In vivo 31P and 1H HR-MAS NMR spectroscopy analysis of the unstarved Aporrectodea caliginosa (Lumbricidae)

This study demonstrates that ³¹P and ¹H high resolution-magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR), a recently developed NMR technique, can be applied to the in vivo analysis of metabolites from an unstarved earthworm. The endogeic earthworm Aporrectodea caliginosa was cut into lengths and then the different body parts (anterior, middle, posterior) with the gut content were analyzed. With the HR-MAS NMR, metabolites show well-resolved signals, whereas, with conventional NMR, spectra are highly dependent on the gut content. ³¹P HR-MAS NMR has been used to evaluate the effect of an acute exposure of the earthworm to glyphosate. Our observations support a low toxicity of the herbicide and suggest that glyphosate could be trapped in the cutaneous mucus. Earthworms could therefore play a role in horizontal dispersion and stabilization of glyphosate in the drilosphere. Phosphorylated metabolites, such as phospholombricine and lombricine, were clearly identified and their amount measured during experiments. The ¹H HR-MAS NMR method offers the opportunity to measure, on the same sample and simultaneously, both the hydrosoluble metabolites and lipids. The data on lipid location and relative succinate concentration shed light on the physiological and metabolic functions of the different body parts of the earthworm.     

Origin of the high-frequency resonances in 1H NMR spectra of muscle tissue: an in vitro slow magic-angle spinning study

High-resolution slow magic-angle spinning (150 Hz) 1H PASS NMR spectroscopy is performed on intact excised rat m. tibialis anterior. Untreated muscles and muscles in vitro incubated in Krebs-Ringers buffer based on deuterium oxide are investigated. In the high-frequency region of the 1H NMR spectra, resonances from H4 (approximately 7.1-7.2 ppm) and H2 (approximately 8.2-8.5 ppm) in histidine are observed. In addition, a resonance appears at 6.7 ppm for the untreated muscles. However, this resonance is absent in muscles following incubation in deuterium oxide. On the basis of its behavior in deuterium oxide combined with supplementary measurements for creatine solutions, the 6.7 ppm resonance is ascribed to the amino protons in creatine. Moreover, the present study demonstrates that the observation of the 6.7 ppm resonance depends on pH, which explains earlier reports stating its occasional appearance. Finally, measurements on solutions of ATP/AMP and histidine indicate that both ATP/AMP and histidine contribute to the resonances at approximately 8.2-8.5 ppm in the 1H NMR spectra of muscle tissue.     

Studies of litter and acid insoluble soil organic matter fractions using 13C-cross polarization nuclear magnetic resonance spectroscopy with magic angle spinning

Cross polarization carbon-13 nuclear magnetic resonance spectroscopy with magic angle spinning (CP-MASS) was used to analyse for various forms of carbon in soils, a moss peat, hydrochloric acid insoluble residues from soils and peat, and litter from beech and pine trees. The chemical composition of the litters was also investigated by conventional techniques. The results show that hydrolysis with hydrochloric acid extraction removes nearly all oxygenated alkyl carbon from the soils used. It is shown that humification pathways in which carbohydrates are incorporated into humic substances via nonhydrolysable linkages are not important for the soils investigated in this work. CP-MASS data suggest that the percentage of aromatic carbon in pine leaves increases with increase in ageing time. The results for the beech leaves are not sharply defined.     

Revealing the metabonomic variation of rosemary extracts using 1H NMR spectroscopy and multivariate data analysis

The molecular compositions of rosemary ( Rosmarinus officinalis L.) extracts and their dependence on extraction solvents, seasons, and drying processes were systematically characterized using NMR spectroscopy and multivariate data analysis. The results showed that the rosemary metabonome was dominated by 33 metabolites including sugars, amino acids, organic acids, polyphenolic acids, and diterpenes, among which quinate, cis-4-glucosyloxycinnamic acid, and 3,4,5-trimethoxyphenylmethanol were found in rosemary for the first time. Compared with water extracts, the 50% aqueous methanol extracts contained higher levels of sucrose, succinate, fumarate, malonate, shikimate, and phenolic acids, but lower levels of fructose, glucose, citrate, and quinate. Chloroform/methanol was an excellent solvent for selective extraction of diterpenes. From February to August, the levels of rosmarinate and quinate increased, whereas the sucrose level decreased. The sun-dried samples contained higher concentrations of rosmarinate, sucrose, and some amino acids but lower concentrations of glucose, fructose, malate, succinate, lactate, and quinate than freeze-dried ones. These findings will fill the gap in the understanding of rosemary composition and its variations.     

