HR-MAS Nuclear Magnetic Resonance Research Articles

Evaluation of the Rotating-Frame Relaxation (T1ρ) Filter and Its Application in Metabolomics as an Alternative to the Transverse Relaxation (T2) Filter.

Nuclear magnetic resonance (NMR)-based metabolomic studies commonly involve the use of T2 filter pulse sequences to eliminate or attenuate the broad signals from large molecules and improve spectral resolution. In this paper, we demonstrate that the T1ρ filter-based pulse sequence represents an interesting alternative because it allows the stability and the reproducibility needed for statistical analysis. The integrity of the samples and the stability of the instruments were assessed for different filter durations and amplitudes. We showed that the T1ρ filter pulse sequence did not induce sample overheating for a filter duration of up to 500 ms. The reproducibility was evaluated and compared with the T2 filter in serum and liver samples. The implementation is relatively simple and provides the same statistical and analytical results as those obtained with the standard filters. Regarding tissues analysis, because the duration of the filter is the same as that of the spin-lock, the synchronization of the echo delays with the magic angle spinning (MAS) rate is no longer necessary as for T2 filter-based sequences. The results presented in this article aim at establishing a new protocol to improve metabolomic studies and pave the way for future developments on T1ρ alternative filters, in liquid and HR-MAS NMR experiments.

Tracking lipid profiles of Jatropha curcas L. seeds under different pruning types and water managements by low-field and HR-MAS NMR spectroscopy

Jatropha curcas L. seeds have been used as a promising source of oil for biodiesel production. In the present work, the impacts of different pruning procedures and water management on the content and lipid profile of the Jatropha seed oils were evaluated via nuclear magnetic resonance (NMR). The experiment was conducted at Research Station of ESALQ/USP, located in Piracicaba (São Paulo), Brazil. Jatropha plants were cultivated over four years under two different water conditions (irrigation – I and rainfed – R) and three pruning types (without pruning – P0, with removal of twigs at a height of 1.5m from the ground and 0.75m from the central stem – P1, and with the pruning performed at a height of 2.0m from the ground and 0.75m from the central stem – P2). The evaluation of seed oil contents was performed via low-field NMR, while the fatty acid composition was assessed by HR-MAS NMR. Low-field NMR results showed no changes in the oil content of Jatropha seeds regardless the treatment employed. 1H HR-MAS NMR spectral profile of seeds revealed the presence of well-known signals of triacylglycerols, with the predominance of signals coming from oleic acid (C18:1) and linoleic acid (C18:2). Quantitative analyses of J. curcas seeds showed that the combination of irrigation and pruning managements influenced statistically the contents of unsaturated fatty acids. Among the evaluated conditions, the lipid profile achieved with pruning at 2.0m height and 1.5m canopy diameter (named P2) under irrigation (I) conditions might be considered the most effective for biodiesel production.

1H HR-MAS NMR-based metabolomics analysis for dry-fermented sausage characterization

Proton high-resolution magic angle spinning (1H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy in combination with principal component analysis (PCA) was employed to characterize dry-fermented sausages salchichón type throughout the manufacturing process. 1H HR-MAS NMR metabolite profiling was achieved from a small sample of intact sausage after 0, 2, 4, 7, 11 and 14days of drying. Intriguingly, the obtained results enabled the identification of the three main stages in the traditional production of salchichón. Formulation, fermentation and drying-ripening periods showed distinct and characteristic metabolomic profiles. Compositional changes related to microbial activity, as well as proteolytic and lipolytic phenomena, decisive steps in such a ripening process, could be monitored through the NMR spectra. This study shows the potential of 1H HR-MAS as a rapid method for probing metabolomic profiles and compositional changes during sausages processing.

Characterization of lipid rafts in human platelets using nuclear magnetic resonance: A pilot study

