121
Session Assigned: W&Th P
Title: Exploring the mechanism of ion transport regulation by solid state NMR

Institute: Dept. of Biochemistry, University of Oxford, U.K.
Keywords: magic-angle spinning, rotational resonance, REDOR, Na+/K+-ATPase, ouabain
Abstract:
Ion transportation accross membranes by the P-type ATPases is crucial to many fundamental physiological and pathological events. Understanding the molecular mechanisms of ion transporter regulation is hampered by the continuing lack of high-resolution structural information on these proteins, even though the discovery of the Na+/K+-ATPase was made over 40 years ago. Solid state NMR is now beginning to answer key questions relating to the regulation of the sodium pump and a highly homologous protein, the gastric H+/K+-ATPase, by small molecule inhibitors. Novel NMR experiments will be described which estimate the rates of exchange of reversible inhibitors with these proteins, thereby giving information on their relative binding affinities. Solid state NMR distance (13C-13C and 13C-19F) measurements and angle measurements on inhibitors such as ouabain bound to their sites of action in the functionally and structurally intact proteins are helping to determine their active binding conformations and accumulate structure-activity relationship data. Finally, new methods for isotope labelling unique sites in the proteins themselves are enabling the protein structure within 10 A of the inhibitor binding site to be explored using experiments to recouple the dipole interactions between labels on the the ligand and protein.

122
Session Assigned: W&Th P
Title: Indirect coupling tensors in para-difluorobenzene: NMR experiments in liquid crystals and ab initio MCSCF calculations

Institute: University of Oulu, Oulu, Finland
Keywords: indirect coupling ; ab initio calculation; LCNMR; para-difluorobenzene
Abstract:
The indirect coupling tensors J_XF (X = H, C, F) in para-difluorobenzene were determined by NMR experiments in liquid-crystalline solutions and ab initio multiconfiguration self-consistent field linear response calculations. In the calculations, all the physical contributions to the tensors were obtained at consistent theoretical level. The experiments were performed in the so-called good mixtures of nematic solvents in order to minimize the deformation due to anisotropic forces. The effects of vibrational motion and solvent induced deformation of molecular geometry of the solute were taken into account in the data analysis.

In the present case, the information derived from experimental NMR spectra is insufficient for the determination of all the tensorial properties of indirect couplings. Therefore, some of the results from the theoretical calculations had to be utilized in the analysis of experimental data. The remaining anisotropy and asymmetry parameters related to the ⁿJ_CF (n = 1,2,3,4) and ⁵J_FF tensors were obtained in good agreement with the ab initio results.

This study indicates that the tensorial properties of the indirect fluorine couplings may affect the corresponding anisotropic couplings, D^exp, by even several percent. In particular, in molecules with D_2h or lower point group symmetry, in which cases the orientation of the molecule is decribed by two or more order parameters, the indirect coupling may even dominate D^exp. Consequently, this contribution must be taken into account when using anisotropic couplings in accurate determinations of geometry and orientation of molecules containing, for example, fluorine-substituted phenyl rings.

123
Session Assigned: W&Th P
Title: Purple membrane induced alignment of biological macromolecules in the magnetic field

Institute: Forschungszentrum Juelich/University Duesseldorf
Keywords: electric interaction; steric interaction; phase transition; colloid
Abstract:
The general possibility to align biological macromolecules in the magnetic field by the presence of orienting agents such as lipid bicelles has led to a wealth of new structural parameters which can be derived from residual tensorial interactions in high resolution NMR. Here, we report that alignment of water soluble biomacromolecules can be achieved in the presence of the naturally occurring two-dimensional crystals (purple membrane) of the membrane protein bacteriorhodopsin. The extent of the alignment is tunable by the concentration of purple membranes and by the addition of salt which reduces the strength of the electric interaction between solute macromolecule and the membrane. At very high salt concentrations the purple membrane suspension undergoes a phase transition to a very viscous state. In this state, rotation of the membranes is hindered, such that the orientation of the membrane patches persists even in the absence of a magnetic field. The induced alignment of solute molecules is shown for the two proteins, ubiquitin and p53, with residual dipolar one bond 1H-15N couplings in the order of 20 Hz. A concept for the description of the irreducible components of the alignment tensors as a linear vector space is presented. In the case of ubiquitin, the direction of the alignment tensor differs strongly from the alignment tensor observed in DMPC/DHPC bicelles. This offers the possibility of an accurate triangulation of the bond vector direction from a combination of the two alignment experiments.

124
Session Assigned: W&Th P
Title: Phase Transitions, Molecular Packing and Motion in Reentrant Nematic Liquid Crystals: A Xe-129 NMR Study

Institute: Chem. Dept. and NHMFL, U. Florida, Gainesville, FL
Keywords: reentrance, nematic, xenon NMR, liquid crystal
Abstract:
The ability to detect and characterize nematic reentrance by xenon-129 NMR chemical shift and spin relaxation data is demonstrated in several different reentrant nematic liquid crystal mixtures. The activation energies of the dominating spin relaxation process in the nematic and reentrant nematic phases are derived from the temperature dependence of the spin relaxation times. The results for the binary and tertiary reentrant nematic liquid crystal mixtures are compared on the basis of molecular packing and dynamical modes responsible for nuclear spin relaxation.

support: NSF CHE-9724635, NHMF IHRP (CRB).

125
Session Assigned: W&Th P
Title:Lipid Bilayer Dynamics from Deuterium NMR Relaxation

Institute: University of Arizona, Tucson, Arizona 85721, USA
Keywords: liquid crystals; membranes; molecular dynamics; order fluctuations
Abstract:
Measurement and analysis of the NMR spin relaxation rates of molecular solids, liquid crystals, proteins, and lipid membranes constitute a powerful approach for evaluating the properties of these materials. The various relaxation rates depend on the spectral density of the stochastic fluctuations, and manifest their amplitudes and time-scales (1). As a rule, the mean-squared amplitudes are related to the average structure; whereas the correlation times represent the dynamics. In the case of membrane lipids, relaxation measurements as a function of both frequency and bilayer orientation allow one to disentangle the rates and amplitudes of the various fluctuations (2).

