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

Abstract

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...

New Methodology For Use in Rotating Field Nuclear MagneticResonance

“…Figure 7.5 c) shows more closely the region between 0-4ppm in which distinct resonances can be assigned based on previous studies, given in the caption. [54,62] The projected spectrum, Figure 7.5 d), shows the same resonances which are in the MAS spectrum.…”

High-resolution NMR of anisotropic samples with spinning away from the magic angle

“…Rotor synchronized techniques, such as Magic Angle Hopping [21] or Turning [22][23][24] have been developed in the past and could be easily combined into the framework of experiments away from the magic angle. This type of techniques eliminate the presence of sidebands even in the case of very slow spinning [25]. This does not represent a serious problem for the technique and we plan to show that with the appropriate hardware such combined techniques can recover the isotropic information even in the presence of the very slow spinning.…”

Metabolite identification in human liver needle biopsies by high-resolution magic angle spinning1H NMR spectroscopy

“…As mentioned above, HR-MAS NMR spectroscopy has been proven to be an appropriate technique for the study of intact tissues. In previous studies, HR-MAS NMR spectroscopy has been used for extensive intact rat liver tissue metabolic characterization (19)(20)(21). Resonances of most significant metabolites, such as choline, free fatty acids, monomers and short chains of -and -glucose, glycogen and glycerol, were assigned and the applicability of the technique to the study of pathological conditions was established.…”