Determination of arylglycerol-beta-aryl ethers and other linkages in lignins using DFRC/(31)P NMR

An analytical method for lignins has been developed that involves derivatization followed by reductive cleavage (DFRC), depolymerization, and quantitative (31)P NMR spectroscopy. This technique detects and quantifies the various ether linkages present in softwood residual kraft lignins (RKL) and milled wood lignins (MWL). In addition, the technique supplies new quantitative information about beta-aryl ethers linked to condensed and noncondensed aromatic moieties, including dibenzodioxocins. Within RKL, beta-aryl ether bonds connected to condensed phenolic moieties predominated over those connected to noncondensed phenolic moieties. In addition, the amount of DFRC monomers determined by gas chromatography was minute in the RKL but large in the MWL. This indicates that almost all noncondensed beta-aryl ether linkages were cleaved during kraft pulping. The method offers new avenues for the detailed investigation of the bonding patterns of native and technical lignins.     

Study of interactions between food phenolics and aromatic flavors using one- and two-dimensional (1)H NMR spectroscopy

Changes in flavor release and aroma characteristics in a medium including food phenolics may be attributed to an intermolecular interaction between flavor compounds and phenolics. To investigate the interaction, one- and two-dimensional NMR studies have been carried out on the binding of two phenolics, gallic acid and naringin, with three aroma compounds, 2-methylpyrazine, vanillin, and ethyl benzoate. Evaluation of thermodynamic parameters and intermolecular nuclear Overhauser effects reveals that gallic acid can interact more strongly with aromatic flavors than naringin. The supramolecular complexation is also dependent on the structural nature of the flavors, with 2-methylpyrazine and vanillin interacting more strongly than ethyl benzoate. The interaction is principally pi-pi stacking between the galloyl ring and the aromatic ring of the aroma compounds, but secondary hydrogen-bonding effects help to stabilize the complex and enhance the specificity.     

Geographical characterization of greek virgin olive oils (cv. Koroneiki) using 1H and 31P NMR fingerprinting with canonical discriminant analysis and classification binary trees

This work deals with the prediction of the geographical origin of monovarietal virgin olive oil (cv. Koroneiki) samples from three regions of southern Greece, namely, Peloponnesus, Crete, and Zakynthos, and collected in five harvesting years (2001-2006). All samples were chemically analyzed by means of 1H and 31P NMR spectroscopy and characterized according to their content in fatty acids, phenolics, diacylglycerols, total free sterols, free acidity, and iodine number. Biostatistical analysis showed that the fruiting pattern of the olive tree complicates the geographical separation of oil samples and the selection of significant chemical compounds. In this way the inclusion of the harvesting year improved the classification of samples, but increased the dimensionality of the data. Discriminant analysis showed that the geographical prediction at the level of three regions is very high (87%) and becomes (74%) when we pass to the thinner level of six sites (Chania, Sitia, and Heraklion in Crete; Lakonia and Messinia in Peloponnesus; Zakynthos). The use of classification and binary trees made possible the construction of a geographical prediction algorithm for unknown samples in a self-improvement fashion, which can be readily extended to other varieties and areas.     

Classification of edible oils by employing 31P and 1H NMR spectroscopy in combination with multivariate statistical analysis. A proposal for the detection of seed oil adulteration in virgin olive oils

A combination of (1)H NMR and (31)P NMR spectroscopy and multivariate statistical analysis was used to classify 192 samples from 13 types of vegetable oils, namely, hazelnut, sunflower, corn, soybean, sesame, walnut, rapeseed, almond, palm, groundnut, safflower, coconut, and virgin olive oils from various regions of Greece. 1,2-Diglycerides, 1,3-diglycerides, the ratio of 1,2-diglycerides to total diglycerides, acidity, iodine value, and fatty acid composition determined upon analysis of the respective (1)H NMR and (31)P NMR spectra were selected as variables to establish a classification/prediction model by employing discriminant analysis. This model, obtained from the training set of 128 samples, resulted in a significant discrimination among the different classes of oils, whereas 100% of correct validated assignments for 64 samples were obtained. Different artificial mixtures of olive-hazelnut, olive-corn, olive-sunflower, and olive-soybean oils were prepared and analyzed by (1)H NMR and (31)P NMR spectroscopy. Subsequent discriminant analysis of the data allowed detection of adulteration as low as 5% w/w, provided that fresh virgin olive oil samples were used, as reflected by their high 1,2-diglycerides to total diglycerides ratio (D > or = 0.90).     