However, in the case of membrane lipids there is a problem: models for molecular motions that are successful in reproducing the angular anisotropy of the relaxation fail to describe the frequency dependence. Likewise, models for collective bilayer dynamics that reproduce the frequency dependence of the relaxation fail to explain the angular anisotropy. Hence, a composite membrane deformation model has been developed (3), which includes collective order-director fluctuations (ODF) plus rotations of the lipid chains. The model has been successfully fit to angular-dependent ²H spin-lattice (R_1Z) and quadrupolar order (R_1Q) ²H relaxation rates for DMPC-d₅₄ in the liquid-crystalline state, together with frequency-dependent ²H R_1Z data for specifically ²H-labeled DMPC. The results suggest the dynamics of lipid bilayers in the MHz regime involve nematic-like excitations of the membrane interior (free membrane limit), together with axial rotations of the ²H-labeled chain segments.

1. M. F. Brown and S. I. Chan, in Encyclopedia of Nuclear Magnetic Resonance, Wiley, New York, p. 871 (1995).
2. A. A. Nevzorov and M. F. Brown, J. Chem. Phys. 107, 10288 (1997).
3. A. A. Nevzorov, T. P. Trouard, and M. F. Brown, Phys. Rev. E 58, 2259 (1998).

126
Session Assigned: W&Th P
Title: NMR Relaxation and Diffusion Studies of Fluorocarbon Surfactant Systems

Institute: Royal Institute of Technology, Stockholm, Sweden
Keywords:
Abstract:
Fluorocarbon surfactants are in many respects different from their hydrocarbon-based cousins. For example, they have larger surface activity and present rather different phase diagrams. This variation in properties can be traced back to their differences in molecular properties, such as conformational flexibility and charge distribution. When studied by NMR, fluorocarbon surfactants present some advantages. Most importantly, the large chemical shift dispersion of ¹⁹F makes the signals from the different fluoroalkyl segments along the surfactant tail well separated from each other. ¹⁹F chemical shifts are also sensitive to the solvent environment.

¹⁹F and ¹⁹F-decoupled ¹³C NMR spectroscopy at several different field strengths has been used to measure the spin relaxation in perfluoroalkanoate soaps and perfluoroalkanoate-based polysoaps (perfluoroalkyl chains attached to a polymer backbone). Simple perfluoroalkanoate micelles present conventional high-resolution ¹⁹F and ¹³C spectra which indicates fast (~ms) exchange of the surfactant molecules among different micellar aggregates. The obtained field-dependent ¹³C longitudinal relaxation rates and ¹³C-¹⁹F cross-relaxation rates yield the order parameter profile of the fluorocarbon chain, which is both flatter and of larger values than the order parameter profiles in corresponding hydrocarbon-based surfactant micelles. In the polysoap systems, the ¹⁹F NMR spectra consist of broad and split lines, indicating slow (~s) exchange of the hydrophobic chains among different aggregates. Within these long-lived aggregates, however, the molecular dynamics is rather similar to that in simple micelles. One significant difference, though, is that the polysoap aggregates cannot include all sidechains. This feature is caused by the steric constraint imposed on by the polymer backbone which leaves some hydrophobic sidechains excluded into the solvent. This internal structure is rapidly reorganizing, leading to a fast (~ms) exchange of the sidechains between aqueous and hydrophobic environments, as inferred from ¹⁹F transverse relaxation rates.

Lowering the temperature transforms the isotropic micellar solution of perfluoroalkanoates into various lyotropic liquid crystal phases. Often, a nematic phase is followed by a defective lamellar one and the nature of the phase transition between these two phases remains a controversial issue. In particular, despite extensive scattering and NMR studies it remains unclear when, if ever, do the individual discotic micelles of the nematic phase start to fuse into a defective lamellar bilayer. Here we show how to use PGSE-type ¹⁹F-decoupled ¹³C NMR experiments for measuring the anisotropic diffusion of the surfactant molecules in those phases. In particular, the in-layer diffusion coefficient is sensitive to the topology of the aggregates and thus can be used to pinpoint the onset of micellar fusion.

127
Session Assigned: W&Th P
Title: Fast Molecular Reorientations in Electric Fields investigated by Solid-state NMR

Institute: Universität Leipzig, 04103 Leipzig, Germany
Keywords:
Abstract:
NMR techniques have been modified in order to investigate in-situ fast reorientation processes which are induced by externally applied electric fields. The experiments have been carried out on thin capacitor cells at relatively high voltages which provides some technical problems. The synchronization of the NMR pulse programs and of the switching of voltage allows, in combination with the reversibility of the reorientation, the access to fast processes up to reorientation times shorter than 1 ms. Results are discussed for low molecular liquid crystals and liquid crystalline polymers exploiting information obtained from the time dependence of dipolar interaction and chemical shift anisotropy as well. Some material parameters can be obtained by the external manipulation of orientation states and of reorientational dynamics.

Institute: Massachusetts Institute of Technology
Keywords:
Abstract:
The study of spatially heterogeneous samples (e.g. emulsion, biological and geologic specimens) by NMR presents unique challenges due to the presence of local background magnetic field gradients that are introduced by the sample itself. Even for a diamagnetic sample, variations in bulk magnetic susceptibility, when placed in a strong magnetic field, lead to small distortions of the electron orbits that introduce local dipole fields. While the shape and size of the overall field distribution is very sample geometry dependent, the field may locally be taken as dipolar. Since the background gradients have simple dipolar symmetry, magic angle sample spinning (MAS) is able to remove this broadening through coherent averaging, as shown by Garroway (J. Magn. Reson. 49, 168, 1982). Here it is helpful to recall that the dipolar interaction is traceless, and so MAS returns a high resolution like spectrum. This realization, along with recent designs for MAS probes capable of high resolution (Maas et al. J. Am. Chem. Soc. 118, 13085,1996), has led to a number of NMR studies on semi-solid state heterogeneous samples, including swollen resins for combinatorial chemistry, lipids, tissues, and cultured cells.

