Unsupervised principal component analysis of NMR metabolic profiles for the assessment of substantial equivalence of transgenic grapes (Vitis vinifera).

Substantial equivalence is a key concept in the evaluation of unintended and potentially harmful metabolic impact consequent to a genetic modification of food. The application of unsupervised multivariate data analysis to the metabolic profiles is expected to improve the effectiveness of such evaluation. The present study uses NMR spectra of hydroalcoholic extracts, as holistic representations of the metabolic profiles of grapes, to evaluate the effect of the insertion of one or three copies of the DefH9-iaaM construct in plants of Silcora and Thompson Seedless cultivars. The comparison of the metabolic profiles of transgenic derivatives with respect to their corresponding natural lines pointed out that the overall metabolic changes occur in the same direction, independent of the host genotype, although the two cultivars are modified to different extents. A higher number of copies not only produces a larger effect but also modifies the whole pattern of perturbed metabolites.

New isatin derivatives with antioxidant activity.

The reaction between isatin and 2,5-dimethoxyaniline is described. The main product was identified as 3,3-bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one. The antioxidant activity of the compounds isolated was evaluated with two methods. Three published antitumor E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones entered the same tests to search whether they are endowed with antioxidant activity too. 3,3-Bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one and the three antitumor agents showed a good chemical antioxidant activity.

Facile deferration of commercial fertilizers containing iron chelates for their NMR analysis.

Ethylenediamine-N,N'-bis(o-hydroxyphenylacetic) acid (o,o-EDDHA) is widely used in commercial formulations as a Fe(3+) chelating agent to remedy iron shortage in calcareous and alkaline soils. Commercially available o,o-EDDHA-Fe(3+) formulations contain a mixture of EDDHA regioisomers (o,p-EDDHA and p,p-EDDHA), together with other, still uncharacterized, products. NMR spectroscopy can be applied to their study as long as iron is accurately removed prior to the observation. This paper shows that it is possible to obtain a deferrated solution of the organic ligands present in commercial fertilizers containing the EDDHA-Fe(3+) chelate by treating the chelate with ferrocyanide, thus forming Prussian Blue that can be easily removed by centrifugation. This iron removal process does not cause significant losses of the o,o-EDDHA ligand or its minor structural isomers.

Effect of a synbiotic food consumption on human gut metabolic profiles evaluated by (1)H Nuclear Magnetic Resonance spectroscopy.

The capacity of human lactobacilli and bifidobacteria to produce metabolites under conditions that may prevail in the human intestine has been studied "in vitro". However, the effect of systematic probiotic consumption on human metabolic phenotype has not been investigated in faeces. This paper shows the potential for the use of (1)H Nuclear Magnetic Resonance ((1)H NMR) spectroscopy for studying the changes of the metabolic profiles of human faecal slurries. Faeces of 16 subjects, characterized by different natural levels of lactobacilli and bifidobacteria were recovered before and after 1 month of supplementation with a synbiotic food based on Lactobacillus acidophilus, Bifidobacterium longum and fructooligosaccharides, and analyzed by (1)H NMR. Multivariate statistical approach has been applied to the data obtained and particularly Canonical Discriminant Analysis of Principal Coordinates (CAP). More than 150 molecules belonging to short chain fatty acids, organic acids, esters, alcohols and amino acids were detected and quantified in the samples considered. The number and the extent of these molecules in faecal slurries were strongly affected by the synbiotic food consumption and gave rise to characteristic metabolic signature. In particular, the short chain fatty acid concentrations significantly increased while the amino acids contents decreased. The comparison of the data indicated that the intake of the synbiotic food alters the host metabolism in a measure dependent on the initial level of lactobacilli and bifidobacteria detected in the faecal specimens. The analysis of (1)H NMR profiles with CAP allowed a separation of faecal samples of the subjects on the basis of the synbiotic food intake. The multivariate statistical approach used demonstrated the potential of NMR metabolic profiles to provide biomarkers of the gut-microbial activity related to dietary supplementation of probiotics.

Water absorption of freeze-dried meat at different water activities: a multianalytical approach using sorption isotherm, differential scanning calorimetry, and nuclear magnetic resonance.

