostly composed of lignin polymers regardless of catalyst. However, the structures could be slightly modified during pretreatment and mixed with come carbohydrates by chemical bonds and/or physical association.

In vivo and in vitro metabolism of fipronil by larvae of the European corn borer Ostrinia nubilalis.

In vivo and in vitro metabolism of [14C]fipronil was examined in a susceptible European corn borer (Ostrinia nubilalis, Hübner) laboratory strain. [14C]Fipronil penetrated the larval integument slowly, with 71.5% of the applied radioactivity recovered from surface rinses 24 h after topical application. Despite this slow penetration, radioactivity was detected in both the excrement and internal organo-soluble fractions. Radioactivity in the internal aqueous fraction and tissue pellet accounted for less than 0.8% of total radioactivity. The in vivo studies suggest that fipronil oxidation to its sulfone metabolite is the major route of metabolic conversion. In vitro studies were performed using subcellular microsomal fractions isolated from European corn borer larval midguts. Cytochrome P450-dependent monooxygenase activity (methoxyresorufin O-demethylase) was consistently observed in midgut preparations, and formation and detection of the sulfone metabolite in the same midgut preparations was also NADPH-dependent and inhibited by piperonyl butoxide. In vitro metabolism results indicate microsomal monooxygenases are responsible for the conversion of fipronil to its sulfone form in the European corn borer.

Effects of capsaicin on rat liver S9‐mediated metabolism and DNA binding of aflatoxin

At concentrations of 25, 50, and 100 microM, capsaicin, which is the major component in various aspects of Capsicum hot peppers, decreased the binding of aflatoxin (AFB1) to calf thymus DNA by 19%, 44%, and 71%, respectively, in incubations with rat liver S9. At concentrations of 50 and 100 microM, capsaicin decreased the formation of AFB-DNA adducts (AFB1-N7-Gua) by 53% and 75% as determined by high-pressure liquid chromatography (HPLC). HPLC analysis of organo-soluble fractions showed that these effects correlated with a concentration-dependent decrease in S9-mediated metabolism of AFB1 by capsaicin. Capsaicin also altered the formation of water-soluble conjugates of AFB1. This was indicated by a decrease in radioactivity in water-soluble fractions and in glutathione conjugates of AFB1 analyzed by HPLC. These results suggest that capsaicin inhibited the biotransformation of AFB1 by modifying Phase I hepatic enzyme activity.

Effect of glucose administration on hamster liver S9-mediated mutagenesis, metabolism and DNA-binding of benzo[a]pyrene and aflatoxin B1

Hamster liver S9 prepared from control animals and animals given 30% glucose in drinking water 48 h before killing was used in studies of benzo[a]pyrene (BaP) and aflatoxin (AFB1)-induced mutagenesis, metabolism of BaP and AFB1, and metabolite binding to calf thymus DNA. BAP-induced mutagenesis in Salmonella typhimurium TA100 was reduced 38.5% while AFB1-induced mutagenesis was increased 36% by S9 from glucose-treated hamsters. The reduction of [3H]BaP metabolite binding to calf thymus DNA in incubations with S9 from glucose-treated hamsters correlated with a decrease in unknown BP metabolite-deoxyribonucleoside adducts isolated by high performance liquid chromatography (HPLC). Differences in the 7R and 7S-diol epoxide-1 and 2 deoxyguanosine adducts of BaP between control and glucose-treated S9 were not observed. HPLC analysis of AFB1-DNA adducts showed a 25% increase in [3H]AFB1-N7-guanine in incubations of glucose-treated S9 with [3H]AFB1 and calf thymus DNA. HPLC analysis of the organosoluble fraction of incubations with [3H]BaP and [3H]AFB1 indicated a significant effect by glucose-treated S9 on metabolism. The effect of glucose on metabolism was further reflected in the reduction of both BaP and AFB1 metabolite conjugation with glucuronide and glutathione as determined by separation on an alumina column. These results indicate that the oral administration of 30% glucose in drinking water alters hamster liver S9-mediated mutagenesis and binding of BaP and AFB1 metabolites to DNA through an effect on the metabolism of these two carcinogens.

Metabolism of 7,12-dimethylbenz[ a]anthracene by mouse mammary epithelial cells cultured serum-free inside collagen gels