The influence of these local fields on the NMR signal depends on both the strength of the local magnetic field gradient and on the rate at which the molecules diffuse through the gradient. In the case of fast MAS, both the static as well as the dynamic effects of the susceptibility fields are averaged. In the case of slow MAS, the NMR resonance is split into sidebands, whose analysis permits a direct measurement of the strength of the backgrounds field in the sample.

129
Session Assigned: W&Th P
Title: SR-MAS Method as a New Technology for the Structure Determination in Combinatorial Chemistry

Institute: JEOL Ltd. and The University of Tokyo
Keywords: SR-MAS Method; solid-phase synthesis; combinatorial Chemistry
Abstract:
It was shown that the SR-MAS(Swollen-Resin Magic Angle Spinning) method enabled us to determine the structure of solid-phase organic products without cleavage from solid supports. Consequently, characterization of resins containing products was successfully carried out in very short time compared with the conventional methods such as IR or MS. It takes more than 1 day to confirm the structure of solid-phase organic products using conventional methods. SR-MAS technique enables us to confirm it in only 30 minutes. Usefulness of the 13C SR-MAS spectrum has been demonstrated throughout the useful carbon-carbon bond-forming reactions on the solid-phase.

JEOL Lambda400 spectrometer and SR-MAS probe was used for these experiment. The diameter of the sample tube is 6mm. The volume of the sample tube is 166ul. The sample cap is made of Kel-F. The clearance between the tube and the cap is very tight so that there is no leakage of solvent when the sample tube is rotating at the speed of 4kHz. The sampling process is very easy and 13C sensitivity is very high, because of the big volume sample tube.

S.kobayashi, et al. Molecules Online, 1998, 2, 35-39.

130
Session Assigned: W&Th P
Title: Applications of Magic Angle Spinning in Solid Phase Synthesis and Combinatorial Chemistry, SPMAS.

Institute: Affymax Research Institute,Santa Clara, California
Keywords:
Abstract:
Solid phase organic synthesis suffers from a lack of routine analytical tools for following reactions and assessing product identity, purity and yield. In particular, conventional NMR spectra of organic molecules covalently attached to solid surfaces or solid lattices are difficult to interpret due to large line widths caused by magnetic inhomogeneities at the solid interface. During the last four years we have succeeded in overcoming a number of these difficulties and now applying the SPMAS technique routinely. This poster illustrates applications in the following areas.

1. Monitoring large scale syntheses on not so NMR friendly supports, on polystyrene beads.
2. NMR spectra of small molecules covalently bonded to PTFA membranes.
3. NMR spectra of small molecules covalnetly bonded to ordinary filter paper.

Solid phase organic synthesis suffers from a lack of routine analytical tools for following reactions and assessing product identity, purity and yield. In particular, conventional NMR spectra of organic molecules covalently attached to solid surfaces or solid lattices are difficult to interpret due to large line widths caused by magnetic inhomogeneities at the solid interface. During the last four years we have succeeded in overcoming a number of these difficulties and now applying the SPMAS technique routinely.

This poster illustrates applications in the following areas.

1. Monitoring large scale syntheses on not so NMR friendly supports, on polystyrene beads.
2. NMR spectra of small molecules covalently bonded to PTFA membranes.
3. NMR spectra of small molecules covalnetly bonded to ordinary filter paper.

131
Session Assigned: W&Th P
Title: On Bead Analysis of Peptides and other Solid Phase Synthesis Products using High Resolution MAS NMR

Institute: University of Chicago, Chicago, IL, USA
Keywords:
Abstract:
Using the proper swelling agent the residual mobility of polymer bound molecules is high enough that magic angle spinning using a High Resolution MAS or Nano probe leads to spectra approaching the quality of solution state NMR.

The poster gives an overview of the results achieved from homo- and heteronuclear 1D/2D HRMAS-NMR experiments for:

A) the reaction control of a Sharpless asymmetric dihydroxylation,
B) a partly 15N-labeled peptide as model for peptides/proteins attached to biomolecular surfaces.

132
Session Assigned: W&Th P
Title: Spin Echoes for Diffusion in a Constant Gradient in a Restricted Geometry

Institute: Schlumberger-Doll Research
Keywords: CPMG, PGSE, Restricted Diffusion, Edge Enhancement
Abstract:
The spin echo response of magnetization of spins diffusing in abounded region of size L_Sin the presence of a magnetic field inhomogeneity is charachterized by a dephasing length, L_G, a diffusion length during half-echo time, L_D, and a length L_&r due to surface relaxation. The relaxation exponent, depends on the echo number n, and dimensionless parameters which are given by combinations of these lengths. Depending on the length scales, three main regimes of decay have been identified: free, localization and motionally averaging regimes. In the localization regime, the decay exponent depends on a fractional power of gradient, denoting a break-down of the cumulant expansion or the Guassian phase approximation (GPA). In the other two regimes, the GPA works well.

