Robert D. O'Connor

Intramyocardial triglyceride quantification by magnetic resonance spectroscopy: In vivo and ex vivo correlation in human subjects

Accumulation of triglycerides (TG) in heart tissue has been associated with changes in left ventricular function. Proton magnetic resonance spectroscopy is currently the only noninvasive in vivo method to measure myocardial triglycerides content. The primary aim of this study was to determine if these in vivo measurements are specific to myocardial triglycerides in human subjects. Thus, in vivo proton magnetic resonance spectroscopy measurements were conducted on orthotopic heart transplant patients (n = 8) immediately before they underwent routine biopsies and ex vivo measurements were made on the endomyocardial biopsy samples. The correlation coefficient between the two measurements was 0.97, with P < 0.005, demonstrating for the first time the specificity of the in vivo measurement in human heart. From accompanying reliability experiments, the standardized typical error for the in vivo proton magnetic resonance spectroscopy method was estimated to be 7.0%, with a 95% confidence interval from 5.5 to 9.4%. These results suggest that proton magnetic resonance spectroscopy provides a specific and reliable measurement of myocardial triglycerides content and is suitable for routine studies. Magn Reson Med, 2011.

Plant Cell-Wall Cross-Links by REDOR NMR Spectroscopy

We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[ε-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase.

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase–S3P–Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native 31P in S3P and Glp, biosynthetically 13C-labeled S3P, specifically 13C and 15N labeled Glp, and a variety of protein-15N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.

Quantification of global myocardial oxygenation in humans: initial experience

PurposeTo assess the feasibility of our newly developed cardiovascular magnetic resonance (CMR) methods to quantify global and/or regional myocardial oxygen consumption rate (MVO2) at rest and during pharmacologically-induced vasodilation in normal volunteers.MethodsA breath-hold T2 quantification method is developed to calculate oxygen extraction fraction (OEF) and MVO2 rate at rest and/or during hyperemia, using a two-compartment model. A previously reported T2 quantification method using turbo-spin-echo sequence was also applied for comparison. CMR scans were performed in 6 normal volunteers. Each imaging session consisted of imaging at rest and during adenosine-induced vasodilation. The new T2 quantification method was applied to calculate T2 in the coronary sinus (CS), as well as in myocardial tissue. Resting CS OEF, representing resting global myocardial OEF, and myocardial OEF during adenosine vasodilation were then calculated by the model. Myocardial blood flow (MBF) was also obtained to calculate MVO2, by using a first-pass perfusion imaging approach.ResultsThe T2 quantification method yielded a hyperemic OEF of 0.37 ± 0.05 and a hyperemic MVO2 of 9.2 ± 2.4 μmol/g/min. The corresponding resting values were 0.73 ± 0.05 and 5.2 ± 1.7 μmol/g/min respectively, which agreed well with published literature values. The MVO2 rose proportionally with rate-pressure product from the rest condition. The T2 sensitivity is approximately 95% higher with the new T2 method than turbo-spin-echo method.ConclusionThe CMR oxygenation method demonstrates the potential for non-invasive estimation of myocardial oxygenation, and should be explored in patients with altered myocardial oxygenation.

Isotopically Labelled and Unlabelled β‐Peptides with Geminal Dimethyl Substitution in 2‐Position of Each Residue: Synthesis and NMR Investigation in Solution and in the Solid State

The preparation of (S)-β2,2,3-amino acids with two Me groups in the α-position and the side chains of Ala, Val, and Leu in the β-position (double methylation of Boc-β-HAla-OMe, Boc-β-Val-OMe, and Boc-β-Leu-OMe, Scheme 2) is described. These β-amino acids and unlabelled as well as specifically 13C- and 15N-labelled 2,2-dimethyl-3-amino acid (β2,2-HAib) derivatives have been coupled in solution (Schemes 1, 3 and 4) to give protected (N-Boc, C-OMe), partially protected (N-Boc/C-OH, N-H/C-OMe), and unprotected β2,2- and β2,2,3-hexapeptides, and β2,2- and β2,2,3-heptapeptides 1–7. NMR Analyses in solution (Tables 1 and 2, and Figs. 2–4) and in the solid state (2D-MAS NMR measurements of the fully labelled Boc-(β2,2-HAib)6-OMe ([13C30, 15N6]-1e; Fig. 5), and TEDOR/REDOR NMR investigations of mixtures (Fig. 6) of the unlabelled Ac-(β2,2-HAib)7-OMe (4) and of a labelled derivative ([13C4,15N2]-5; Figs. 7–11, and 19), a molecular-modeling study (Figs. 13–15), and a search in the Cambridge Crystallographic Data Base (Fig. 16) allow the following conclusions: i) there is no evidence for folding (helix or turn) or for aggregation to sheets of the geminally dimethyl substituted peptide chains in solution; ii) there are distinct conformational preferences of the individual β2,2- and β2,2,3-amino acid residues: close to eclipsing around the C(O)C(Me2(CHR)) bond (τ1,2), almost perfect staggering around the C(2)C(3) ethane bond (τ2,3), and antiperiplanar arrangement of H(C3) and H(N) (τ3,N; Fig. 12) in the solid state; iii) the β2,2-peptides may be part of a turn structure with a ten-membered H-bonded ring; iv) the main structure present in the solid state of F3CCO(β2,2-HAib)7-OMe is a nonfolded chain (>30 A between the termini and >20 A between the N-terminus and the CH2 group of residue 5) with all CO bonds in a parallel alignment (±10°). With these structural parameters, a simple modelling was performed producing three (maybe four) possible chain geometries: one fully extended, two with parallel peptide planes (with zick-zack and crankshaft-type arrangement of the peptide bonds), and (possibly) a fourth with meander-like winding (D–G in Figs. 17 and 18).

