Hyperpolarized 13C NMR studies of glucose metabolism in living breast cancer cell cultures.

The recent development of dissolution dynamic nuclear polarization (DNP) gives NMR the sensitivity to follow metabolic processes in living systems with high temporal resolution. In this article, we apply dissolution DNP to study the metabolism of hyperpolarized U-(13)C,(2)H7-glucose in living, perfused human breast cancer cells. Spectrally selective pulses were used to maximize the signal of the main product, lactate, whilst preserving the glucose polarization; in this way, both C1-lactate and C3-lactate could be observed with high temporal resolution. The production of lactate by T47D breast cancer cells can be characterized by Michaelis-Menten-like kinetics, with K(m) = 3.5 ± 1.5 mM and V(max) = 34 ± 4 fmol/cell/min. The high sensitivity of this method also allowed us to observe and quantify the glycolytic intermediates dihydroxyacetone phosphate and 3-phosphoglycerate. Even with the enhanced DNP signal, many other glycolytic intermediates could not be detected directly. Nevertheless, by applying saturation transfer methods, the glycolytic intermediates glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, phosphoenolpyruvate and pyruvate could be observed indirectly. This method shows great promise for the elucidation of the distinctive metabolism and metabolic control of cancer cells, suggesting multiple ways whereby hyperpolarized U-(13)C,(2)H7-glucose NMR could aid in the diagnosis and characterization of cancer in vivo.

Heteronuclear recoupling in solid-state magic-angle-spinning NMR via overtone irradiation.

A heteronuclear dipolar recoupling scheme applicable to I-S spin pairs undergoing magic-angle-spinning (MAS) is introduced, based on the overtone irradiation of one of the coupled nuclei. It is shown that when I is a quadrupole, for instance (14)N, irradiating this spin at a multiple of its Larmor frequency prevents the formation of MAS dipolar echoes. The ensuing S-spin signal dephasing is significant and dependent on a number of parameters, including the I-S dipolar coupling, the magnitude of I's quadrupolar coupling, and the relative orientations between these two coupling tensors. When applied to a spin-1 nucleus, this overtone recoupling method differs from hitherto proposed recoupling strategies in that it involves only the +/-1> I(z) eigenstates. Its dephasing efficiency becomes independent of first-order quadrupolar effects yet shows a high sensitivity to second-order offsets. A constant-time/variable-offset recoupling sequence thus provides a simple route to acquire, in an indirect fashion, (14)N overtone spectra from rotating powders. The principles underlying this kind of S-(14)N experiments and different applications involving S = (13)C, (59)Co sites are presented.

Spin-1/2 and beyond: a perspective in solid state NMR spectroscopy.

Novel applications of solid state nuclear magnetic resonance (NMR) to the study of small molecules, synthetic polymers, biological systems, and inorganic materials continue at an accelerated rate. Instrumental to this uninterrupted expansion has been an improved understanding of the chemical physics underlying NMR. Such deeper understanding has led to novel forms of controlling the various components that make up the spin interactions, which have in turn redefined the analytical capabilities of solid state NMR measurements. This review presents a perspective on the basic phenomena and manipulations that have made this progress possible and describes the new opportunities and challenges that are being opened in the realms of spin-1/2 and quadrupole nuclei spectroscopies.

Spectral editing in (13)C MAS NMR under moderately fast spinning conditions.

Novel procedures for the spectral assignment of peaks in high-resolution solid-state (13)C NMR are discussed and demonstrated. These methods are based on the observation that at moderate and already widely available rates of magic-angle spinning (10--14 kHz MAS), CH and CH(2) moieties behave to a large extent as if they were effectively isolated from the surrounding proton reservoir. Dipolar-based analogs of editing techniques that are commonly used in liquid-state NMR such as APT and INEPT can then be derived, while avoiding the need for periods of homonuclear (1)H--(1)H multipulse decoupling. The resulting experiments end up being very simple, essentially tuning-free, and capable of establishing unambiguous distinctions among CH, CH(2), and --C--/-CH(3) carbon sites. The principles underlying such sequences were explored using both numerical calculations and experimental measurements, and once validated their editing applications were illustrated on a number of compounds.

