Application of 1H and 23Na magic angle spinning NMR spectroscopy to define the HRBC up-taking of MRI contrast agents

The up-take of Gd(III) complexes of BOPTA, DTPA, DOTA, EDTP, HPDO3A, and DOTP in HRBC has been evaluated by measuring the lanthanide induced shift (LIS) produced by the corresponding dysprosium complexes (DC) on the MAS-NMR resonances of water protons and free sodium ions. These complexes are important in their use as MRI contrast agents (MRI-CA) in diagnostics. 1H and 23Na MAS-NMR spectra of HRBC suspension, collected at 9.395T, show only one signal due to extra- and intra-cellular water (or sodium). In MAS spectra, the presence of DC in a cellular compartment produces the LIS of only the nuclei (water proton or sodium) in that cellular compartment and this LIS can be related to the DC concentrations (by the experimental curves of LIS vs. DC concentrations) collected in the physiological solution. To obtain correct results about LIS, the use of MAS technique is mandatory, because it guarantees the only the nuclei staying in the same cellular compartment where the LC is present show the LIS. In all the cases considered, the addition of the DC to HRBC (100% hematocrit) produced a shift of only the extra-cellular water (or sodium) signal and the gradient of concentration (GC) between extra- and intra-cellular compartments resulted greater than 100:1, when calculated by means of sodium signals. These high values of GC are direct proofs that none of the tested dysprosium complexes crosses the HRBC membrane. Since the DC are iso-structural to the gadolinium complexes the corresponding gadolinium ones (MRI-CA) do not cross the HRBC membrane and, consequently, they are not up-taken in HRBC. The GC values calculated by means of water proton signals resulted much lower than those obtained by sodium signals. This proves that the choice of the isotope is a crucial step in order to use this method in the best way. In fact, GC value depends on the lowest detectable LIS which, in turn, depends on the nature of the LC (lanthanide complex) and the observed isotopes.     

Angular Flow in Toroid Cavity Probes

NMR signals from samples that rotate uniformly about the central conductor of a TCD (toroid cavity detector) exhibit frequency shifts that are directly proportional to the sample's angular velocity. This newly observed effect is based on the unique radiofrequency field inside TCDs, which is variable in direction. If a liquid sample is pumped through a capillary tube wound about the central conductor, the frequency shift is proportional to the flow rate. A mathematical relationship between a volumetric flow rate and the frequency shift is established and experimentally verified to high precision. Additionally, two-dimensional flow-resolved NMR spectroscopy for discrimination between components with different flow velocities yet retaining chemical shift information for structural analysis is presented. The application of the two-dimensional method in chromatographic NMR is suggested. Furthermore, utilization of the frequency-shift effect for rheologic studies if combined with toroid-cavity rotating-frame imaging is proposed.     

A subspace time-domain algorithm for automated NMR spectral normalization

Recently, two methods have been proposed for quantitatively comparing NMR spectra of control and treated samples, in order to examine the possible occurring variations in cell metabolism and/or structure in response to numerous physical, chemical, and biological agents. These methods are the maximum superposition normalization algorithm (MaSNAl) and the minimum rank normalization algorithm (MiRaNAl). In this paper a new subspace-based time-domain normalization algorithm, denoted by SuTdNAl (subspace time-domain normalization algorithm), is presented. By the determination of the intersection of the column spaces of two Hankel matrices, the common signal poles and further on the components having proportionally varying amplitudes are detected. The method has the advantage that it is computationally less intensive than the MaSNAl and the MiRaNAl. Furthermore, no approximate estimate of the normalization factor is required. The algorithm was tested by Monte Carlo simulations on a set of simulation signals. It was shown that the SuTdNAl has a statistical performance similar to that of the MiRaNAl, which itself is an improvement over the MaSNAl. Furthermore, two samples of known contents are compared with the MiRaNAl, the SuTdNAl, and an older method using a standard. Finally, the SuTdNAl is tested on a realistic simulation example derived from an in vitro measurement on cells.     

