ABSTRACT Here we report the first 1H NMR metabolomics studies on excised lungs and bronchoalveolar lavage fluid (BALF) from mice exposed to crystalline silica. High-resolution 1H NMR metabolic profiling on intact excised lungs was performed using slow magic angle sample spinning (slow-MAS) 1H PASS (phase-altered spinning sidebands) at a sample spinning rate of 80 Hz. Metabolic profiling on BALF was completed using fast magic angle spinning at 2 kHz. Major findings are that the relative concentrations of choline, phosphocholine (PC), and glycerophosphocholine (GPC) were statistically significantly increased in silica-exposed mice compared to sham controls, indicating an altered membrane choline phospholipids metabolism (MCPM). The relative concentrations of glycogen/glucose, lactate, and creatine were also statistically significantly increased in mice exposed to silica dust, suggesting that cellular energy pathways were affected by silica dust. Elevated levels of glycine, lysine, glutamate, proline, and 4-hydroxyproline were also increased in exposed mice, suggesting the activation of a collagen pathway. Furthermore, metabolic profiles in mice exposed to silica dust were found to be spatially heterogeneous, consistent with regional inflammation revealed by in vivo magnetic resonance imaging (MRI).

The sections in this article are 1 Introduction 2 Theory 3 Applications of DNP NMR 4 Summary, Conclusions, and Perspectives 5 Biographical Sketch Related Articles

NMR spectroscopy has been applied to cells and tissues analysis since its beginnings, as early as 1950. We have attempted to gather here in a didactic fashion the broad diversity of data and ideas that emerged from NMR investigations on living cells. Covering a large proportion of the periodic table, NMR spectroscopy permits scrutiny of a great variety of atomic nuclei in all living organisms non-invasively. It has thus provided quantitative information on cellular atoms and their chemical environment, dynamics, or interactions. We will show that NMR studies have generated valuable knowledge on a vast array of cellular molecules and events, from water, salts, metabolites, cell walls, proteins, nucleic acids, drugs and drug targets, to pH, redox equilibria and chemical reactions. The characterization of such a multitude of objects at the atomic scale has thus shaped our mental representation of cellular life at multiple levels, together with major techniques like mass-spectrometry or microscopies.NMR studies on cells has accompanied the developments of MRI and metabolomics, and various subfields have flourished, coined with appealing names: fluxomics, foodomics, MRI and MRS (i.e. imaging and localized spectroscopy of living tissues, respectively), whole-cell NMR, on-cell ligand-based NMR, systems NMR, cellular structural biology, in-cell NMR… All these have not grown separately, but rather by reinforcing each other like a braided trunk. Hence, we try here to provide an analytical account of a large ensemble of intricately linked approaches, whose integration has been and will be key to their success.We present extensive overviews, firstly on the various types of information provided by NMR in a cellular environment (the “why”, oriented towards a broad readership), and secondly on the employed NMR techniques and setups (the “how”, where we discuss the past, current and future methods). Each subsection is constructed as a historical anthology, showing how the intrinsic properties of NMR spectroscopy and its developments structured the accessible knowledge on cellular phenomena. Using this systematic approach, we sought i) to make this review accessible to the broadest audience and ii) to highlight some early techniques that may find renewed interest. Finally, we present a brief discussion on what may be potential and desirable developments in the context of integrative studies in biology.

In a previous work ( 1 ), the susceptibility broadening in the 1 H NMR metabolite spectrum obtained in a live mouse was separated from the isotropic information, which significantly increased the spectral resolution. This was achieved using ultraslow magic angle spinning (MAS) of the animal combined with a modified phase‐corrected magic angle turning (PHORMAT) pulse sequence. However, PHORMAT cannot be used for spatially selective spectroscopy. This article introduces a modified sequence called localized magic angle turning (LOCMAT) that makes this possible. Proton LOCMAT spectra were obtained from the liver and heart of a live mouse while the animal was spun at a speed of 4 Hz in a 2 Tesla field. It was found that even in this relatively low field, LOCMAT provided isotropic line widths that were a factor of 4–10 times smaller than those obtained in a stationary animal. Furthermore, the susceptibility broadening of the heart metabolites showed unusual features that are not observed in dead animals. The limitations of LOCMAT and possible ways to improve the technique are discussed. It is concluded that in vivo LOCMAT can significantly enhance the utility of NMR spectroscopy for biomedical research. Magn Reson Med, 2006. Published 2005 Wiley‐Liss, Inc.