133
Session Assigned: W&Th P
Title: Ammonia diffusion in aqueous solutions: a 14N study

Institute: University of California, Davis, CA 95616
Keywords: N-14 NMR; diffusion
Abstract:
Flammability and toxicity of ammonia resulting from release of gases from the waste tanks at Hanford (Washington) have recently been the subject of several studies at the Pacific Northwest National Laboratories (PNNL). Given that diffusion is a crucial parameter quantifying transport of hazardous compounds in the waste, we have conducted preliminary studies to characterize the diffusivity of ammonia in model systems. The diffusion of ammonium ions in aqueous solutions was measured by Pulsed Field Gradient (PFG) methods directly on the nitrogen nuclei (14N). The ammonium solutions were obtained by dissolving ammonium salts and/or ammonium hydroxide in distilled water. Despite the low gyromagnetic ratio of nitrogen, results show that it is possible to measure the diffusion of ammonium ions in aqueous solutions for concentrations as low as 0.18 M. Typical values of the diffusion coefficient of the ammonium ion are around 2.1 x.10^-5 cm2/s (+/-10%) at 20 deg. C. The experiments conducted at the University of California, Davis have allowed the diffusion of ammonia to be characterized over a wide range of conditions similar to those within the Hanford tanks: high pH, low concentration of ammonia and solutions within porous media.

134
Session Assigned: W&Th P
Title: Microstructural Investigations of Monoglyceride Gels by PFG NMR

Institute: Unilever Research Vlaardingen, The Netherlands
Keywords:
Abstract:
Monoglycerides can act as surfactants and when mixed with water they can occur in several liquid crystal forms. Because of their ability to bind large volumes of water these molecules can be employed as fat-replacers in food technology. The macroscopic properties of these materials governed by the molecular and microstructural arrangement of the monoglyceride surfactants. Expanding our knowledge in structure-property relations at the molecular and microstructural level will permit us to have better understanding of these water-structured products.

NMR PFG water self-diffusion measurements were used for the microstructural investigation of these materials and it has been demonstrated that this provides a method for the determination of several microstructural parameters, i.e. network tortuosity, surface-to-volume ratio, network heterogeneity, and network dimensionality. Below a threshold value, both tortuosity and surface-to-volume ratio vary proportional with concentration, This is due to the formation of more crystal plates in the network when more monoglyceride is added. At higher monoglyceride concentrations the diffusional properties of water become invariant: no additional plates are formed. The microstructural parameters of monoglyceride gels, as assessed with PFG NMR, also vary with monoglyceride type and processing conditions. TD and PFG NMR experiments were employed in order to extract information regarding network heterogeneity and dimensionality. The conclusions drawn for the PFG NMR results are in accordance with electron and light microscopy observations.

135
Session Assigned: W&Th P
Title: Solid State 15N-1H 2D HETCOR Studies of Diphenylmethane Diisocyanate Reaction in Wood

Institute: Department of Chemistry, The University of Akron
Keywords: solid state; 2D-NMR; polyurethane; wood composites; cellulose
Abstract:
The N-15/H-1 solid state HETCOR NMR experiments were employed to study the products from the reaction of N-15 enriched diphenylmethane diisocyanate(MDI) in cellulose, lignin, water, aspen and pine samples. Both urea and biuret structures can be clearly recognized in the wood/MDI composites. Two peaks around 104-106 ppm result from different chemical environment of urea structure. Both intramolecular hydrogen bonding of pMDI and intermolecular hydrogen bonding between pMDI and wood molecules are observed.

136
Session Assigned: W&Th P
Title: Solid-State NMR Studies of Environmental Photocatalysis

Institute: Purdue University, West Lafayette, IN 47907
Keywords:
Abstract:
Solid-State NMR is used to probe the surface chemical processes during the degradation of environmental pollutants on various semiconductor photocatalysts. Our experiments are focused on understanding the varying ability of TiO2, V2O5, and WO3 to oxidize TCE and ethanol into less harmful products. Utilizing in-situ irradiation allows us to identify reaction intermediates and monitor reaction pathways. Initial work using powdered TiO2 to decompose TCE showed the presence of dark regions within the sample that reduced the catalytic efficiency by allowing intermediates to persist during the photoreaction. The use of porous Vycor glass and microscopic quartz fibers as catalyst supports eliminated the dark regions and improved the photoefficiency. We have also used porous Vycor glass to support vanadium and tungsten oxide catalysts in the photodegradation of ethanol into carbon dioxide. Supported TiO2 was found to be the most efficient catalyst while V2O5 was moderately effective and WO3 was least effective. Cross polarization experiments show that this trend is related to the formation of surface bound ethoxy species by a small fraction of the ethanol molecules which are much more reactive than hydrogen bonded species.

137
Session Assigned: W&Th P
Title: In Situ Surface Charge Characterization by Capillary Array Electrophoretic NMR

Institute: Department of Chemistry, University of Connecticut
Keywords: Electrophoretic NMR
Abstract:
Proteins, DNA molecules, and other polyions can be organized in layers on surfaces according to detailed "molecular architecture" plans. With few exceptions of using specific enzyme/substrate interactions as assembly forces, most films were fabricated according to electrostatic interactions between oppositely charged polyions. UV-visible spectroscopy, small angle X-ray reflectivity analysis and quartz crystal microbalance (QCM) have been used to characterize the film molecular structures, the amount of adsorbed material and the total film thickness. Few techniques, however, offer in situ surface charge characterization of these films in aqueous solutions. Here we report a simple procedure for such measurement by capillary array electrophoretic NMR. Alternate layer-by-layer assembly of charged films of Poly(styrensulphonate) sodium salt (PSS), poly(allylamine) hydrochloride (PAH) or branched poly(ethylenimine) (PEI) were coated on capillary glass surfaces of electrophoretic NMR sample cells. The surface charges of the films were probed by electroosmotic flow of water and electrophoretic motion of small electrolytes. To our surprise, highly charged polyions in films frequently give no electroosmotic flow of water and are, therefore, neutral at the plane of shear in aqueous solutions. In the presence of a microemulsion solution, we also observed a slow, dynamic change of the glass surface charge.