Picoliter (1)H NMR spectroscopy

In this study, a 267-microm-diameter solenoid transceiver is used to acquire localized (1)H NMR spectra and the measured signal-to-noise ratio (SNR) at 500 MHz is shown to be within 20--30% of theoretical limits formulated by considering only its resistive losses. This is illustrated using a 100-microm-diameter globule of triacylglycerols (approximately 900mM) that may be an oocyte precursor in young Xenopus laevis frogs and a water sample containing choline at a concentration often found in live mammalian cells (approximately 33 mM). In chemical shift imaging (CSI) experiments performed using a few thousand total scans, the choline methyl line is shown to have an acceptable SNR in resolved volume elements containing only 50 pL of sample, and localized spectra are resolved from just 5 pL in the Xenopus globule. These findings demonstrate the feasibility of performing (1)H NMR on picoliter-scale sample volumes in biological cells and tissues and illustrate how the achieved SNR in spectroscopic images can be predicted with reasonable accuracy at microscopic spatial resolutions.     

Solid-state NMR spectroscopy of paramagnetic metallocenes

The paramagnetic metallocenes and decamethylmetallocenes (C(5)H(5))(2)M and (C(5)Me(5))(2)M with M=V (S=3/2), Mn (S=5/2 or 1/2), Co (S=1/2), and Ni (S=1) were studied by (1)H and (13)C solid-state MAS NMR spectroscopy. Near room temperature spinning sideband manifolds cover ranges of up to 1100 and 3500 ppm, and isotropic signal shifts appear between -260 and 300 ppm and between -600 and 1640 ppm for (1)H and (13)C NMR spectra, respectively. The isotropic paramagnetic signal shifts, which are related to the spin densities in the s orbital of ligand atoms, were discussed. A Herzfeld--Berger spinning sideband analysis of the ring carbon signals yielded the principal values of the paramagnetic shift tensors, and for metallocenes with a small g-factor anisotropy the electron spin density in the ligand pi system was determined from the chemical shift anisotropy. The unusual features of the (1)H and (13)C solid-state NMR spectra of manganocene were related to its chain structure while temperature-dependent (1)H MAS NMR studies reflected antiferromagnetic interaction between the spin centers.     

Sensitivity enhancement in structural measurements by solid state NMR through pulsed spin locking

Free induction decay (FID) signals in solid state NMR measurements performed with magic angle spinning can often be extended in time by factors on the order of 10 by a simple pulsed spin locking technique. The sensitivity of a structural measurement in which the structural information is contained in the dependence of the integrated FID amplitude on a preceding evolution period can therefore be enhanced substantially by pulsed spin locking in the signal detection period. We demonstrate sensitivity enhancements in a variety of solid state NMR techniques that are applicable to selectively isotopically labeled samples, including 13C-15N rotational echo double resonance (REDOR), 13C-13C dipolar recoupling measurements using the constant-time finite-pulse radio-frequency-driven recoupling (fpRFDR-CT) and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) techniques, and torsion angle measurements using the double quantum chemical shift anisotropy (DQCSA) technique. Further, we demonstrate that the structural information in the solid state NMR data is not distorted by pulsed spin locking in the detection period.     

NMR Relaxation and Diffusion Measurements on Iron(III)-Doped Kaolin Clay

Kaolin clay samples were mixed with various amounts of Fe₂O₃ powder. The influence of this magnetic impurity on NMR relaxation and diffusion measurements on the water in this porous material was investigated. The NMR relaxation measurements showed a nearly mono-exponential decay, leading to the conclusion that the pore size distribution of the clay samples is either narrow and/or that the pores are interconnected very well. Both the longitudinal and the transverse relaxation rate depend linearly on the concentration of the Fe₂O₃ impurity. The NMR diffusion measurements revealed that the Fe₂O₃ causes internal magnetic field gradients that largely exceed the maximum external gradient that could be applied by our NMR apparatus (0.3 T/m). Additional SQUID measurements yielded the magnetization and magnetic susceptibility of the samples at the magnetic field strength used in the NMR measurements (0.8 T). A theoretical estimate of the internal magnetic field gradients leads to the conclusion that the water in the porous clay samples cannot be described by the commonly observed motional averaging regime. Probably an intermediate or a localization regime is induced by the large internal gradients, which are estimated to be on the order of 1 to 10 T/m in the pore volume and may exceed 1000 T/m at the pore surface.     

