Application of high-resolution 1H MAS NMR spectroscopy to the analysis of intact bones from mice exposed to gamma radiation.

Herein we demonstrate that high-resolution magic angle spinning (MAS) 1H NMR can be used to profile the pathology of bone marrow rapidly and with minimal sample preparation. The spectral resolution obtained allows several metabolites to be analyzed quantitatively. The level of NMR-detectable metabolites in the epiphysis + metaphysis sections of mouse femur were significantly higher than that observed in the diaphysis of the same femur. The major metabolite damage to bone marrow resulting from either 3.0 Gy or 7.8 Gy of whole-body gamma radiation 4 days after exposure were (1) decreased total choline content, (2) increased fatty acids in bone marrow, and (3) decreased creatine content. These results suggest that the membrane choline phospholipid metabolism (MCPM) pathway and the fatty acid biosynthesis pathway were altered as a result of radiation exposure. We also found that the metabolic damage induced by radiation in the epiphysis + metaphysis sections of mouse femur was higher than that of the diaphysis of the same femur. Traditional histopathology analysis was also carried out to correlate radiation damage with changes in metabolites. Importantly, the molecular information gleaned from high-resolution MAS 1H NMR complements the pathology data.

Metabolomics in lung inflammation:a high-resolution (1)h NMR study of mice exposedto silica dust.

ABSTRACT Here we report the first (1)H NMR metabolomics studies on excised lungs and bronchoalveolar lavage fluid (BALF) from mice exposed to crystalline silica. High-resolution (1)H NMR metabolic profiling on intact excised lungs was performed using slow magic angle sample spinning (slow-MAS) (1)H 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).

(1)H relaxation times of metabolites in biological samples obtained with nondestructive ex-vivo slow-MAS NMR.

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.

Dynamic high-resolution 1H and 31P NMR spectroscopy and 1H T2 measurements in postmortem rabbit muscles using slow magic angle spinning.

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).

High-resolution 1H NMR spectroscopy in a live mouse subjected to 1.5 Hz magic angle spinning.

It is demonstrated that the resolution of the (1)H NMR metabolite spectrum in a live mouse can be significantly enhanced by an ultraslow magic angle spinning of the animal combined with a modified phase-corrected magic angle turning (PHORMAT) pulse sequence. Proton NMR spectra were measured of the torso and the top part of the belly of a female BALBc mouse in a 2 T field while spinning the animal at a speed of 1.5 Hz. It was found that even in this relatively low field, with PHORMAT an isotropic spectrum is obtained with line widths that are a factor of 4.6 smaller than those obtained in a stationary mouse. It is concluded that in vivo PHORMAT has the potential to significantly increase the utility of (1)H NMR spectroscopy for biochemical and biomedical animal research.

High-resolution 1H NMR spectroscopy in rat liver using magic angle turning at a 1 Hz spinning rate.

It is demonstrated that a high-resolution (1)H NMR spectrum of excised rat liver can be obtained using the technique of magic angle turning (MAT) at a sample spinning rate of 1 Hz. A variant of the phase-corrected MAT (PHORMAT) pulse sequence that includes a water suppression segment was developed for the investigation. The spectral resolution achieved with PHORMAT approaches that obtained from a standard magic angle spinning (MAS) experiment at a spinning rate of several kHz. With such ultra-slow spinning, tissue and cell damage associated with the standard MAS experiment is minimized or eliminated. The technique is potentially useful for obtaining high-resolution (1)H spectra in live animals.