138
Session Assigned: W&Th P
Title: A New Method for Characterizing Solid Acids

Institute: University of Florida, Gainesville, FL, USA
Keywords: Solid Acidity Scale
Abstract:
Accurately measuring the strength of solid acids is the key to furthering the understanding of acid catalyzed reactions. Techniques based on solvatochromism of probe molecules to establish scales of solution acidity have been highly successful. Magic angle spinning (MAS) NMR has been previously used to characterize several solid acids based on chemical shift changes that depend on the nature of the acid site. However, the choice of probe molecule and nuclear spin species used in this earlier work suffers from several shortcomings. The most severe drawback is that the low concentration of acid sites in or on the solid requires a probe molecule with high sensitivity. To overcome this problem, we have developed a method based on the change in ³¹P isotropic chemical shift of chemisorbed triethylphosphine oxide (TEPO) that is both fast and highly accurate. We have adsorbed TEPO onto solid acid standards with very different acidities and have found that the isotropic ³¹P signal of TEPO is well suited for the characterization of solid acids. The correlation of this scale to other methods of characterization serves to validate this technique.

Institute: GlaxoWellcome Medicines Research Centre Verona (I)
Keywords:
Abstract:
To investigate the mechanism of permeation through the lipid bilayers the High Resolution MAS technique (Nano Probe) were applied to Toluene, Rodamine B and to the fluoroquinolone Ciprofloxacin.

The different mechanism are investigate in order to discriminate relative membrane location for the three model compounds.

comparison between fluorescence and NMR results are deeply analysed and some hypothesis on the distribution of Ciprofloxacin in liposomes are added to help understand the interaction with biological membranes.

141
Session Assigned: W&Th P
Title: Assesment of Drug Toxicity Using ¹H Magic Angle Spinning NMR Spectroscopy of Biological Tissues.

Institute: London, UK
Keywords: HMQC HMBC TOCSY
Abstract:
Traditionally Magic Angle Spinning (MAS) NMR spectroscopy has been applied to the analysis of rigid solids. However, with the new generation of high resolution MAS probes, ¹H MAS NMR spectroscopy of biological tissue samples weighing as little as 8mg has become feasible, thereby making the direct assessment of toxicological consequences in target tissues and organs possible. The relationship between toxin-induced perturbations in the NMR spectral profiles of biofluids and tissue specific lesions has generally been established by analysis of tissue extracts. MAS NMR spectroscopy of intact tissues offers a more direct also bringing insight into mobility and compartmentalisation of metabolites in biomaterials. We have established ¹H MAS profiles for a range of control tissues such as liver, kidney, prostate, brain and red blood cells. 400 MHz MAS NMR spectra, obtained from blood and erythrocytes, MAS spectra were superior in resolution to 800 MHz solution state spectra obtained using a standard probe. We have also evaluated various methods of sample preparation and assessed the effects of changing sample spinning rates and temperature. Perfusion of tissues with D2O saline was shown to significantly enhance spectral resolution. An increase in spin rate from 4KHz to 12 KHz resulted in increased contributions from membrane lipids. This increase in spin rate was also found to induce a corresponding (frictional) increase in temperature which was measured directly from the differences in the chemical shifts of the water and anomeric glucose protons. We have applied MAS NMR spectroscopy to the study of the metabolic consequences of toxic states including nephrotoxicity (using 2-bromoethanamine), hepatotoxicity (using hydrazine) and erythrocyte toxicity (using phenylhydrazine). Combinations of endogenous biomarkers relating to the tissue or mechanism of toxicity were established for each toxin. Application of MAS NMR spectroscopy to the analysis of biological tissues has shown promise in the evaluation of toxicological episodes and disease states and resulted in the biochemical characterisation of chemically-induced damage in tissues which may lend insight into mechanisms of drug toxicity.

142
Session Assigned: W&Th P
Title: MRI measurement of cell volume fraction in the perfused rat hippocampal slice

Institute: University of Florida, Gainesville, Florida, USA
Keywords: contrast agent, ischemia
Abstract:
The mammalian brain slice is an established model for the measurement of water distribution and diffusion. Recently, we demonstrated the utility of the model with NMR imaging [1]. Here we describe a method of determining the cell volume fraction of perfused rat hippocampal slices using gadodiamide and T1 imaging. Measures of tissue compartmentation are important for determining the origin of NMR image contrast in biological tissues.

NMR data were acquired using a home-built perfusion chamber and a Doty microimaging probe interfaced to a Varian 600 MHz instrument [1]. The relaxivity of the perfusate, artificial cerebrospinal fluid (aCSF), was measured using an inversion recovery sequence and samples of aCSF doped with gadodiamide to a maximum concentration of 4 mM. Rat hippocampii were isolated using standard methods and cut into 0.5 mm thick slices. The slices were perfused with aCSF at 292 K. Perfusion was suspended during the acquisition of 11 images, each with an increasing inversion recovery period. Each slice was then perfused for 10 minutes with aCSF containing 1 mM gadodiamide. In experiments on four slices the aCSF was further doped to 4 mM gadodiamide while the remaining slices were perfused with 60 mM mannitol. Data were extracted from regions encompassing the brain slices in each image. All data from each slice were fit simultaneously using an inversion recovery equation to obtain estimates of baseline T1 and intracellular spin fraction, f(IC) (= 1 - f(EC)).

The relaxivity of gadodiamide in aCSF was estimated as 3.75 +/- 0.02 /mM/s. The average value of f(IC) estimated from 8 slices was 0.59 +/- 0.05. When mannitol was added to three slices f(IC) decreased by 36% (p<0.001). All data exhibited monoexponential T1 relaxation and a linear increase in relaxation rate with gadodiamide concentration supporting the fast proton exchange assumption. The addition of mannitol produced the anticipated decrease in f(IC). However, f(IC) does not directly represent cell volume fraction. The spin density of the ECS is greater than that of the ICS. If we assume relative densities of 0.95 of 0.71, respectively [2] the cell volume fraction becomes 0.66. This value is similar to that previously obtained in the hippocampal slice using radiotracer techniques [3]. The NMR imaging method, however, has the significant advantage of allowing us to produce images representing the local values of cell fraction by calculating the change in relaxation rate on a pixel-by-pixel basis. These images can be used to estimate changes in cell volume throughout the slice following osmotic, chemical or physical perturbation. These results further demonstrate the utility of the brain slice model for NMR studies.

