Applications of NMR Spectroscopy

Traditionally, food has not only been the source of human nutrition but also an excellent factor for deriving nutraceuticals and pharmaceuticals. The biopolymers such as protein and polysaccharides derived from food sources have special significance as they are important part of human diet. Several important bioactive peptides have been discovered from food protein hydrolysates and fermented products that have shown to possess a spectrum of beneficial pharmacological activities. This chapter deals with food protein derived bioactive peptides and their structural characterization employing NMR spectroscopy. It is well known from the vast literature on bioactive peptides that NMR spectroscopy is an indispensable tool to fully characterize the structures of bioactive peptides in their inherent physiological state. However, limited literature exists on detailed structural characterization of food derived bioactive peptides. It is therefore very important to review the literature on structural aspects of food peptides and provide an appraisal of NMR techniques suitable for comprehensive structural characterization of this class of bioactive peptides.

This chapter is aimed to introduce NMR spectroscopy and its applications to a wide range of readers in the field of food chemistry in general and to the food peptide researchers in particular. The main objective is to review the literature on food derived bioactive peptides and highlight the potential of NMR spectroscopy to understand structure-activity relationship of bioactive peptides in order to boost further developments in this important field. With a view to make this chapter readable to beginners in the field, basic principles of NMR spectroscopy will also be included.

Application of solution NMR spectroscopy to bioactive peptides will then be described. Salient features including the size and structural differences of food bioactive peptides when compared to the other classes of bioactive peptides will then be discussed in order to make an informed assessment of choice of NMR techniques suitable for their structural characterization.

Following high-level sound exposure, central auditory changes are evident long afterward. Homeostatic central mechanisms appear to compensate, and often overcompensate, for loss of peripheral sensitivity resulting from insults. Overcompensation may produce the sensation of sound without a physical correlate, i.e., tinnitus. However not everyone exposed to auditory trauma develops tinnitus. Similarly, in a controlled laboratory environment, not every animal exposed to highlevel sound develops tinnitus. Despite more than two decades of effort, tinnitus pathophysiology is incompletely understood. Contributing is the unexpected complexity of tinnitus’ central nervous system profile. Compensatory neural changes, such as increased spontaneous activity, have been identified, but they occur in the context of many other changes. Underlying mechanisms are also poorly understood. They may involve down-regulation of inhibitory neurotransmission mediated by γ-amino butyric acid (GABA), and/or up-regulation of excitatory neurotransmission, mediated by glutamic acid (Glu), or modulation by other systems, such as acetylcholine, involved in functions such as attention. Neural systems are integrated and well-regulated.

Therefore compensatory changes in one system can produce reactive changes in others. Some or all may be relevant to tinnitus, and they may contribute to the failure to develop generally effective tinnitus therapeutics. In this context the potential roles of GABA, Glu and acetylcholine (indirectly indicated via choline, Cho) were quantified in the auditory pathway of rats with and without tinnitus, using high-resolution proton magnetic resonance spectroscopy (1H-MRS). Brain volumes of interest (VOI) were the dorsal cochlear nucleus (DCN), inferior colliculus (IC), medial geniculate body (MGB), and primary auditory cortex (A1). VOI spectra were obtained using a vertical bore Varian Unity/Inova 600 mHz NMR spectrometer with a 14.1 T magnet. A hybrid short-pulse and short echo time sequence was used for microvolume localized 1H-MRS. The pulse sequence was optimized for signal acquisition in the spectral band containing the neurochemicals of interest. Signals were further optimized using a tunable pickup coil. Brain spectra were compared to external calibration spectra for determination of GABA, Glu, and Cho concentrations (mM) in each VOI. Chronic tinnitus was produced by a single high-level unilateral sound exposure, and was quantified using a psychophysical procedure sensitive to tinnitus. Contrary to expectation, significant decreases in GABA (i.e., loss of inhibition) were not found in tinnitus animals. Glu levels were found to be significantly elevated in the contralateral A1, as were GABA levels. In exposed animals without tinnitus, GABA levels were uniquely elevated in the ventral MGB, suggesting that in those animals inhibitory compensation in the MGB might counter overcompensation. Cho levels were also found to be elevated in the contralateral A1 of tinnitus animals. The observed local concentrations of GABA and Glu may reflect a distributed alteration of inhibitory-excitatory equilibrium. These results suggest that targeting multiple neurotransmitter systems when developing therapeutics could improve outcomes.

Nuclear Magnetic Resonance (NMR) spectroscopy is one of the most simple and reliable techniques for structural elucidation of polymers. This chapter summarizes the application of NMR spectroscopy for identifying monomers and polymers, and also elucidation of constitutional, configuration and conformational structures of polymers and copolymers. The determination of various polymer structures includes polymer tacticity, polymer molecular weights and molecular weight distributions, copolymer composition, monomer reactivity ratios, monomer sequence distribution in the copolymer chains. Examples are cited in most of the cases to explain the polymer characterization from the NMR spectra. It also illustrates the characterization of polymers containing nanomaterials like fullerene (C60) and carbon nanotubes (CNTs).