Methods suitable for measuring (1)H relaxation times such as T(1), T(2) and T(1rho) of metabolites in small, intact biological objects including live cells, excised organs and tissues, oil seeds etc. are developed in this work. This was achieved by combining inversion-recovery, spin-echo, or a spin-lock segment with the phase-adjusted spinning sideband (PASS) technique, which was applied at low sample-spinning rates. Here, PASS was used to produce high-resolution (1)H spectra in a nondestructive way so that the relaxation parameters of individual metabolite could be determined. The methodologies were demonstrated by measuring (1)H T(1), T(2), and T(1rho) of metabolites in excised rat liver at a spinning rate of 40 Hz.

A simple approach is reported that yields high resolution, high sensitivity ¹H NMR spectra of biofluids with limited mass supply. This is achieved by spinning a capillary sample tube containing a biofluid at the magic angle at a frequency of about 80Hz. A 2D pulse sequence called ¹H PASS is then used to produce a high-resolution ¹H NMR spectrum that is free from magnetic susceptibility induced line broadening. With this new approach a high resolution ¹H NMR spectrum of biofluids with a volume less than 1.0 µl can be easily achieved at a magnetic field strength as low as 7.05T. Furthermore, the methodology facilitates easy sample handling, i.e., the samples can be directly collected into inexpensive and disposable capillary tubes at the site of collection and subsequently used for NMR measurements. In addition, slow magic angle spinning improves magnetic field shimming and is especially suitable for high throughput investigations. In this paper first results are shown obtained in a magnetic field of 7.05T on urine samples collected from mice using a modified commercial NMR probe.

Novel procedures and instrumentation are described for nuclear magnetic resonance (NMR) spectroscopy and imaging studies of live, in situ microbial films. A perfused NMR/optical microscope sample chamber containing a planar biofilm support was integrated into a recirculation/dilution flow loop growth reactor system and used to grow in situ Shewanella oneidensis strain MR-1 biofilms. Localized NMR techniques were developed and used to non-invasively monitor time-resolved metabolite concentrations and to image the biomass volume and distribution. As a first illustration of the feasibility of the methodology an initial 13C-labeled lactate metabolic pathway study was performed, yielding results consistent with existing genomic data for MR-1. These results represent progress toward our ultimate goal of correlating time- and depth-resolved metabolism and mass transport with gene expression in live in situ biofilms using combined NMR/optical microscopy techniques.

Obtaining detailed in vivo metabolic information has been identified as key elements of better understanding the efficacy and toxicity of new therapies. A new nuclear magnetic resonance (NMR) technology called LOCMAT is reported in this paper that yields substantially increased spectral resolution in spatially localized in vivo 1H NMR metabolite spectra, as illustrated by measurements in the liver of a live mouse. LOCMAT promises to significantly enhance the utility of NMR spectroscopy for biomedical research.

The scope of applications of magnetic resonance to food science continues to expand. Recently, the focus has turned to the way in which the interpretation and quantification of magnetic resonance data of complex food systems increasingly requires the application of multivariate data analytical protocols. This book provides an up-to-date, global perspective of the latest developments in the field, including methods of studying metabolic processes both in vivo and in vitro, functional MRI and the sensory perception of food. Content is divided into five sections: sensory science; aroma and flavour; authenticity and quantification of food; functionality, structure and ingredients; applications of solid-state methods; and new NMR methods and instrumentation. Magnetic Resonance in Food Science: The Multivariate Challenge is ideal for graduates and researchers as well as for academics and professionals in the field.

We are developing novel nuclear magnetic resonance (NMR) microscopy, spectroscopy and combined NMR/optical techniques for the study of biofilms under known, controlled growth conditions. Objectives include: time and depth-resolved metabolite concentrations with isotropic spatial resolution on the order of 10 microns, metabolic pathways and flux rates, mass transport and ultimately their correlation with gene expression by optical microscopy in biofilms. We describe the implementation of ex-situ grown biofilms to improve growth environment control and NMR analysis. In-situ NMR depth resolved metabolite profiling techniques are introduced and demonstrated for a Shewanella oneidensis strain MR-1 biofilm. Finally, initial combined confocal fluorescence and magnetic resonance images are shown for a GFP-labeled Shewanella biofilm. These methods are equally applicable to other biofilm systems of interest; thus they may provide a significant contribution toward the understanding of adherent cell metabolism.