[1] Blackband SJ, et al., Magn. Reson. Med. 38, 1012-1015, (1997).
[2] Pirkle JL, Ashley DL, Goldstein JH, Biophys J. 25, 389-406, (1979).
[3] Newman GC, et al., J. Neurosci. Meth. 61, 33-46, (1995).

144
Session Assigned: W&Th P
Title: Fast collection of post-surgical prostate tissue guided by MRI and in vivo 3D ¹H MR spectroscopy.

Institute: University of California San Francisco
Keywords:
Abstract:
Radical resection of the prostate remains a common treatment option for prostate cancer. In addition, resection of the gland provides researchers with valuable opportunities to study human tissues. For many tests, collection and fixation of tissue in a timely fashion is necessitated as degradation may begin quickly. Many prostate tumors are not palpable after surgery and are rarely visually identifiable, so in order to be assured that the tissue collected is either normal or cancerous, microscopic pathologic diagnosis must first be made. Typically, information from pre-surgical biopsy is used to provide an initial guide to disease localization. However, transrectal ultrasound guided biopsies usually provide at best sextant resolution. In addition, because they suffer from large sampling errors and low sensitivity and specificity, it may be difficult to locate tumors post surgically and valuable time may be wasted. Combined high resolution in vivo MRI and ¹H MR spectroscopy has demonstrated the ability to localize cancer within the prostate with both high sensitivity and specificity. In the current study, we demonstrate that the three dimensional information provided by combined MRI/MRS can aid in guiding pathologic identification for quick post-surgical prostate tissue collection..

In 15 patients awaiting prostate gland resection, an endorectal MRI/MRS staging examination was performed. Axial T₁ and T₂ weighted and coronal T₂ weighted images were collected followed by 3D PRESS CSI (0.24 to 0.34 cm³ resolution) using dual band selective inversion with gradient dephasing (BASING) pulses for water and lipid suppression. After the exam, an analytical correction was performed on the images to remove the near-field high signal intensity caused by the endorectal coil. To spatially correlate spectral data sets with prostatic zonal anatomy and MRI images, spectral arrays were overlaid on corresponding T₂ weighted axial images. Spectra were categorized as normal, possible cancer, or cancer based on the peak area ratio of (choline + creatine)/citrate. A radiologic diagnosis of the combined imaging and spectroscopy information was then performed. After surgical removal of the prostate, alignment of the prostate with the MRS overlays was done visually. An incision was then made into the gland to a region found to be most abnormal by MRI/MRS. Cells were scraped from this site and observed microscopically..

In all 15 cases, cancer cells were detected pathologically from the MR guided location, representing a marked improvement over the use of biopsy data to guide tissue collection. Tissue was then collected from the region of the incision for further analysis by various researchers including ourselves. The total time elapsed from removal of the prostate to tissue fixation was typically less than 15 minutes. This represents a novel application for in vivo MRI/MRS of the prostate and provides us with the unique ability to collect tissue from a specific location for future biochemical and high resolution NMR (MAS and extract) analysis..

145
Session Assigned: W&Th P
Title: Changes in Apparent Proton Diffusion in Rat Piriform Cortex Following Status Epilepticus

Institute: University of Saskatchewan
Keywords: temporal lobe epilepsy, astrocytes, NMR,
Abstract:
Status epilepticus similar to that seen in humans can be induced in rodents by injection of pilocarpine, a cholinergic agonist. The prolonged seizures result in extensive neuronal loss in the piriform cortex with moderate neuronal loss in the hippocampus. Histological staining (cresyl violet, silver degeneration) revealed marked cellular loss in the piriform cortex as early as 6 hr after seizures and appeared to be complete by 7 days post injection. Staining for glial cells with glial fibrillary acidic protein (GFAP) suggested that reactive glial cell activation and/or proliferation accompanied the neuronal cell loss. Thus, overall tissue density appeared to remain constant despite the underlying qualitative changes in the cell population.

Brain tissues were examined using diffusion weighted (b=17776 s/cm²) magnetic resonance imaging (MRI) at 6 hr, 12 hr, 24 hr, 7 and 30 days after seizures. Diffusion maps were generated then manually segmented to isolate the piriform cortex as the region of interest. Mean diffusion values were calculated as unweighted averages over the region of interest.

In control rats, the mean apparent diffusion coefficient (ADC) in the piriform cortex was 103 X 10^-7 cm²/s. At six hours post seizures a reduction to 75 X 10^-7 cm²/s ADC was observed. ADC values were reduced until 7 days after seizure induction when the ADC returned to control values. The reduction in ADC is understandable considering the heterogeneous cell population in the piriform cortex. Histological data indicated that the rapid neuronal cell loss in the piriform cortex was accompanied by an equally rapid glial cell response. Thus, while T2 weighted images suggested an increase in bulk phase tissue water, the mean diffusion path for this water was circumscribed by restrictive (e.g. glial cell) boundaries. At later time points when glial cell proliferation was apparent there was an increase in ADC. We speculate that the ADC changes are due to a pathophysiological hypertrophy of glial cells.

Variable strength (b= 5000 - 122819 s/cm²) diffusion gradients (Q-slice) were applied in an effort to refine our understanding of the spin motion associated with the evolving glial cell population. Our preliminary results suggest that in microglial cells (prior to injury activation) water is less mobile than in healthy neuronal cells. Activated glial cells appear to have ADC values similar to those of healthy neuronal cells. The Q-slice approach permitted selection of spin sub-populations such that it is possible to prospectively distinguish glial and neuronal cell populations based upon their diffusion characteristics.

