Caratterizzazione dei composti fenolici in tessuti ed essudati radicali di Parietaria judaica sottoposta a condizioni di Fe carenza dirette ed indotte

La ferro carenza nelle piante \ue8 causa di una notevole riduzione delle rese agricole. Il sintomo pi\uf9 evidente della carenza di ferro (Fe) \ue8 la clorosi ferrica, ovvero l'ingiallimento internervale delle foglie pi\uf9 giovani che denota una diminuzione del contenuto in clorofilla. Il principale fattore che incide sull'acquisizione del Fe da parte delle piante \ue8 il pH del suolo. Infatti, pH elevati rendono il Fe, che in suoli ben aereati \ue8 presente nella sua forma ferrica, meno disponibile portando la concentrazione del Fe nella soluzione del suolo ben al di sotto di quella richiesta per una crescita ottimale. La carenza di Fe diventa particolarmente importante nei suoli calcari dove la sua biodisponibilit\ue0 diminuisce in modo drastico. In questi ambienti alcalini le forme ferriche sono estremamente insolubili creando una condizione di carenza funzionale del micronutriente. Le piante dicotiledoni attivano i meccanismi della Strategia I per facilitare l\u2019acquisizione di Fe che si basano fondamentalmente sull\u2019acidificazione della rizosfera e sulla riduzione del Fe3+ e successivo trasporto. Studi precedenti hanno confermato l\u2019induzione di tutti i meccanismi della Strategia I da parte di P. judaica sottoposta a condizioni di Fe carenza (Dell\u2019Orto et al. 2003), l\u2019aumento del contenuto di acidi organici e polifenoli (Donnini et al. 2012) e il relativo adeguamento metabolico (Tato et al. 2013). In particolare l\u2019elevato contenuto in polifenoli gioca senza dubbio un ruolo importante nella strategia adattativa di P. judaica agli ambienti calcarei. Per meglio determinare il ruolo svolto dai polifenoli \ue8 stata condotta la caratterizzazione dei composti fenolici in P. judaica su campioni di tessuti radicali prelevati in campo e su campioni di radici e di essudati di piante allevate in idroponica in condizioni di bassa disponibilit\ue0 di Fe per carenza diretta o indotta dalla presenza di bicarbonato e di un sistema tampone alcalino. L'analisi HPLC ha confermato l'incremento quantitativo dei composti fenolici indotto sia dalla mancanza di Fe sia dalla ridotta biodisponibilit\ue0 dovuta ai trattamenti alcalini (bicarbonato e buffer alcalino). Inoltre ha messo in evidenza le caratteristiche qualitative di tale incremento nelle radice e nei rispettivi essudati

Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane

The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of 31P- and 1H-NMR analyses of the lipid extract. In total, 10 lipid molecules per retinal were found to be present in the PM lipid extract: 2–3 molecules of phosphatidylglycerophosphate methyl ester (PGP-Me), 3 of glycolipid sulfate, 1 of phosphatidylglycerol, 1 of archaeal glycocardiolipin (GlyC), 2 of squalene plus minor amounts of phosphatidylglycerosulfate (PGS) and bisphosphatidylglycerol (archaeal cardiolipin) (BPG) and a negligible amount of vitamin MK8. The novel data of the present study are necessary to identify the lipids in the electron density map, and to shed light on the structural relationships of the lipid and protein components of the PM. —Corcelli, A., V. M. T. Lattanzio, G. Mascolo, P. Papadia, and F. Fanizzi. Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane. J. Lipid Res. 2002. 43: 132–140

Critical evaluation of LC-MS-based methods for simultaneous determination of deoxynivalenol, ochratoxin A, zearalenone, aflatoxins, fumonisins and T-2/HT-2 toxins in maize