Lipid microdomains (‘lipid rafts’) are plasma membrane subregions, enriched in cholesterol and glycosphingolipids, which participate dynamically in cell signaling and molecular trafficking operations. One strategy for the study of the physicochemical properties of lipid rafts in model membrane systems has been the use of nuclear magnetic resonance (NMR), but until now this spectroscopic method has not been considered a clinically relevant tool. We performed a proof-of-concept study to test the feasibility of using NMR to study lipid rafts in human tissues. Platelets were selected as a cost-effective and minimally invasive model system in which lipid rafts have previously been studied using other approaches. Platelets were isolated from plasma of medication-free adult research participants (n=13) and lysed with homogenization and sonication. Lipid-enriched fractions were obtained using a discontinuous sucrose gradient. Association of lipid fractions with GM1 ganglioside was tested using HRP-conjugated cholera toxin B subunit dot blot assays. 1H high resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectra obtained with single-pulse Bloch decay experiments yielded spectral linewidths and intensities as a function of temperature. Rates of lipid lateral diffusion that reported on raft size were measured with a two-dimensional stimulated echo longitudinal encode-decode NMR experiment. We found that lipid fractions at 10–35% sucrose density associated with GM1 ganglioside, a marker for lipid rafts. NMR spectra of the membrane phospholipids featured a prominent ‘centerband’ peak associated with the hydrocarbon chain methylene resonance at 1.3ppm; the linewidth (full width at half-maximum intensity) of this ‘centerband’ peak, together with the ratio of intensities between the centerband and ‘spinning sideband’ peaks, agreed well with values reported previously for lipid rafts in model membranes. Decreasing temperature produced decreases in the 1.3ppm peak intensity and a discontinuity at ~18°C, for which the simplest explanation is a phase transition from Ld to Lo phases indicative of raft formation. Rates of lateral diffusion of the acyl chain lipid signal at 1.3ppm, a quantitative measure of microdomain size, were consistent with lipid molecules organized in rafts. These results show that HRMAS NMR can characterize lipid microdomains in human platelets, a methodological advance that could be extended to other tissues in which membrane biochemistry may have physiological and pathophysiological relevance.

The role of water in the degradation process of paper using 1H HR-MAS NMR spectroscopy.

The thermodynamic properties of water are essential for determining the corresponding properties of every biosystem it interacts with. Indeed, the comprehension of hydration mechanisms is fundamental for the understanding and the control of paper degradation pathways induced by natural or artificial aging. In fact, the interactions between water and cellulose at the accessible sites within the fibres' complex structure are responsible for the rupture of hydrogen bonds and the consequent swelling of the cellulose fibres and consumption of the amorphous regions. In this paper we study the hydration process of cellulose in naturally and artificially aged paper samples by measuring the proton spin-lattice (T1) and spin-spin (T2) relaxation times of the macroscopic magnetization through nuclear magnetic resonance (NMR) experiments. The observed behaviour of T1 and T2 is quite complex and strictly dependent on the water content of paper samples. This has been interpreted as due to the occurrence of different mechanisms regulating the water-cellulose interaction within the fibres. Furthermore, we have measured T1 as a function of the artificial aging time comparing the results with those measured on three paper samples dated back to the 15th century. We found that the evolution of T1 in model papers artificially aged is correlated with that of ancient paper, providing therefore a way for estimating the degradation of cellulosic materials in terms of an equivalent time of artificial aging. These results provide fundamental information for industrial applications and for the preservation and restoration of cultural heritage materials based on cellulose such as ancient paper or textiles.

Correction: A novel approach for testing the teratogenic potential of chemicals on the platform of metabolomics: studies employing HR-MAS nuclear magnetic resonance spectroscopy

Correction for ‘A novel approach for testing the teratogenic potential of chemicals on the platform of metabolomics: studies employing HR-MAS nuclear magnetic resonance spectroscopy’ by Nikunj Sethi et al., RSC Adv., 2015, 5, 26027–26039.

The Effect of Antitumor Glycosides on Glioma Cells and Tissues as Studied by Proton HR-MAS NMR Spectroscopy

The effect of the treatment with glycolipid derivatives on the metabolic profile of intact glioma cells and tumor tissues, investigated using proton high resolution magic angle spinning (1H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy, is reported here. Two compounds were used, a glycoside and its thioglycoside analogue, both showing anti-proliferative activity on glioma C6 cell cultures; however, only the thioglycoside exhibited antitumor activity in vivo. At the drug concentrations showing anti-proliferative activity in cell culture (20 and 40 µM), significant increases in choline containing metabolites were observed in the 1H NMR spectra of the same intact cells. In vivo experiments in nude mice bearing tumors derived from implanted C6 glioma cells, showed that reduction of tumor volume was associated with significant changes in the metabolic profile of the same intact tumor tissues; and were similar to those observed in cell culture. Specifically, the activity of the compounds is mainly associated with an increase in choline and phosphocholine, in both the cell cultures and tumoral tissues. Taurine, a metabolite that has been considered a biomarker of apoptosis, correlated with the reduction of tumor volume. Thus, the results indicate that the mode of action of the glycoside involves, at least in part, alteration of phospholipid metabolism, resulting in cell death.