Application of (31)P NMR spectroscopy in food analysis. 1. Quantitative determination of the mono- and diglyceride composition of olive oils

This paper introduces a facile method to determine the amount of mono- and diglycerides in virgin olive oils. This method is based on the phosphorylation of the free hydroxyls of the mono- and diglycerides with 2-chloro-4,4,5,5-tetramethyldioxaphospholane and the integration of the appropriate peaks in the (31)P NMR spectrum. Quantitative (31)P NMR spectroscopy can be extended to the quantification of other constituents of olive oils bearing functional groups with labile protons.     

Effects of companion crops and tillage on soil phosphorus in a Brazilian oxisol: a chemical and 31P NMR spectroscopy study

Journal of Soils and Sediments - Phosphorus (P) is often the main limiting factor for plant growth in highly weathered tropical soils. Phosphate use efficiency and crop yields could be increased in...     

Identification and quantification of major steviol glycosides in Stevia rebaudiana purified extracts by 1H NMR spectroscopy

The use of (1)H NMR spectroscopy for the characterization of Stevia rebaudiana extracts is presented. The developed method allows qualitative and quantitative determination of the major steviol glycosides in purified extracts and fractions obtained from various stages of the purification process. Moreover, it proved to be a powerful tool to differentiate between glycosides which are naturally occurring in the stevia plant and artifacts formed in the course of the manufacturing process. Identification of steviol glycosides was achieved by the use of 2D NMR techniques, whereas quantification is based on qHNMR using anthracene as internal standard. The solvent mixture pyridine-d(5)-DMSO-d(6) (6:1) enabled satisfactory separation of the signals to be integrated. Validation of the method was performed in terms of specificity, precision, accuracy, linearity, robustness, and stability. Quantitative results were compared to those obtained with the JECFA HPLC-UV method and were found to be in reasonable agreement. NMR analysis does not rely on the use of reference compounds and enables significantly faster analysis compared to HPLC-UV. Thus, NMR represents a feasible alternative to HPLC-based methods for the quality control of Stevia rebaudiana extracts.     

Mobility and distribution of water in cassava and potato starches by 1H and 2H NMR

Mobility and distribution of water in cassava (rainy and drought crops) and potato starches were studied by solid state and NMR relaxometry as a function of H(2)O and D(2)O contents ranging from 0 to 44% (dry basis). Measurements of relative mobility derived from (2)H solid state NMR were based on relative area and line shape analysis. The narrow peak (mobile component) started to show at 5% and increased with increasing D(2)O content. This increase in mobile fraction was accompanied by a line narrowing. The mobile fractions of deuterons reached a >98% level above 15% D(2)O, which is well below the water holding capacity of starch ( approximately 27%) and the previously assigned "glassy-rubbery transition point" (24.3%; Jouppila, K.; Roos, Y. H. The physical state of amorphous corn starch and its impact on crystallization. Carbohydr. Polym. 1997, 32, 95-104). This reconfirms the liquidlike nature of water in the so-called glassy state of starch granules. The plasticization effect of water on starch chains was observed at 14-17% for cassava and potato starches as indicated by the T(1) minimum. This, however, did not seem to relate to the difference observed in swelling among the starches studied.     