The present review summarizes current applications of the NMR phenomenon in paramagnetic systems for studying structure and intramolecular dynamics of multielectron molecular systems. The potential of spin labels for molecular identification was demonstrated in the example of structural biology issues. Discussion of molecular structure of specific organic heteroatomic compounds and their complexes is preceded by the section, where peculiarities of the NMR phenomenon in paramagnetic systems are considered. In opinion of the authors of the review, such consideration is needed for researchers who are not experts in the NMR theory. The separate section of the review is devoted to a method for investigation of structure and dynamics of complex molecular systems. This method is based on application of the NMR spectra transformed by complexes, first of all, paramagnetic ones (spin labels).

Magnetic resonance spectroscopy (MRS) is a diagnostic technique that permits non-invasive measurement of metabolites in tissues.

Brain Spectroscopy for diagnostic use mainly relies on two techniques: multivoxel and single voxel acquisitions. The first allows a wider extension of the analysis of tumoral and peritumoral brain tissue, with a better mapping of metabolites distribution. This technique is time consuming and can be affected by several artifacts.

The single-voxel technique allows a more robust acquisition, more repeatable, and a better visualization of metabolites spectrum but is limited by the size and position of the voxel of acquisition, based upon operator choice.

Brain MR Spectroscopy is often an essential tool in differential diagnosis of undetermined brain mass. The cases in which spectroscopy can help in differential diagnosis are: gliomas versus metastasis, gliomas versus non-neoplastic lesions and glioma versus lymphoma. The neuronal metabolite pattern and the ratios between NAcetyl- Aspartate, Creatine and Choline add diagnostic and functional information to the classic MR morphologic features of the brain lesion.

A lymphoma and a Glioblastoma have very different natural history, prognosis and also therapeutic options. Grading assessment is fundamental in the subsequent decision and therapeutic menagement.

The WHO classifies brain tumors from the least aggressive (benign) to the most aggressive (malignant). Glioblastomas belong to astrocytic tumors, ranging from Grade I (least malignant, e.g., I grade, pilocytic astrocytoma) to Grade IV (most malignant, e.g., IV grade Glioblastoma)

Determining the classification and grade of a brain tumor can predict its likely behaviour.

The histopathological assessment after the biopsy or surgery is fundamental for characterizing the brain tumors. Not in every case biopsy is possible, depending on the localization of the tumor and conditions of the patient; in these cases, Brain spectroscopy can be as a non-invasive tool to hypothesize the tumor grading based on neuronal metabolites ratios. In the follow-up of low-grade gliomas, Spectroscopy allows, with diffusion and perfusion techniques, to find early signs of transformation into higher grades, with a consequent change in patient treatment and care. Moreover, after neurosurgery and chemo-radiotherapy, Spectroscopy has an important role in the differential diagnosis between radionecrosis, pseudoprogression and pseudoresponse.

In conclusion, Spectroscopy, although must be considered as a complementary MRI technique, gives an important contribution to the diagnosis, characterization, follow-up of brain gliomas.

Nuclear magnetic resonance (NMR) is widely applied in chemistry and biochemistry, as well as in materials science. The capacity to elucidate new compounds or recognize structures by a fingerprint are common uses of liquid state NMR. Solid state NMR provides another added advantage to the analytical flux, as it can be performed without multistep sample preparation and in a non-destructive way; these aspects greatly speed up sample analysis. Research on mechanics and electronics in the recent years has led to the development of hardware for solid state NMR and a more versatile technique, cross polarization - magical angle spectroscopy (13C CP MAS). These techniques are robust, operationally less expensive and reliable even when performed by non-highly trained experts. These developments have allowed this technique to be used for the analysis of food. Here, we present some technical considerations for the implementation of solid state NMR as a reproducible technique, considering typical NMR parameters such as the length of pulses and spectral calibrations. The potential target compounds in food analysis are discussed regarding the type of results that can be obtained; changes to the protein and lipid content and quality lead to different spectra as well as physical-chemical characteristics with an impact on the organoleptic and taste experience for consumers. The structure, crystal arrangement and uses of the starch flour were reviewed to explain the role of materials science tools and structural knowledge. Many techniques are used to perform state-o- -the-art starch analysis and to assess its role in flour products; solid state NMR spectroscopy has considerable potential as a quick, reliable and cost-beneficial way to routinely validate processes and products. Finally, we suggest parameters for future standardized experiments and the possible comparison with other analytical methods.