Novel procedures and instrumentation are described for nuclear magnetic resonance (NMR) spectroscopy and imaging studies of live, in situ microbial films. A perfused NMR/optical microscope sample chamber containing a planar biofilm support was integrated into a recirculation/dilution flow loop growth reactor system and used to grow in situ Shewanella oneidensis strain MR-1 biofilms. Localized NMR techniques were developed and used to non-invasively monitor time-resolved metabolite concentrations and to image the biomass volume and distribution. As a first illustration of the feasibility of the methodology an initial 13C-labeled lactate metabolic pathway study was performed, yielding results consistent with existing genomic data for MR-1. These results represent progress toward our ultimate goal of correlating time- and depth-resolved metabolism and mass transport with gene expression in live in situ biofilms using combined NMR/optical microscopy techniques.

In vitro and in vivo 1H NMR spectroscopy is widely used to measure metabolic profiles in cells, tissues, animals, and humans and to use them, e.g., for diagnosis and therapy response evaluations. However, the spectra often suffer from poor resolution due to variations in the isotropic bulk magnetic susceptibility present in biological objects, resulting in a broadening of the NMR lines. In principle this broadening can be averaged to zero by the technique of magic angle spinning (MAS), where the sample is rotated about an axis making an angle of 54o44’ relative to the external magnetic field. However, a problem is that in a standard MAS experiment spinning speeds of a kHz or more are required in order to avoid the occurrence of spinning sidebands (SSBs) in the spectra, which renders analysis of the spectra difficult again. At these spinning speeds the large centrifugal forces cause severe structural damage in larger biological objects, so that this method cannot be used to study metabolic processes in intact samples. In solid state NMR several methods have been developed where slow MAS is combined with special radio frequency pulse sequences to eliminate spinning side bands or separate them from the isotropic spectrum somore » that a SSB-free high-resolution isotropic spectrum is obtained. It has been shown recently that two methods, phase-adjusted spinning sidebands (PASS) and phase-corrected magic angle turning (PHORMAT), can successfully be modified for applications in biological materials (1, 2). With PASS MAS speeds as low as 40 Hz can be employed, allowing non or minimally invasive in vitro studies of excised tissues and organs. This method was used, amongst other things, to study post mortem changes in the proton metabolite spectra in excised rabbit muscle tissue (3). With PHORMAT the NMR sensitivity is reduced and longer measuring times are required, but with this methodology the MAS speed can be reduced to ~1 Hz. This makes PHORMAT amenable for in vivo applications, as was demonstrated in a living mouse (4). In this presentation the principles and limitations of PASS and PHORMAT will be briefly discussed and illustrated with spectra obtained on a variety of biological objects. Moreover, it will be shown that PASS can be employed to study metabolic processes in biofilms and other living microbial systems. Finally, on-going work to develop PASS and PHORMAT spectroscopic imaging and to improve the performance of both techniques will be discussed. It is anticipated that slow- and ultra-slow-MAS will significantly enhance the utility of proton MR spectroscopy for biomedical research in living cell systems and live animals, and in future perhaps even in the clinic, in the latter case by rotating the external magnetic field rather than the patient.« less

We induced apoptosis and necrosis in monolayer cultures of Chinese hamster ovary cells using okadaic acid and hydrogen peroxide (H2O2), respectively, and examined the effect on water diffusion and compartmentalization using pulsed-field-gradient (PFG) 1H-NMR and simultaneous confocal microscopy. In PFG experiments characterized by a fixed diffusion time (<4.7 ms) and variable b-values (0-27000 s/mm2), 1H-NMR data collected with untreated cells exhibited multiexponential behavior. Analysis with a slow-exchange model revealed two distinct cellular water compartments with different apparent diffusion coefficients (ADCs; 0.56, 0.06 x 10(-3) mm2/s) and volume fractions (0.96 and 0.04). During the first 12 hr of necrosis or apoptosis, the amount of water in the smallest compartment increased twofold before significant changes in cell density or plasma membrane integrity occurred. Over the same period, water content in the largest compartment decreased by a factor of >2 in apoptotic cells, in accordance with observed cell shrinkage, and changed little in necrotic counterparts, where only slight swelling was evident. These results indicate that PFG 1H-NMR serves as a sensitive indicator of early cell death in monolayer cultures, and can be used to distinguish apoptosis from necrosis. Measurements of restricted diffusion and water exchange are presented to elucidate the compartment origins and justify the model assumptions.