Imaging the long-range dipolar field in structured liquid state samples

We describe imaging experiments in which the pattern of the dipolar field generated by spatially modulated nuclear magnetization is directly visualized in simply structured phantoms. Two types of experiment have been carried out at 11.7 T using (1)H NMR signals. In the first, the field from a single spin species is imaged via its own NMR signal. In the second, the NMR signal from one spin species is used to image the field generated by a second species. The field patterns measured in these experiments correspond well with those calculated using simple theoretical expressions for the dipolar field. The results also directly demonstrate the spatial sensitivity of the signal generated using dipolar field effects, indicating that the range of the field depends upon the inverse of the spatial frequency with which the magnetization is modulated.     

GAMMA simulations of stray field responses: slice thickness and pulse calibration

A stray field (STRAFI) module has been added to the GAMMA magnetic resonance simulation platform in order to facilitate computational investigations of NMR experiments in large static field gradients that are on the order of 50 T/m. The package has been used to examine system response during echo trains generated by the application of shaped pulses. The associated echo amplitude maxima and effective slice thickness are presented. A new accurate method for STRAFI pulse calibration based on relative echo amplitudes is proposed.     

Changes in Porcine Muscle Water Characteristics during Growth—An in Vitro Low-Field NMR Relaxation Study

This study investigates the effects of developmental stage and muscle type on the mobility and distribution of water within skeletal muscles, using low-field ¹H-NMR transverse relaxation measurements in vitro on four different porcine muscles (M. longissimus dorsi, M. semitendinosus, M. biceps femoris, M. vastus intermedius) from a total of 48 pigs slaughtered at various weight classes between 25 kg and 150 kg. Principal component analysis (PCA) revealed effects of both slaughter weight and muscle type on the transverse relaxation decay. Independent of developmental stage and muscle type, distributed exponential analysis of the NMR T₂ relaxation data imparted the existence of three distinct water populations, T_2b, T₂₁, and T₂₂, with relaxation times of approximately 1–10, 45–120, and 200–500 ms, respectively. The most profound change during muscle growth was a shift toward faster relaxation in the intermediate time constant, T₂₁. It decreased by approx. 24% in all four muscle types during the period from 25 to 150 kg live weight. Determination of dry matter, fat, and protein content in the muscles showed that the changes in relaxation time of the intermediate time constant, T₂₁, during growth should be ascribed mainly to a change in protein content, as the protein content explained 77% of the variation in the T₂₁ time constant. Partial least squares (PLS) regression revealed validated correlations in the region of 0.58 to 0.77 between NMR transverse relaxation data and muscle development for all the four muscle types, which indicates that NMR relaxation measurements may be used in the prediction of muscle developmental stage.     

Analysis of protein/ligand interactions with NMR diffusion measurements: the importance of eliminating the protein background

Pulsed-field gradient nuclear magnetic resonance (PFG-NMR) is a well-established method for the determination of translational diffusion coefficients. Recently, this method has found applicability in the combinatorial arena with the introduction of affinity NMR for characterizing protein/ligand interactions. Although affinity NMR has been reported to be an effective method for the identification of active compounds in a complex mixture, there are limitations of this method. We have developed a simple mathematical model to predict optimum concentration ratios of the ligand and protein in order to observe maximum changes in the ligand diffusion coefficient upon protein binding. The ligand/protein systems of L-tryptophan and ibuprofen binding to human serum albumin were chosen to demonstrate the usefulness of this model. However, even when the conditions of the mathematical model are satisfied, the spectral background arising from the protein in proton-detected experiments can be problematic. To this end, we have employed spectral subtraction of the protein spectrum to yield ligand diffusion coefficients that are in agreement with those predicted by simulation.     