Two-dimensional dynamic-director (13)C NMR of liquid crystals.

A novel nuclear magnetic resonance (NMR) experiment for facilitating the resolution and assignment of liquid crystalline (13)C NMR spectra is described. The method involves the motor-driven reorientation of the liquid crystalline director, in synchrony with the acquisition of a 2D chemical shift correlation spectrum. By monitoring in this fashion the (13)C NMR evolution of spins in the liquid crystal at two different director orientations with respect to the magnetic field, the method distinguishes anisotropic from isotropic displacements and can be utilized for assigning the resonances and estimating local degrees of order. Of various potential pairs of angles suitable for such a correlation, the (0 degrees, 90 degrees ) choice was found to be most convenient, as it avoids line broadening complications that may otherwise originate from heterogeneities of the oriented phase. The technique thus derived was employed in the analysis of a series of monomeric and polymeric liquid crystal systems.

Multidimensional dipolar exchange-assisted recoupling measurements in solid-state NMR.

Aseries of uni- and multidimensional variants of the dipolar exchange-assisted recoupling (DEAR) NMR experiment is described and applied to determinations of (13)C-(14)N dipolar local field spectra in amino acids and dipeptides. The DEAR protocol recouples nearby nuclei by relying on differences in their relative rates of longitudinal relaxation, and has the potential to give quantitative geometric results without requiring radiofrequency pulsing on both members of a coupled spin pair. One- and two-dimensional variants of this recoupling strategy on generic I-S pairs are discussed, and measurements of (13)C-(14)N distances and 2D local field experiments sensitive to the relative orientation of CN vectors with respect to the (13)C shielding tensor are presented. Since these measurements did not involve pulsing on the broad nitrogen resonance, their results were independent of the quadrupolar parameters of this nucleus. High-resolution 3D NMR versions of the 2D experiments were also implemented in order to separate their resulting local field patterns according to the isotropic shifts of inequivalent (13)C sites. These high-resolution 3D acquisitions involved collecting a series of 2D DEAR NMR data sets on rotating samples as a function of spinning angle, and then subjecting the resulting data to a processing akin to that involved in variable-angle correlation NMR. Once successfully tested on l-alanine this experiment was applied to the analysis of a series of dipeptides, allowing us to extract separate local field (13)C-(14)N spectra from this type of multisite systems.

Heteronuclear local field NMR spectroscopy under fast magic-angle sample spinning conditions

The acquisition of bidimensional heteronuclear nuclear magnetic resonance local field spectra under moderately fast magic-angle spinning (MAS) conditions is discussed. It is shown both experimentally and with the aid of numerical simulations on multispin systems that when sufficiently fast MAS rates are employed, quantitative dipolar sideband patterns from directly bonded spin pairs can be acquired in the absence of (1)H-(1)H multiple-pulse homonuclear decoupling even for "real" organic solids. The MAS speeds involved are well within the range of commercially available systems (10-14 kHz) and provide sidebands with sufficient intensity to enable a reliable quantification of heteronuclear dipolar couplings from methine groups. Simulations and experiments show that useful information can be extracted in this manner even from more tightly coupled -CH(2)- moieties, although the agreement with the patterns simulated solely on the basis of heteronuclear interactions is not in this case as satisfactory as for methines. Preliminary applications of this simple approach to the analysis of molecular motions in solids are presented; characteristics and potential extensions of the method are also discussed. Copyright 2000 Academic Press.