Determination of the diglyceride content in greek virgin olive oils and some commercial olive oils by employing (31)P NMR spectroscopy

In this study, the diglyceride contents of 96 samples of virgin olive oils from the regions of Crete, Lesvos, Messinia, Pilion, Zakynthos, Halkidiki, and Ilia, 15 samples of commercial extra virgin and pure olive oils, and 3 samples each of refined olive oils and pomace oils were determined by a facile method introduced in a previous publication. This method is based on the phosphitylation of the free hydroxyls of the diglycerides with 2-chloro-4,4,5,5-tetramethyldioxaphospholane and the integration of the appropriate peaks in the (31)P NMR spectra. This preliminary study showed interesting trends in the diglyceride content of the virgin olive oils from the various regions of Greece that can be used as simple criteria to assess the olive oil characteristics. Analysis of variance has been carried out for the diglyceride content of each region in an attempt to detect possible differences in the diglyceride levels among the various regions. Finally, the relationship between the ratio of 1,2-diglycerides to the total amount of diglycerides and the total amount of diglycerides has been used to monitor the quality of virgin olive oils, commercial olive oils, refined olive oils, and pomace oils.     

Analysis of monoglycerides, diglycerides, sterols, and free fatty acids in coconut (Cocos nucifera L.) oil by 31P NMR spectroscopy

Phosphorus-31 nuclear magnetic resonance spectroscopy ( (31)P NMR) was used to differentiate virgin coconut oil (VCO) from refined, bleached, deodorized coconut oil (RCO). Monoglycerides (MGs), diglycerides (DGs), sterols, and free fatty acids (FFAs) in VCO and RCO were converted into dioxaphospholane derivatives and analyzed by (31)P NMR. On the average, 1-MG was found to be higher in VCO (0.027%) than RCO (0.019%). 2-MG was not detected in any of the samples down to a detection limit of 0.014%. On the average, total DGs were lower in VCO (1.55%) than RCO (4.10%). When plotted in terms of the ratio [1,2-DG/total DGs] versus total DGs, VCO and RCO samples grouped separately. Total sterols were higher in VCO (0.096%) compared with RCO (0.032%), and the FFA content was 8 times higher in VCO than RCO (0.127% vs 0.015%). FFA determination by (31)P NMR and titration gave comparable results. Principal components analysis shows that the 1,2-DG, 1,3-DG, and FFAs are the most important parameters for differentiating VCO from RCO.     

Analysis of acid-soluble glycogen in pork extracts of two PRKAG3 genotypes by 1H liquid-state NMR spectroscopy and biochemical methods

Meat extracts with acid-soluble glycogen (macroglycogen) from M. longissmus dorsi of carriers and noncarriers of the PRKAG3 mutation (RN(-) and rn(+) genotype) were analyzed by both (1)H liquid-state NMR spectroscopy and a biochemical method. The (1)H NMR analysis revealed that shorter polymers (dimers, trimers, etc.) of α-1,4-linked glucose were generated 24-48 h post-mortem. This is not possible to elucidate with the biochemical method, by which only the total amount of hydrolyzed glucose residues is determined. The shorter polymers were primarily formed in carriers of the PRKAG3 mutation, suggesting different post-mortem glycogen degradation mechanisms in the two genotypes.     

Kinetics of diglyceride formation and isomerization in virgin olive oils by employing 31P NMR spectroscopy. Formulation of a quantitative measure to assess olive oil storage history

Diacylglycerol isomers and free acidity were determined for five extra virgin olive oils of different initial acidities by employing a facile (31)P NMR methodology as a function of storage time and storage conditions. The kinetic treatment of the hydrolysis of triacylglycerols (TGs) and the isomerization of 1,2-diacylglycerols (1,2-DGs) to 1,3-diacylglycerols (1,3-DGs) during storage of 18 months at ambient temperature in the dark and light and at 5 degrees C in the dark showed that the isomerization is strongly dependent on the rate of the TGs hydrolysis, the initial free acidity (H(0)) of the virgin olive oil samples, and storage conditions. Although the time-evolution of the diacylglycerols (DGs) depends on the TGs hydrolysis, the ratio D of the concentration of 1,2-DGs to the total amount of DGs was found to be independent of this factor. From the kinetic expression of the ratio D, a quantitative measure was formulated that allows the estimation of the storage time or age of virgin olive oils. Application of this quantitative measure to several olive oil samples of known and unknown storage history resulted in a very good agreement with respect to the actual storage time for up to 10-12 months of storage. For a longer storage period, where the isomerization of DGs is close to its equilibrium state, the calculated age index is only indicative.     