The results of a proficiency test for the LC-MS/(MS) determination of up to 11 mycotoxins (aflatoxins B1, B2, G1 and G2, fumonisins B1 and B2, ochratoxin A, deoxynivalenol, T-2 and HT-2 toxins and zearalenone) in maize were evaluated to identify possible strengths and weaknesses of various methodologies used by the 41 participating laboratories. The majority of laboratories (56%) used mixtures of acetonitrile:water for extraction. Other laboratories used methanol:water mixtures (17%) or performed two consecutive extractions with phosphate buffer solution (PBS) followed by methanol (15%). Few laboratories used mixtures of acetonitrile:water:methanol (7%), water:ethyl acetate (2.5%) or PBS alone (2.5%). The majority of laboratories (58%) used a clean-up step prior to chromatography. The remaining laboratories analysed crude extracts (37%) or used a mixed approach (5%). The amount of sample equivalent injected into LC-MS/(MS) ranged between 0.1-303 mg for purified extracts and 0.08-20 mg for directly analysed crude extracts. External (54%), matrix-matched (22%) or stable isotope-labelled internal standards calibration (24%) were used for toxin quantification. In general, extraction mixtures of water with acetonitrile, methanol or both provided good results for quantitative extraction of mycotoxins from maize. Laboratories using sample extract clean-up reported acceptable results for the majority of mycotoxins. Good results were also obtained by laboratories that analysed crude extracts although a high variability of results was observed for all tested mycotoxins. Matrix-matched calibration or isotope-labelled internal standards efficiently compensated matrix effects whereas external calibration gave reliable results by injecting =10 mg of matrix equivalent amounts. Unacceptable high recovery and high variability of fumonisin results were obtained by the majority of laboratories, which could not be explained and thus require further investigation. These findings provide the basis for the optimization and selection of methods to be used in future interlaboratory validation studies to derive their performance characteristics for simultaneous determination of mycotoxins in maize

Results of a proficiency test for multi-mycotoxin determination in maize by using methods based on LC-MS/(MS)

Liquid chromatography coupled with single or tandem mass spectrometry (LC-MS/(MS)) is routinely used for the simultaneous determination of mycotoxins in food and feed although official methods using this technique have not yet been adopted by the European Committee for Standardization and the Association of Analytical Communities. A proficiency test (PT) was conducted for the simultaneous determination of up to 11 mycotoxins (aflatoxin B-1 (AFB(1)), aflatoxin B-2 (AFB(2)), aflatoxin G(1) (AFG(1)), aflatoxin G(2) (AFG(2)), ochratoxin A (OTA), deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEA), fumonisin B-1 (FB1) and fumonisin B-2 (FB2)) in maize using LC-MS/(MS) to benchmark laboratories currently using this technique and to obtain information on currently used methodologies and method-related performances. Each participant received the following: instructions; a comprehensive questionnaire; a mixed mycotoxins calibration solution; a spiking solution (AFB(1), AFB(2), AFG(1) and AFG(2), OTA, DON, T-2, HT-2, ZEA, FB1 and FB2); and two test materials, namely a contaminated maize sample and a blank maize sample to be spiked with a spiking solution containing 11 mycotoxins. Laboratory results were rated with z-scores. Of the 64 laboratories enrolled in the PT, 41 laboratories from 14 countries returned 43 sets of results for various combinations of analytes. The majority of laboratories (61%) reported results for all 11 mycotoxins, whereas the remaining laboratories reported results for a restricted combination (from 2 to 10 analytes). For contaminated maize and spiked maize the percentage of satisfactory z-score values (vertical bar z vertical bar 3) was obtained for FB1 (31%), FB2 (32%), AFB(1) (32%) and AFB(2) (32%) in contaminated maize and for DON (35%), FB1 (63%) and FB2 (52%) in spiked maize. Mean recovery results were acceptable for all mycotoxins (74% to 109%), except for fumonisins, where these were unacceptably high (159% for FB1 and 163% for FB2). A robust and reliable method for simultaneous determination of 11 mycotoxins in maize could not be identified from the results of this PT. Additional experimental work is necessary to set up a method suitable for inter-laboratory validation. The results of this PT and the relevant method's details can be useful to identify methodology strengths and weaknesses

Critical evaluation of LC-MS-based methods for simultaneous determination of deoxynivalenol, ochratoxin A, zearalenone, aflatoxins, fumonisins and T-2/HT-2 toxins in maize