Quadrupolar and chemical shift tensors characterized by 2D multiple-quantum NMR spectroscopy

The present work discusses a new 2D NMR method for characterizing the principal values and relative orientations of the electric field gradient and the chemical shift tensors of half-integer quadrupolar sites. The technique exploits the different contributions that quadrupolar and shielding interactions impart on the evolution of multiple-quantum and of single-quantum coherences, in order to obtain 2D powder lineshapes that are highly sensitive to these nuclear spin coupling parameters. Different spinning variants of this experiment were assayed, but it was concluded that a static version can yield the highest sensitivity to the values of the principal components and to the relative geometries of the local coupling tensors. It was found that correlating the central transition evolution with the highest available order of the spin coherence was also helpful for maximizing this spectral information. Good agreement between data obtained on 87Rb (S = 32) and 59Co (S = 72) samples and ideal theoretical lineshape predictions of this experiment was obtained, provided that heterogeneities in the multiple-quantum excitation and conversion processes were suitably accounted by procedures similar to those described in the spin-(1/2) multiple-quantum NMR literature. Copyright 1999 Academic Press.

Orientational alignment in solids from bidimensional isotropic-anisotropic nuclear magnetic resonance spectroscopy: applications to the analysis of aramide fibers.

The use of two-dimensional isotropic-anisotropic correlation spectroscopy for the analysis of orientational alignment in solids is presented. The theoretical background and advantages of this natural-abundance 13C NMR method of measurement are discussed, and demonstrated with a series of powder and single-crystal variable-angle correlation spectroscopy (VACSY) experiments on model systems. The technique is subsequently employed to analyze the orientational distributions of three polymer fibers: Kevlar 29, Kevlar 49 and Kevlar 149. Using complementary two-dimensional NMR data recorded on synthetic samples of poly(p-phenyleneterephthalamide), the precursor of Kevlar, it was found that these commercial fibers possess molecules distributed over a very narrow orientational range with respect to the macroscopic director. The widths measured for these director distribution arrangements were (12 +/- 1.5) degrees for Kevlar 29, (15 +/- 1.5) degrees for Kevlar 49, and (8 +/- 1.5) degrees for Kevlar 149. These figures compare well with previous results obtained for non-commercial fiber samples derived from the same polymer.

Solid and liquid phase 59Co NMR studies of cobalamins and their derivatives.

We describe the application of 59Co NMR to the study of naturally occurring cobalamins. Targets of these investigations included vitamin B12, the B12 coenzyme, methylcobalamin, and dicyanocobyrinic acid heptamethylester. These measurements were carried out on solutions and powders of different origins, and repeated at a variety of magnetic field strengths. Particularly informative were the solid-state central transition NMR spectra, which when combined with numerical line shape analyses provided a clear description of the cobalt coupling parameters. These parameters showed a high sensitivity to the type of ligands attached to the metal and to the crystallization history of the sample. 59Co NMR determinations also were carried out on synthetic cobaloximes possessing alkyl, cyanide, aquo, and nitrogenated axial groups, substituents that paralleled the coordination of the natural compounds. These analogs displayed coupling anisotropies comparable to those of the cobalamins, as well as systematic up-field shifts that can be rationalized in terms of their stronger binding affinity to the cobalt atom. Cobaloximes also displayed a higher regularity in the relative orientations of their quadrupole and shielding coupling tensors, reflecting a higher symmetry in their in-plane coordination. For the cobalamines, poor correlations were observed between the values measured for the quadrupole couplings in the solid and the line widths observed in the corresponding solution 59Co NMR resonances.

Pulsed-gradient spin-echo measurements of anisotropic diffusion by dipole-decoupled 13C nuclear magnetic resonance.

A new approach for monitoring diffusion in anisotropic phases is proposed and demonstrated. The method relies on the observation of dilute spins (e.g., 13C) in the presence of heteronuclear high-power dipolar decoupling, a procedure which can yield time-domain nuclear magnetic resonance (NMR) signals lasting over three orders of magnitude longer than their 1H counterparts. This allows one to apply conventional 90-180 degrees pulsed-gradient spin-echo (PGSE) schemes on organic systems without having to employ complex and highly sensitive multiple-pulse 1H irradiation schemes. Instrumental aspects of this 13C method are discussed, and an application to the variable-temperature determination of anisotropic self-diffusion in a thermotropic liquid crystal is illustrated.

Interactions between natural polyamines and tRNA: an 15N NMR analysis.