Time course of the evolution of malic and lactic acids in the alcoholic and malolactic fermentation of grape must by quantitative 1H NMR (qHNMR) spectroscopy

Quantitative NMR can be used to monitor several processes that take place in the transformation of the must of wine grapes. The study described here focused attention on monitoring of the malic and lactic acid levels during the alcoholic and malolactic fermentation processes. The method allows the simultaneous quantification of both acids through a range of 1-3.2 mmol/L. The effectiveness of each process was assessed and compared by carrying out precise analyses using enzymatic methods.     

Detection of primary and secondary oxidation products by Fourier transform infrared spectroscopy (FTIR) and 1H nuclear magnetic resonance (NMR) in sunflower oil during storage

The oxidation of sunflower oil, stored in closed receptacles at room temperature for a period of 10 years, was monitored using Fourier transform infrared spectroscopy (FTIR) and 1H nuclear magnetic resonance (NMR). The objective was to understand the evolution of the oxidation process in sunflower oil under the conditions above mentioned. These techniques provide information about the oxidative status of several oil samples and the primary and some of the secondary oxidation products formed in the oxidation process. The results obtained show that, under these conditions, sunflower oxidation takes place in a different way to that at higher temperatures with aeration. The 1H NMR spectra show that in the first oxidation stages of the process only hydroperoxides supporting cis, trans-conjugated double bonds are formed and that at more advanced stages hydroperoxides having trans, trans-conjugated double bonds are generated, with the latter always being in a smaller proportion than the former. In addition, the presence of hydroxy derivatives supporting cis, trans-conjugated double bonds among the primary oxidation compounds is shown for the first time. Also, from early oxidation stages onward and unlike the process at 70 degrees C with aeration, it is noticeable that 4-hydroxy- trans-2-alkenals are formed in much higher proportions than 4-hydroperoxy- trans-2-alkenals. This fact could be associated with the presence of hydroxy derivatives with cis, trans-conjugated double bonds among the primary oxidation products and the limited concentration of oxygen during the oxidation. Furthermore, relationships between some oxidation conditions and the oxidation level of the samples were statistically analyzed.     

Bioactive compounds in cod (Gadus morhua) products and suitability of 1H NMR metabolite profiling for classification of the products using multivariate data analyses

This work investigates the suitability of (1)H NMR spectroscopy to identify the fate of some bioactive compounds in seafood submitted to several processing conditions and examines the possibility of using (1)H NMR spectroscopy profiling to classify such products. Perchloric acid extracts of cod white muscle from newly killed and (i) unprocessed, (ii) boiled, and (iii) fried fillets and from (iv) frozen fillets, (v) the frozen fillets after thawing, and (vi) their drip loss and from (vii) rehydrated cod klippfish (n = 5) were analyzed by 500 MHz (1)H NMR spectroscopy. It was possible to identify taurine, betaine, anserine, creatine, and trimethylamine oxide (TMAO) in all extracts examined, and frozen fish was recognizable by the presence of dimethylamine (DMA). None of the heating procedures seemed to induce the loss of bioactive compounds from the fillet, but freezing and thawing did: the compounds were lost in what is known as drip loss. About 80% of the samples were correctly classified using a probabilistic neural network procedure having as inputs the scores of the first 20 principal components of the principal component analysis of a selected region of the NMR spectra.     

Tumor-promoting effect of GGN-MRP extract from the Maillard reaction products of glucose and glycine in the presence of sodium nitrite in C3H10T1/2 cells.

GGN-MRP is an extract from the Maillard reaction products of nitrite with glucose and glycine in the Maillard browning system. No genotoxicity of GGN-MRP in culture hepatocyte was found. A two-stage transformation protocol was used to transform chemically mouse embryo fibroblast C3H10T1/2 cells. To initiate transformation, the cells were treated with benzo[a]pyrene [B(a)P; 0.1 microg/mL], and GGN-MRP (0.01, 0.1, and 1.0 mg/mL) was employed to subsequently complete the transformation process. Malignant transformed foci were formed in B(a)P-initiated and GGN-MRP-promoted C3H10T1/2 cells after 8 weeks. Cells treated with GGN-MRP alone failed to induce transformation. However, cells initiated with B(a)P and promoted by GGN-MRP demonstrated oncogenic properties. Transformed colonies derived from GGN-MRP-treated cells exhibited enhanced growth rate, anchorage independence, and tumorgenicity in animals relative to parent cells. These results indicated that GGN-MRP contains a tumor promoter and may induce tumor promotion by two-stage oncogenesis.