It is shown that high-resolution 1 H NMR spectra of intact excised tissues and organs can be obtained by rotating the sample slowly about an axis at the magic angle of 54°44 with the external magnetic field. In this way tissue and cellular damage invoked by standard magic angle spinning (MAS) experiments, where spinning speeds of several kHz are typically employed, are minimized. Special RF pulse sequences, developed originally in solid state NMR, can be used to produce a spinning sideband-free isotropic spectrum. In this article the first results are shown of the brain, heart, liver, gluteus muscle, and kidney excised from mice using the 2D-phase-altered spinning sidebands (PASS) technique and employing MAS spinning speeds of 43-125 Hz. It was found that with slow sample spinning similar, and in some cases even better, spectral resolutions are obtained as compared with fast MAS. Magn Reson Med 46: 213-218,

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).

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. Magn Reson Med 50: 1113-1119, 2003.

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. In vitro and in vivo 1 H NMR spectroscopy is widely used for investigating biochemical processes in cells, tissues, animals, and humans (1-5). However, the 1 H spectrum obtained from a static sample often suffers from poor resolution due to the various line broadening mechanisms inherent with a biological system. This hampers a quantitative analysis of the spectra (4). It has been found that in biological samples the major mechanism responsible for line broadening is the variation in the isotropic bulk magnetic susceptibility present in intact cells and tissues (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 with an angle of 54°44Ј relative to the external magnetic field (8 -10). High-resolution 1 H MAS spectroscopy at spinning rates from several kHz to more than 10 kHz has been used to study metabolites in intact tissues and cells, avoiding the necessity of utilizing cell and tissue extracts (3,7,(11)(12)(13)(14)(15). However, the large centrifugal force associated with these spinning rates destroys the tissue structure and even some of the cells (7), which makes the method unusable for chemical shift imaging or localized spectroscopy in live cell systems or intact tissues. Hence, for these applications it is important to develop methods in which the spinning speed can be reduced.A problem with slow MAS is the occurrence of spinning sidebands (SSBs) in the spectrum, which renders analysis of the spectra difficult or impossible. In solid state NMR, where similar problems exist, several methods have been developed to overcome this problem. These methods include 1D total sideband suppression (TOSS) (16), 2D-phase-altered spinning sidebands (PASS) (17), and 2D phase-corrected magic angle turning (PHORMAT) (18), which is a variant of Gan's original MAT (19). It was found that the SSB suppression with TOSS failed at spinning speeds of 1 kHz and lower (20). However, in a previous work (21) it was shown that with PASS, sideband-free isotropic spectra could be obtained in a variety of excised intact organs at spinning speeds as low as 43 Hz, with a spectral resolution comparable to or better than the resolution obtained with fast MAS. While this frequency is already 2 orders of magnitude lower than the frequencies employed in fast MAS, it is still too large to keep largersize soft tissues and organs undamaged, or to apply in in vivo applications. However, the spinning speed cannot arbitrarily be reduced in a PASS experiment. In PASS the magneti...

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).