146
Session Assigned: W&Th P
Title: Multiple component diffusion tensor imaging in excised fixed CNS tissue

Institute: University of Florida, Gainesville, FL USA
Keywords: CNS; multiexponential; diffusion; anisotropy; MRI
Abstract:
Diffusion-weighted imaging has been used extensively as a contrast mechanism to characterize normal and pathological nervous tissues. Most imaging and spectroscopy diffusion studies have been done at low pulsed-gradient diffusion weighting (<1000 s/mm²), and been characterized by a single diffusion rate or single diffusion rate tensor representing an average rate of diffusion across the various diffusion regimes (1, 2). With the availability of very large magnetic field gradients, more recent studies have been performed with pulsed-gradient spin-echo (PGSE) spectroscopy at much higher diffusion weightings. From these studies it was realized that data taken to high diffusion weightings no longer fits a single diffusion rate model. Instead the measured diffusion weighting data more closely resembles a multiexponential curve, allowing the diffusion rate to be separated into different diffusion regimes, i.e. a fast and a slow diffusion regime (3).

In this work, the ability to reach high gradient weightings was employed to explore the application of multiple component diffusion tensor imaging in excised, fixed central nervous system (CNS) tissue. The work involves two in vitro studies: one using PGSE spectroscopy on a sample of excised, fixed human corpus callosum and a second using a PGSE imaging sequence on a sample of excised, fixed rat spinal cord. Only two unique components of diffusion were observed for the limits of the gradients utilized (~ 10000 s/mm²). The spectroscopy data was fit as a full biexponential diffusion matrix, and fit very well to the anisotropic biexponential model. The imaging data was fit pixelwise to the full biexponential diffusion model to get a full tensor of directional information in both the fast and slow diffusing regimes. These results can be interpreted in terms of extracellular (fast) and intracellular (slow) diffusion within the CNS tissue.

1. E.O. Stejkal and J.E. Tanner, J Chem Phys 42, 288 (1965).
2. P.J. Basser, et al. Biophys J 66, 259 (1994).
3. J. Karger, et al. Adv in Mag Reson 12, 1 (1988).

147
Session Assigned: W&Th P
Title: Relaxivity of Paramagnetically Labeled Proteins in Cartilage

Institute: Wright State University, Dayton, OH
Keywords:
Abstract:
The T₁ relaxivity of paramagnetic compounds in agarose gel, healthy and trypsinized calf articular cartilage has been measured. The paramagnetic compounds studied include small molecules, such as copper ion or Gd(DTPA), and protein sized molecules with chelated Gd. The experiments to analyze relaxivity include T₁ measurements after long-term equilibration, and dynamic experiments during the equilibration period. Previous studies have indicated that the relaxivity of smaller paramagnetic compounds is relatively unchanged when in cartilage if one bases relaxivity on the concentration of the paramagnetic in the water space (1). This finding is confirmed in these studies for the smaller paramagnetic compounds.

However, when protein sized compounds chelated to Gd are studied, the relaxivity in cartilage appears to be enhanced relative to relaxivity in saline solution. The enhancement is greater in healthy cartilage than in trypsinized cartilage. A small protein (lysozyme) experienced almost no enhancement in trypsinized cartilage, but significant enhancement of a factor of 3 or more in healthy cartilage. A larger protein (BSA) experienced enhancement of 50% in trypsinized cartilage. A measurement of BSA enhancement in healthy cartilage was not possible due to the very small penetration of BSA into these samples. Preliminary experiments indicate that the enhancement is not a result of the permanent dissociation of Gd from the protein while in the cartilage. Possible physical mechanisms for the relaxivity enhancement will be discussed.

(1) Donahue KM, Burstein D, Manning WJ, Gray ML. Magn. Reson. Med. 32:66-76,1994.

148
Session Assigned: W&Th P
Title: Proton NMR Spectroscopy and Sodium Imaging of Single Cells

Institute: University of Florida
Keywords: Microscopy; Localized Spectroscopy; Microcoils; Neuron
Abstract:
NMR microscopy is now an established subset of NMR imaging, able to image the coarse cellular structure of large cells. To date, the frog ova (&lt;1-mm diameter) and the more difficult to image Aplysia Californica L7 neuron (&lt;300-&micro;m diameter) are the only single animal cells that have been examined, due mainly to their relatively large size [1]. The information from NMR microimaging studies is limited, allowing spatial discrimination of the cell nucleus and cytoplasm and quantitative measurements of T₁, T₂ and the ADC in those compartments as a function of a variety of perturbations. These data may be used to aid in the interpretation of NMR signals from macroscopic assemblies of cell, i.e. tissues. In this work, we extend the utility of MR studies on single cells to include localized ¹H spectroscopy and ²³Na imaging on single Aplysia neurons. These improvements were achieved by increasing SNR with the use of higher field strengths (600MHz) and susceptibility-matched RF microcoils. The solenoidal microcoils were 700 &micro;m in diameter with an optimal length-to-diameter relationship to maximize SNR for sodium imaging and to optimize SNR increases against spectral resolution limitations for proton spectroscopy. The RF coils were tuned singly to ¹H or doubly to ¹H/²³Na.

Localized ¹H spectra were obtained on subcellular regions of single neurons using 220-&micro;m isotropic STEAM voxels. In separate experiments on nine cells, three large resonances corresponding to the osmolytes and metabolites betaine and choline were observed in high concentrations (not observed extracellularly) with only a few minutes averaging. These signals decreased in intensity over time, an observation consistent with dilution due to swelling of the unperfused cells. A spectrum on one cell collected over a two-hour average showed additional metabolites, e.g. acetate and lactate. Peak assignments were confirmed by standard high-resolution spectroscopy on several homogenized cells in D₂O.