15N NMR spectroscopy was used to explore the interactions between natural polyamines and Escherichia coli tRNA. It was found that when tRNA is added to solutions of 15N-labeled spermine or spermidine, there is a considerable decrease in the relative heights of the -NH(2+)--resonances with respect to the signals arising from the -NH3+ groups. The presence of tRNA was also found to reduce the longitudinal relaxation times T1 of the nitrogens, mainly those of the -NH(2+)- groups. The longitudinal relaxation times of the nitrogens were used to characterize the temperature dependence of the binding, and they allowed us to calculate the activation energies that determine the correlation times of amino groups in the presence of tRNA. Both the thermodynamic and the relaxation results indicate that (i) spermine binds more strongly to tRNA than spermidine does and (ii) within each of these molecules the -NH(2+)- groups bind more strongly to tRNA than the more electropositive -NH3+ moieties. This specificity suggests that the interaction between polyamines and tRNA cannot be described exclusively in terms of electrostatic forces and that other interactions (most likely, hydrogen bonding) are very important for establishing the polyamine-tRNA link. Some of the factors that may conspire against the binding of -NH3+ groups to tRNA are briefly discussed.

NMR diffraction and spatial statistics of stationary systems.

Nuclear magnetic resonance (NMR) spatial imaging data may be acquired, processed, and interpreted in ways that provide information directly analogous to diffraction experiments, with length scales determined by gradient strengths rather than radiation wavelengths. This approach, originally considered by Mansfield nearly two decades ago, provides access to autocorrelations of sample density that statistically characterize small-scale density variations. These NMR "Patterson functions" can be acquired orders of magnitude more rapidly than comparably resolved NMR images and are suitable for spatial characterization of small features in bulk samples, such as morphology in structural materials. Unlike hindered diffusion approaches, neither mobility, penetrants, nor transport time are required for examining granularity and porosity.

Physical restraint in Polish psychiatric facilities.

Physical restraint in the form of direct force, forced feeding and medication, mechanical restriction of bodily movement, or the threat of force was administered to close to 30% of patients admitted and hospitalized for a period of at least fourteen days. In transporting the patient to the admission room and then to his treatment ward, the dominant form of restraint used was direct force, or the threat of such force. During the following fourteen days of hospitalization, mechanical restraint and forced medication predominated. Restraint was applied most often, across each of the four phases of the study, to patients deemed dangerous to self or others. This relationship became significantly stronger as the period of hospitalization continued. Across the four phases, patients who protested their hospitalization were more likely to be restrained than patients who agreed to it or who did not object. During each of the four phases of the study, numerous patients who did not object to their hospitalization and who were not deemed dangerous were nevertheless subjected to restraint. The pervasiveness of physical restraint, the wide inter-hospital differences in its use, the apparent over-prediction of patient dangerousness, and the use of restraint with patients who were not deemed dangerous and who did not object to their hospitalization, confirms the need for legal and ethical regulation of physical restraint of psychiatric patients.

Psychiatric hospitalization in Poland.

An overview of psychiatric hospitalization in Poland is presented in the context of Polish political and socio-cultural developments. The areas addressed include: the characteristics of the patient population; the organization of Polish mental health service; the nature of psychiatric treatment; psychiatric legislation; patients' rights; and the training and social status of the various mental health professionals. In spite of the meager resources allocated to mental health services, and the consequent staff shortages and overcrowded, drab living conditions in psychiatric facilities, the care afforded patients is generally humane and nonoppressive. Polish psychiatry has succeeded in maintaining its professional autonomy and has assumed a leadership role in the modernization of its service delivery system.

KIE: After first disputing some widely-held Western perceptions concerning mental health care in Eastern Europe and then depicting the sociocultural setting of institutional psychiatry in Poland, the author provides an overview of the patient population, staff, and organization of the psychiatric hospital in Poland. He then discusses involuntary commitment and the development of what is likely to be the next version of Poland's Mental Health Act. He also compares the rights of psychiatric patients in Poland and the United States, and presents an optimistic appraisal of the future of patients' rights in Poland.