Hydration of freeze-dried chicken breast meat was followed in the water activity range of aw=0.12-0.99 by a multianalytical approach comprising of sorption isotherm, differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). The amount of frozen water and the shape of the T2-relaxogram were evaluated at each water content by DSC and NMR, respectively. Data revealed an agreement between sorption isotherm and DSC experiments about the onset of bulk water (aw=0.83-0.86), and NMR detected mobile water starting at aw=0.75. The origin of the short-transverse relaxation time part of the meat NMR signal was also reinvestigated through deuteration experiments and proposed to arise from protons belonging to plasticized matrix structures. It is proved both by D2O experiments and by gravimetry that the extra protons not contributing to the water content in the NMR experiments are about 6.4% of the total proton NMR CPMG signal of meat.

A proton NMR relaxation study of hen egg quality.

A quantitative analysis of NMR proton relaxation in hen egg albumen and yolk is undertaken to research the causes of quality loss during the first few days of storage and to access the feasibility of an on-line NMR sensor of internal egg quality. It is shown that the change in the transverse relaxation in thick egg albumen mainly results from an increase in proton exchange rate resulting from a pH increase attributed to loss of carbon dioxide by diffusion through the eggshell. The results suggest that the low-field T1 is the best relaxation time indicator of albumen quality.

Gradient ion-pair chromatographic method for the determination of iron N,N'-ethylenediamine-di-(2-hydroxy-5-sulfophenylacetate) by high performance liquid chromatography-atmospheric pressure ionization electrospray mass spectrometry.

The most effective remedy for iron deficiency is the use of synthetic iron chelates, specifically chelates derived from polyaminecarboxylic acids as EDDHSA (N,N'-ethylenediamine-di-(2-hidroxy-5-sulfophenylacetic) acid). A gradient ion-pair chromatographic method was developed to quantify the total amount of chelated iron in EDDHSA/Fe3+ fertilizers. Two mobile phases were used containing, respectively, 35 and 75% acetonitrile in a 0.005 M tetrabutylammonium hydroxide aqueous solution at pH 6.0. The stationary phase was a reverse phase C-18 column (150mm x 3.9mm i.d., dp = 5 microm). Two chromatographic peaks appeared and were identified by Electrospray Mass Spectrometry. The first peak corresponds to the monomer of EDDHSA/Fe3+ and the second peak has been assigned to condensation molecules. Quality parameters indicate that the method is suitable for the quantification of iron chelate by EDDHSA fertilizers.

Nature of impurities in fertilizers containing EDDHMA/Fe(3+), EDDHSA/Fe(3+), and EDDCHA/Fe(3+) chelates.

Iron chelates derived from ethylenediaminedi(o-hydroxyphenylacetic) acid (EDDHA), ethylenediaminedi(o-hydroxy-p-methylphenylacetic) acid (EDDHMA), ethylenediaminedi(2-hydroxy-5-sulfophenylacetic) acid (EDDHSA), and ethylenediaminedi(5-carboxy-2-hydroxyphenylacetic) acid (EDDCHA) are remarkably efficient in correcting iron chlorosis in plants growing in alkaline soils. This work reports the determination of impurities in commercial samples of fertilizers containing EDDHMA/Fe(3+), EDDHSA/Fe(3+), and EDDCHA/Fe(3+). The active components (EDDHMA/Fe(3+), EDDHSA/Fe(3+), and EDDCHA/Fe(3+)) were separated easily from other compounds present in the fertilizers by HPLC. Comparison of the retention times and the UV-visible spectra of the peaks obtained from commercial EDDHSA/Fe(3+) and EDDCHA/Fe(3+) samples with those of standard solutions showed that unreacted starting materials (p-hydroxybenzenesulfonic acid and p-hydroxybenzoic acid, respectively) were always present in the commercial products. 1D and 2D NMR experiments showed that commercial fertilizers based on EDDHMA/Fe(3+) contained impurities having structures tentatively assigned to iron chelates of two isomers of EDDHMA. These findings suggest that current production processes of iron chelates used in agriculture need to be improved.