Sodium imaging was performed in separate experiments on six cells. Resolutions of 78 x 78 x 210 &micro;m were achieved in 32 minutes. As with the simultaneously collected ¹H images, the cell nucleus displayed a larger signal than the cell cytoplasm with the short TE used. We speculate that this contrast is primarily due to a longer ²³Na T₂ in the nucleus than in the cytoplasm, though quantitative confirmation is required. However, it is known that the intracellular ²³Na T₂ is very short compared to the extracellular sodium T₂, and thus, these observations are consistent with previous ¹H measurements on the same cells.

In conclusion, we have collected the first ¹H spectra showing the osmolyte and metabolite content in single cells. NMR imaging and localized spectroscopy can now be used to image structure and examine the osmolytic and metabolic content down to the cellular level. We also have collected the first sodium images of single cells, opening up the possibility of exciting, non-invasive studies on cell volume regulation.

[1] N.R.Aiken, et al., &quot;A Review of NMR Microimaging Studies of Single Cells,&quot; J. Magn. Reson. Anal. 1, p. 41, 1995.

149
Session Assigned: W&Th P
Title: Quantitation of Tracer Levels of Deuterium for Human Metabolic Studies by ²H NMR at 92.1 MHz (14.1T)

Institute: U.T. Southwestwern Medical Center
Keywords: clinical; glycogen; non-radioactive; pathways
Abstract:
²H NMR at 92.1 MHz is sufficiently sensitive to detect tracer levels of ²H from 50 &micro;moles of metabolite. Since 50 &micro;moles of glucose can be recovered from 20-30 mls of human blood, ²H NMR could potentially be used to quantitate positional ²H-enrichment of plasma glucose following ingestion of safe levels of D₂O. This measurement provides an estimate of the relative contributions of glycogenolysis and gluconeogensis to hepatic glucose output, providing a key metabolic measurement that is difficult to achieve otherwise. Although positional ²H-enrichment of glucose can be obtained by GC-MS, this technique requires a laborious derivitization procedure prior to analysis. Furthermore, the MS analysis cannot be performed on samples containing other tracers such as ¹³C (often used to measure other pathways simultaneously) because ¹³C and ²H mass isotopomers of glucose cannot be fully resolved by MS.

Since positional ²H-enrichment levels can be potentially determined by ²H NMR even in the presence of tracer levels of ¹³C, dual tracer (²H, ¹³C) measurements are feasible from a single blood sample. Preliminary ²H NMR spectra derived from blood of subjects that had ingested D₂O showed acceptable signal-to-noise but showed poor resolution of all glucose signals except for the H1 anomers. Lactate and 3-hydroxybutyrate signals were also present, causing further crowding of the spectrum. While ¹H-²H coherence spectroscopy could theoretically improve dispersion, in practice we were unable to detect ²H-edited signals using conventional ¹H{²H}HMQC spectroscopy. Editing sequences that better accomodate ²H may be more effective but such methods remain to be developed.

Since we are currently limited to ²H detection our approach has been to simplify the ²H NMR spectrum by isolating glucose and converting it to gluconate. We first separated glucose from lactate and 3-hydroxybutyrate by anion-exchange and then quantitatively oxidized glucose to gluconate using glucose oxidase. ¹H NMR spectroscopy of the gluconate product provided an assessment of sample purity as well as chemical shift registration for the ²H NMR signals. The ²H NMR spectrum of gluconate showed fully resolvable H2 and H3 resonances and the H3/H2 enrichment ratio gives an estimate of the fraction of plasma glucose derived from gluconeogenesis. We applied this method to glucose obtained from 30 mls of blood drawn from healthy, overnight-fasted subjects that had previously ingested tracer levels of D₂O and [U-¹³C]propionate and were also receiving tracer levels of [1,6-¹³C₂]glucose intravenously. In this experiment, plasma glucose is enriched in all possible carbon and hydrogen positions and is therefore intractable to either ²H or ¹³C isotopomer analysis by GC-MS. For 4 normal individuals (2 males and 2 females) the H3/H2 ratio was 0.41 +/- 0.06 indicating that gluconeogensis supplied 41 +/- 6 percent of total glucose output after 14-15 hours of fasting. These data are in good agreement with recent GC-MS measurements from subjects that had ingested similar quantities of D₂O and had fasted for a comparable time.

150
Session Assigned: W&Th P
Title: Changes in Energy Metabolism of Prostate Cancer Cell Lines as Determined by NMR Spectroscopy

Institute: University of Florida, Gainesville, Florida, USA
Keywords:
Abstract:
Lack of a suitable animal model and the difficulty of obtaining homogeneous patient samples have hampered research advances in prostate cancer, a disease that will be diagnosed in over 184,500 men this year. Therefore, new and innovative methods of studying these tumors are urgently needed. We have developed a technique for using NMR to study human prostate cancer cell lines cultured in a defined bioreactor environment. Details of this system, including oxygen perfusion and nutrient conditions will be described. The numerous advantages of this technique over commonly used culture methods include: 3-dimensional growth on a solid support or basement membrane matrix to mimic the in situ environment; ability to study the same population of cells over time, in serial experiments; low cost of materials; repeatability; and the ability to monitor changes in multiple metabolites simultaneously.

We have shown that two human prostate cancer cell lines with very different phenotypes can be successfully cultured in a magnetic field and that NMR can be used to monitor metabolic changes with, for example, ATP abundance. Proton and carbon spectra revealed differences in oxygen and nutrient status. Zinc has been shown to inhibit the Krebs cycle enzyme, m-aconitase, in prostate cells in culture, thereby limiting citrate oxidation and energy production. Our studies confirm that administration of zinc chloride at 1-6 @micro;g/ml media dramatically reduces ATP peaks. Our unique bioreactor system allows us to collect detailed information on the regulation of prostate cancer cell energy metabolism and to pursue a rational approach to new diagnostic and treatment methods.