Mammary epithelial cells from 3–4-month-old BALB c virgin mice were cultured inside collagen gels in the following serum-free media: Dulbecco's Modified Eagle's Medium/Ham's F-12 (1:1) supplemented with (A) insulin, bovine serum albumin, epidermal growth factor; (B) insulin, bovine serum albumin, progesterone, prolactin; (C) insulin, bovine serum albumin, progesterone, prolactin, linoleic acid. Cell number increased with all media used. At day 7 of culture, [ 3H]dimethylbenz[ a]anthracene (DMBA) was added to the cultures and its metabolism to water soluble and organic soluble compounds was determined. Mouse mammary epithelial cells were able to metabolize [ 3H]DMBA to water and organosoluble metabolites. By 72 h, 77– 94% of the added DMBA had been metabolized by the epithelial cells in the three media to water and organosoluble metabolites in equivalent amounts. The distribution between water soluble and organosoluble metabolites was approximately equivalent. The high pressure liquid chromatography profiles of organosoluble fractions from the media indicated that the major products appeared to be the phenols, 2-, 3-, or 4-hydroxydimethylbenz[ a] anthracene, the hydroxymethyl derivatives, 7-methylbenz[ a]anthracene and 7-hydroxymethylbenz[ a]anthracene, trans-3,4-dihydro-3,4-dihydroxydimethylbenz[ a] anthracene and one or two major fractions eluting just behind the marker cis-5,6-dihydro-5,6-dihydroxydimethylbenz[ a]anthracene. The major fraction eluting just ahead of the cis-5,6-dihydro-5,6-dihydroxydimethylbenz[ a] anthracene was most likely trans-8,9-dihydro 8,9-dihydroxydimethylbenz[ a] anthracene. The profiles were similar for the cells cultured in all three serum-free media. The results demonstrate that mouse mammary epithelial cells cultured inside collagen gels with serum-free media can metabolize DMBA to putative carcinogenic forms.

Ellagic acid metabolism and binding to DNA in organ explant cultures of the rat

Ellagic acid (EA) is a plant phenolic compound with postulated antimutagenic and anticarcinogenic activity. In this study, explants of esophagus, forestomach, colon, bladder, trachea, lung and liver from male Sprague-Dawley rats (130–140 g) were incubated in culture medium containing [ 3H]EA (20 μM, 4.5 μCi/ml) for 24 h at 37°C. After extraction, purification and quantitation of explant DNA significant differences in the binding of EA to the DNA was observed. The most binding occurred in esophagus and the least in lung. Analysis of the organosoluble fraction of the culture medium by high performance liquid chromatography yielded 3 metabolites of EA. None of the metabolites were identified. Elution of water-soluble metabolites from an alumina column showed that there were sulfate ester, glucuronide and glutathione conjugates of EA in the explant culture medium from all the organs. The profile of water-soluble conjugates was very similar between colon and forestomach and between trachea and lung. These results indicate that EA binds to DNA in different tissues and that tissues metabolize EA to both organosoluble and water-soluble products.

Disposition and metabolism of [ 14C]chlorpyrifos in the black imported fire ant, Solenopsis richteri Forel

The short-term disposition and metabolism of topically administered [ 14C]chlorpyrifos was assessed in the black imported fire ant ( Solenopsis richteri Forel) in the presence and absence of the mixed-function oxidase inhibitor piperonyl butoxide. Chlorpyrifos is readily absorbed into an internal organosoluble fraction which was quickly converted into a water-soluble fraction. The radioactivity was slowly excreted over a 24-hr period. Piperonyl butoxide slowed the conversion of the internal organosoluble radioactivity to the water-soluble fraction. Thin-layer chromatography indicated that piperonyl butoxide slowed the conversion of chlorpyrifos to material remaining at the origin, presumably water-soluble metabolites. The results of acid hydrolysis studies indicated that the water-soluble radioactivity was comprised mainly of conjugates. Although very little chlorpyrifos oxon was recovered in the metabolism experiments, in vitro studies on fire and head homogenates showed the compound to be an extremely potent anticholinesterase, with an I 50 of 4.6 × 10 −10 M, while a major metabolite, 3,5,6-trichloropyridinol, was an ineffective acetylcholinesterase inhibitor.

Metabolism of dibutyl- 14C-labeled dibutylaminosulfenyl derivative of carbofuran in the cotton plant

The fate of the di- n-butylaminosulfenyl moiety in 2,3-dihydro-2,2-dimethyl-7-benzofuranyl (di- n-butylaminosulfenyl)(methyl)carbamate (DBSC or Marshal) was studied in the cotton plant at 1, 3, 6, and 10 days following foliage treatment with [ di-n-butylamino- 14C]DBSC. Dibutylamine and two major radioactive metabolites were obtained following extraction of the plant tissue with a methanol-buffer containing N-ethylmaleimide (NEM), a sulfhydryl scavenger which was added to prevent the cleavage of the NS bond during the workup procedure. The most adundant radioactive material recovered from plants was identified as a product arising from the reaction between NEM and dibutylamine. Extraction of plant tissue with straight methanol-buffer solution or with methol-buffer containing other sulfhydryl scavengers resulted in 57–86% of the applied radioactivity being recovered as dibutylamine in the organosoluble fraction. When [ 14C]dibutylamine was applied to cotton leaves, most of the radioactivity, i.e., 96% of the total recovered radioactivity, was found in the organosoluble fraction as dibutylamine. Dibutylamine is the major metabolite of [ di-n-butylamino- 14C]DBSC in the cotton plant.