Postmortem changes in rabbit muscle tissue with different glycogen status (normal vs low) were followed continuously from 13 min postmortem until 8 h postmortem and again 20 h postmortem using simultaneous magic angle spinning (1)H and (31)P NMR spectroscopy together with measurement of the transverse relaxation time, T(2), of the muscle water. The (1)H metabolite spectra were measured using the phase-altered spinning sidebands (PASS) technique at a spinning rate of 40 Hz. pH values calculated from the (31)P NMR spectra using the chemical shifts of the C-6 line of histidine in the (1)H spectra and the chemical shifts of inorganic phosphate in the (31)P spectra confirmed the different muscle glycogen status in the tissues. High-resolution (1)H spectra obtained from the PASS technique revealed the presence of a new resonance line at approximately 6.8 ppm during the postmortem period, which were absent in muscles with low muscle glycogen content. This new resonance line may originate from the aminoprotons in creatine, and its appearance may be a result of a pH effect on the exchange rate between the amino and the water protons and thereby the NMR visibility. Alternatively, the new resonance line may originate from the aromatic protons in tyrosine, and its appearance may be a result of a pH-induced protein unfolding exposing hydrophobic amino acid residues to the aqueous environment. Further studies are needed to evaluate these hypotheses. Finally, distributed analysis of the water T(2) relaxation data revealed three relaxation populations and an increase in the population believed to reflect extramyofibrillar water through the postmortem period. This increase was significantly reduced (p < 0.0001) in samples from animals with low muscle glycogen content, indicating that the pH is controlling the extent of postmortem expulsion of water from myofibrillar structures. The significance of the postmortem increase in the amount extramyofibrillar water on the water-holding capacity was verified by centrifugation, which showed a reduced centrifugation loss in muscles with low preslaughter glycogen status (0.9 vs 1.9%, p = 0.07).

The authors show that high resolution H-1 under nuclear magnetic resonance (NMR) spectroscopy can be used to study biofilm metabolism under enviornmentally relevant conditions in a minimally invasive way.

98 Spectroscopy 19(12) December 2004 n vitro and in vivo 1H nuclear magnetic resonance (NMR) spectroscopy increasingly is used for investigating biochemical processes in cells, tissues, animals, and humans (1–5). However, the 1H spectrum often displays a poor resolution, which hampers a quantitative analysis of the spectra (4). It is known that in intact biological systems the susceptibility gradients, arising from variations in the isotropic bulk magnetic susceptibility near boundaries of interand intracellular structures, are the main sources for the observed line widths (6, 7). In principle, this broadening can be averaged to zero by the technique of magic angle spinning (MAS), in which the sample is rotated about an axis making an angle of 54°44’ relative to the external magnetic field (8–10). High-resolution 1H MAS spectroscopy at spinning rates ranging from a few kilohertz to more than 10 kHz has been used successfully to study metabolism in cells and excised tissues (3, 7, 11–15), offering a spectral resolution similar to that of solution NMR using cell extracts. These large spinning speeds are necessary to eliminate the spinning sidebands (SSBs) in the spectra (16), which occur at lower speeds and render the interpretation of the spectra difficult again. However, the large centrifugal forces associated with these spinning speeds destroy tissue and sometimes even cellular structures. Therefore the standard fast MAS approach is not viable for magnetic resonance spectroscopy of larger organisms or tissues that are subject to disruption by centrifugal forces, and methods are needed that yield high-resolution, SSB-free spectra at reduced MAS frequencies. In our laboratory, we have modified two methods that eliminate the SSBs or separate them from the isotropic spectrum so that a SSB-free high-resolution isotropic spectrum is obtained, for 1H NMR metabolite spectroscopy in biological samples (17–22). Both methods originally were developed especially for 13C and 15N solid-state NMR. One is an isotropic-anisotropic chemical shift 2D correlation experiment called 1H PHORMAT (phase corrected magic angle turning) (18–21) and is evolved from the original PHORMAT experiment (23, 24). With 1H PHORMAT, spinning rates as low as 1 Hz can be used (18), making the method viable for in vivo studies, as was recently demonstrated in a live mouse (21). However, a disadvantage of PHORMAT is a reduction in the NMR sensitivity and long measuring times (18–21). The second method is called 1H PASS (phase adjusted spinning sidebands) (17), which is an extension of the original work reported by Antzutkin et al. (25) and Dixon (26, 27). With PASS, the minimum sample spinning rate needs to be High-Resolution 1H NMR Spectroscopy of Metabolically Active Microorganisms Using NonDestructive Magic Angle Spinning The authors show that high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy can be used to study biofilm metabolism under environmentally relevant conditions in a minimally invasive way.