The results of a proficiency test for the LC-MS/(MS) determination of up to 11 mycotoxins (aflatoxins B1, B2, G1 and G2, fumonisins B1 and B2, ochratoxin A, deoxynivalenol, T-2 and HT-2 toxins and zearalenone) in maize were evaluated to identify possible strengths and weaknesses of various methodologies used by the 41 participating laboratories. The majority of laboratories (56%) used mixtures of acetonitrile:water for extraction. Other laboratories used methanol:water mixtures (17%) or performed two consecutive extractions with phosphate buffer solution (PBS) followed by methanol (15%). Few laboratories used mixtures of acetonitrile:water:methanol (7%), water:ethyl acetate (2.5%) or PBS alone (2.5%). The majority of laboratories (58%) used a clean-up step prior to chromatography. The remaining laboratories analysed crude extracts (37%) or used a mixed approach (5%). The amount of sample equivalent injected into LC-MS/(MS) ranged between 0.1-303 mg for purified extracts and 0.08-20 mg for directly analysed crude extracts. External (54%), matrix-matched (22%) or stable isotope-labelled internal standards calibration (24%) were used for toxin quantification. In general, extraction mixtures of water with acetonitrile, methanol or both provided good results for quantitative extraction of mycotoxins from maize. Laboratories using sample extract clean-up reported acceptable results for the majority of mycotoxins. Good results were also obtained by laboratories that analysed crude extracts although a high variability of results was observed for all tested mycotoxins. Matrix-matched calibration or isotope-labelled internal standards efficiently compensated matrix effects whereas external calibration gave reliable results by injecting =10 mg of matrix equivalent amounts. Unacceptable high recovery and high variability of fumonisin results were obtained by the majority of laboratories, which could not be explained and thus require further investigation. These findings provide the basis for the optimization and selection of methods to be used in future interlaboratory validation studies to derive their performance characteristics for simultaneous determination of mycotoxins in maize

Results of a proficiency test for multi-mycotoxin determination in maize by using methods based on LC-MS/(MS)

Liquid chromatography coupled with single or tandem mass spectrometry (LC-MS/(MS)) is routinely used for the simultaneous determination of mycotoxins in food and feed although official methods using this technique have not yet been adopted by the European Committee for Standardization and the Association of Analytical Communities. A proficiency test (PT) was conducted for the simultaneous determination of up to 11 mycotoxins (aflatoxin B-1 (AFB(1)), aflatoxin B-2 (AFB(2)), aflatoxin G(1) (AFG(1)), aflatoxin G(2) (AFG(2)), ochratoxin A (OTA), deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEA), fumonisin B-1 (FB1) and fumonisin B-2 (FB2)) in maize using LC-MS/(MS) to benchmark laboratories currently using this technique and to obtain information on currently used methodologies and method-related performances. Each participant received the following: instructions; a comprehensive questionnaire; a mixed mycotoxins calibration solution; a spiking solution (AFB(1), AFB(2), AFG(1) and AFG(2), OTA, DON, T-2, HT-2, ZEA, FB1 and FB2); and two test materials, namely a contaminated maize sample and a blank maize sample to be spiked with a spiking solution containing 11 mycotoxins. Laboratory results were rated with z-scores. Of the 64 laboratories enrolled in the PT, 41 laboratories from 14 countries returned 43 sets of results for various combinations of analytes. The majority of laboratories (61%) reported results for all 11 mycotoxins, whereas the remaining laboratories reported results for a restricted combination (from 2 to 10 analytes). For contaminated maize and spiked maize the percentage of satisfactory z-score values (vertical bar z vertical bar 3) was obtained for FB1 (31%), FB2 (32%), AFB(1) (32%) and AFB(2) (32%) in contaminated maize and for DON (35%), FB1 (63%) and FB2 (52%) in spiked maize. Mean recovery results were acceptable for all mycotoxins (74% to 109%), except for fumonisins, where these were unacceptably high (159% for FB1 and 163% for FB2). A robust and reliable method for simultaneous determination of 11 mycotoxins in maize could not be identified from the results of this PT. Additional experimental work is necessary to set up a method suitable for inter-laboratory validation. The results of this PT and the relevant method's details can be useful to identify methodology strengths and weaknesses