Unraveling the long-term stabilization mechanisms of organic materials in soils by physical fractionation and NMR spectroscopy

The fundamental mechanisms whereby organic inputs stabilize in soil are poorly resolved, which limits our current capacity to predict the dynamics of soil organic matter (OM) turnover and its influence on soil quality and functioning. Here we fractionated soil OM from long-term experimental field plots either unamended or amended with two organic materials of different quality (i.e., solid cattle manure and crop residues) for 44 years into five measurable and meaningful pools directly related to conceptual preservation mechanisms: dissolved OM, mineral-free particulate OM located outside aggregates (unprotected from decomposition), OM occluded within both macroaggregates and microaggregates (weakly and strongly protected by physical mechanisms, respectively), and OM intimately associated with soil mineral particles (protected by chemical mechanisms). Compared to the unamended soil, the application of cattle manure and crop residues increased total organic C content by 35 and 10%, respectively. Most of these increases (up to 60 and 72% for cattle manure and crop residues, respectively) were explained by the mineral-associated OM pool, followed by the intra-microaggregate OM fraction. In general, the distribution and dynamics of N content paralleled those of C content. As determined by a range of modern nuclear magnetic resonance (NMR) techniques, including 13C cross polarization magic angle spinning (MAS), 1H high resolution (HR)-MAS, and 1H13C heteronuclear single quantum coherence HR-MAS NMR, the mineral-associated OM fraction was found to be predominately of microbial origin, unlike free and intra-aggregate OM pools, which were dominated by plant structures at different stages of decomposition. As a whole, our results indicate that the main mechanism by which organic inputs are stabilized and OM accrues in soils is not the physical and chemical protection of undecayed or partially degraded organic structures, but the adsorption on mineral surfaces of microbial biomass and microbial by-products resulting from microbial growth, transformation, and degradation processes. It is possible that organic amendments increase more than previously thought the microbial populations of the soil, which live, thrive, and die in close association with the mineral surfaces. This mechanism appears to be enhanced with the addition of stable organic materials.

Alginate monomer composition studied by solution- and solid-state NMR – A comparative chemometric study

The potential of using 1H high-resolution (HR) magic angle spinning (MAS) nuclear magnetic resonance (NMR) of alginates suspended in D 2O or 13C cross-polarisation (CP) MAS NMR of alginate powders as an alternative method to the traditional 1H solution-state NMR method for the analysis of the alginate monomer composition (mannuronate (M)/guluronate (G) ratio) has been investigated. The MAS NMR experiments can be performed directly on the intact alginate powders and thereby avoiding the relatively time-consuming and labour intensive sample preparation required for the traditional solution-state experiment. A total of 42 different sodium alginate samples were analysed and it was found that the M/G ratio derived from the HR-MAS NMR spectra were comparable to the M/G ratio calculated from the solution-state NMR spectra. However, both these methods overestimated the M/G ratio of samples with high calcium content. In contrast, the 13C CP-MAS NMR spectra were not affected by residual calcium in the samples and thus provided more consistent measurements independent on physico-chemical phenomena such as partial solubility, viscosity and interactions with ions. It is concluded that 13C CP-MAS NMR is a most suitable candidate for accurate analysis of the M/G ratio of intact alginate powders.

NMR and X-Ray Studies of Starches Derived from Tropical Fruit Seed Gelatinization Process

Gelatinization of the starch obtained from fruit seed such as mango and cumbaru was investigated applying an analytic methodology developed in the polymer nuclear magnetic resonance (NMR) laboratory at IMA/UFRJ, employing high resolution nuclear magnetic resonance spectroscopy in the solid state for hydrogen nucleus, using 1H HR-MAS pulse sequence. The results showed that NMR can be used instead of some techniques normally applied to study the gelatinization process, because 1H HR-MAS and relaxation time allow the evaluation of this process at the molecular level. NMR was also able to indicate the best gelatinization conditions. To start this study the glass transition (Tg) determination was a first step, because the Tg of seed starches are different than the Tg of starches derived from cereal, for example. The 1H HR-MAS NMR showed some useful information on the fruit seed starches gelatinization process. X-ray measurements were also used to support the data obtained from NMR technique. It was verified, from X-ray, that mango starch exhibits crystallinity in the A form and cumbaru showed a predominant amorphous phase. The use of 1H HR-MAS was shown to be a new, powerful method to follow the gelatinization process, because this process can be understood at the molecular level.

Characterization of Polyalkylvinyl Ether Phases by Solid-State and Suspended-State Nuclear Magnetic Resonance Investigations

Pure organic polyalkylvinyl ether phases were synthesized by suspension polymerization using different ratios and compositions of n-butylvinyl ether (C4VE) and n-octadecylvinyl ether (C18VE) with triethylene glycol divinyl ether or divinylbenzene as crosslinkers, respectively. These phases were investigated by means of solid-state 13C cross-polarization magic angle spinning nuclear magnetic resonance (NMR) spectroscopy and 1H high-resolution magic angle spinning (HR MAS) NMR spectroscopy in suspended-state. A comparison of these two methods showed the substantial advantages of 1H HR MAS NMR measurements. Structure elucidation was achieved using a 2D H,H-COSY NMR experiment performed under MAS conditions enabling full peak assignment of the 1H NMR spectra of these phases. The dynamic behavior of the polyalkylvinyl ether phases was determined by employing temperature-dependent measurements of spin–lattice relaxation times (T1) as well as accumulation of a 2D wide line separation NMR spectrum.