H Spectroscopy without Solvent Suppression: Characterization of Signal Modulations at Short Echo Times

While most proton (¹H) spectra acquired in vivo utilize selective suppression of the solvent signal for more sensitive detection of signals from the dilute metabolites, recent reports have demonstrated the feasibility and advantages of collecting in vivo data without solvent attenuation. When these acquisitions are performed at short echo times, the presence of frequency modulations of the water resonance may become an obstacle to the identification and quantitation of metabolite resonances. The present report addresses the characteristics, origin, and elimination of these sidebands. Sideband amplitudes were measured as a function of delay time between gradient pulse and data collection, as a function of gradient pulse amplitude, and as a function of spatial location of the sample for each of the three orthogonal gradient sets. Acoustic acquisitions were performed to demonstrate the correlation between mechanical vibration resonances and the frequencies of MR sidebands. A mathematical framework is developed and compared with the experimental results. This derivation is based on the theory that these frequency modulations are induced by magnetic field fluctuations generated by the transient oscillations of gradient coils.     

An Efficient NMR Experiment for Analyzing Sugar-Puckering in Unlabeled DNA: Application to the 26-kDa Dead Ringer–DNA Complex

We present a new NMR experiment for estimating the type and degree of sugar-puckering in high-molecular-weight unlabeled DNA molecules. The experiment consists of a NOESY sequence preceded by a constant-time scalar coupling period. Two subexperiments are compared, each differing in the amount of time the (3)J(H3'H2') and (3)J(H3'H2") couplings are active on the H3' magnetization. The resultant data are easy to analyze, since a comparison of the signal intensities of any resolved NOE cross peak originating from H3' atoms of the duplex can be used to estimate the sum of the (3)J(H3'H2') and (3)J(H3'H2") couplings and thus the puckering type of the deoxyribose ring. Isotope filters to eliminate signals of the (13)C-labeled component in the F1-dimension are implemented, facilitating analyses of high-molecular-weight protein-DNA complexes containing (13)C-labeled protein and unlabeled DNA. The utility of the experiment is demonstrated on the 26-kDa Dead Ringer protein-DNA complex and reveals that the DNA uniformly adopts the S-type configuration when bound to protein.     

Measuring the longitudinal NMR relaxation rates of fast relaxing nuclei using a signal eliminating relaxation filter

A new experiment for selective determination of the relaxation rates of fast relaxing NMR signals is presented. The experiment is derived from the conventional inversion recovery experiment by substituting the 180 degrees inversion pulse of this experiment with a signal eliminating relaxation filter (SERF) consisting of three 180 degrees pulses separated by two variable delays, Delta1 and Delta2. The SERF experiment allows a selective suppression of signals with relaxation rates below a given limit while monitoring the relaxation of faster relaxing signals. The experiment was tested on a sample of 20% oxidized plastocyanin from Anabaena variabilis, where the fast exchange of an electron between the reduced (diamagnetic) and the oxidized (paramagnetic) form results in a series of average signals with widely different relaxation rates. To ensure an optimum extraction of information from the experimental data, the relaxation rates were obtained from the SERF experiment by a simultaneous analysis of all the FIDs of the experiment using a fast linear prediction model method developed previously. The reliability of the relaxation rates obtained from the SERF experiment was confirmed by a comparison of the rates with the corresponding rates obtained from a conventional inversion recovery experiment.     

Obtaining molecular and structural information from 13C-14N systems with 13C FIREMAT experiments

The effect of dipolar coupling to 14N on 13C FIREMAT (five pi replicated magic angle turning) experiments is investigated. A method is developed for fitting the 13C FIREMAT FID employing the full theory to extract the 13C-14N dipolar and 13C chemical shift tensor information. The analysis requires prior knowledge of the electric field gradient (EFG) tensor at the 14N nucleus. In order to validate the method the analysis is done for the amino acids alpha-glycine, gamma-glycine, l-alanine, l-asparagine, and l-histidine on FIREMAT FIDs recorded at 13C frequencies of 50 and 100 MHz. The dipolar and chemical shift data obtained with this analysis are in very good agreement with the previous single-crystal 13C NMR results and neutron diffraction data on alpha-glycine, l-alanine, and l-asparagine. The values for gamma-glycine and l-histidine obtained with this new method are reported for the first time. The uncertainties in the EFG tensor on the resultant 13C chemical shift and dipolar tensor values are assessed.     

Determination of the chemical shielding tensor orientation from two or one of the three conventional rotations of a single crystal

Chemical shift anisotropy (CSA) is an immensely useful interaction to study the structure, dynamics, and function of a wide variety of chemical and biological molecules. Traditionally the only unambiguous way to determine both the principal values and the orientation of the principal axes of the CSA tensor has been to follow the chemical shift frequency changes as a crystal of known structure is rotated relative to the direction of the external magnetic field. This classic method employs rotations about three mutually orthogonal axes of a single crystal. It is shown here that just two, or one, of the above rotations suffice to determine the CSA tensor orientation by borrowing, the easy to obtain, principal values of CSA from an independent source. Methods for using two rotation patterns or even a single rotation pattern are described and illustrated with known chemical shielding tensors.     

Separating Structure and Dynamics in CSA/DD Cross-Correlated Relaxation: A Case Study on Trehalose and Ubiquitin

We present two new sensitivity enhanced gradient NMR experiments for measuring interference effects between chemical shift anisotropy (CSA) and dipolar coupling interactions in a scalar coupled two-spin system in both the laboratory and rotating frames. We apply these methods for quantitative measurement of longitudinal and transverse cross-correlation rates involving interference of ¹³C CSA and ¹³C–¹H dipolar coupling in a disaccharide, α,α- -trehalose, at natural abundance of ¹³C as well as interference of amide ¹⁵N CSA and ¹⁵N–¹H dipolar coupling in uniformly ¹⁵N-labeled ubiquitin. We demonstrate that the standard heteronuclear T₁, T₂, and steady-state NOE autocorrelation experiments augmented by cross-correlation measurements provide sufficient experimental data to quantitatively separate the structural and dynamic contributions to these relaxation rates when the simplifying assumptions of isotropic overall tumbling and an axially symmetric chemical shift tensor are valid.     

An open magnet utilizing ferro-refraction current magnification

Ferro-refraction is the field magnification that is obtained when a current segment is near a high magnetic permeable boundary. It is shown that ferro-refraction may be used in the design of magnets for NMR or MRI to increase the efficiency of these magnets. The field may be modeled analytically with the Biot--Savart law and the inclusion of mirror image currents. Ferro-refraction is particularly useful in the design of monohedral magnets, magnets producing a remote homogeneous region which have the magnetic sources arranged to one side. These magnets have also been called planar magnets. Two designs for a monohedral magnet which produce good agreement between experimental and analytic results are presented.     

Probing Proteins in Solution by Xe NMR Spectroscopy

The interaction of xenon with different proteins in aqueous solution is investigated by ¹²⁹Xe NMR spectroscopy. Chemical shifts are measured in horse metmyoglobin, hen egg white lysozyme, and horse cytochrome c solutions as a function of xenon concentration. In these systems, xenon is in fast exchange between all possible environments. The results suggest that nonspecific interactions exist between xenon and the protein exteriors and the data are analyzed in term of parameters which characterize the protein surfaces. The experimental data for horse metmyoglobin are interpreted using a model in which xenon forms a 1:1 complex with the protein and the chemical shift of the complexed xenon is reported (Locci et al., Keystone Symposia “Frontiers of NMR in Molecular Biology VI”, Jan. 9–15, 1999, Breckenridge, CO, Abstract E216, p. 53; Locci et al., XeMAT 2000 “Optical Polarization and Xenon NMR of Materials”, June 28–30, 2000, Sestri Levante, Italy, p. 46).