Crystallization of poly(ε-caprolactone) under nanoparticle confinement

Solid-state 13C-NMR spectroscopy has been used to characterize the conformation of the hydrophobic poly(e-caprolactone) core of a nanoparticle having a cross-linked hydrophilic poly(acrylic acid)/polyacrylamide shell. The amphiphilic nanoparticles were synthesized from the diblock copolymer, poly(e-caprolactone)121-b-poly(acrylic acid)165 by self-assembly into polymer micelles, followed by cross-linking via condensation reactions between the carboxylic acid groups of the hydrophilic shell and the amine groups of 2,2′-(ethylenedioxy)bis(ethylamine). NMR Experiments performed at −30° on nanoparticles rapidly quenched from 60° show that the core is largely noncrystalline and locally disordered. Heating to 25° results in some crystallization, although far less than that observed for bulk poly(e-caprolactone) homopolymer. Storage at −30° results in further crystallization and conversion of most rubbery, mobile regions into more rigid, locally ordered amorphous domains. The absence of dipolar coupling between natural-abundance 13C in the poly(e-caprolactone) core of the nanoparticle, and 15N labels dispersed throughout the cross-linked shell show that the interface between core and shell is sharp. The dipolar coupling measurements were accomplished by 13C{15N} rotational-echo double resonance.

Chain packing in polycarbonate glasses

Chain packing in homogeneous blends of carbonate (13)C-labeled bisphenol A polycarbonate with either (i) CF(3)-labeled bisphenol A polycarbonate or (ii) ring-F-labeled bisphenol A polycarbonate has been characterized using (13)C{(19)F} rotational-echo double-resonance (REDOR) nuclear magnetic resonance. In both blends, the (13)C observed spin was at high concentration, and the (19)F dephasing or probe spin was at low concentration. In this situation, an analysis in terms of a distribution of isolated heteronuclear pairs of spins is valid. Nearest-neighbor separation of (13)C and (19)F labels was determined by accurately mapping the initial dipolar evolution using a shifted-pulse version of REDOR. Based on the results of this experiment, the average distance from a ring-fluorine to the nearest (13)C=O is more than 1.2 A greater than the corresponding CF(3)-(13)C=O distance. Next-nearest and more-distant-neighbor separations of labels were measured in a 416-rotor-cycle constant-time version of REDOR for both blends. Statistically significant local order was established for the nearest-neighbor labels in the methyl-labeled blend. These interchain packing results are in qualitative agreement with predictions based on coarse-grained simulations of a specially adapted model for bisphenol A polycarbonate. The model itself has been previously used to determine static and dynamic properties of polycarbonate with results in good agreement with those from rheological and neutron scattering experiments.

T2 preparation method for measuring hyperemic myocardial O2 consumption: in vivo validation by positron emission tomography

To validate a new T2‐prepared method for the quantification of regional myocardial O2 consumption during pharmacologic stress with positron emission tomography (PET).

Bariatric Surgery-Induced Cardiac and Lipidomic Changes in Obesity-Related Heart Failure with Preserved Ejection Fraction: Gastric Bypass: HFpEF and Lipidomic Effects

To determine the effects of gastric bypass on myocardial lipid deposition and function and the plasma lipidome in women with obesity and heart failure with preserved ejection fraction (HFpEF).

A versatile custom cryostat for dynamic nuclear polarization supports multiple cryogenic magic angle spinning transmission line probes

Dynamic nuclear polarization (DNP) with cryogenic magic angle spinning (MAS) provides significant improvements in NMR sensitivity, yet presents unique technical challenges. Here we describe a custom cryostat and suite of NMR probes capable of manipulating nuclear spins with multi-resonant radiofrequency circuits, cryogenic spinning below 6 K, sample exchange, and microwave coupling for DNP. The corrugated waveguide and six transfer lines needed for DNP and cryogenic spinning functionality are coupled to the probe from the top of the magnet. Transfer lines are vacuum-jacketed and provide bearing and drive gas, variable temperature fluid, two exhaust pathways, and a sample ejection port. The cryostat thermally isolates the magnet bore, thereby protecting the magnet and increasing cryogen efficiency. This novel design supports cryogenic MAS-DNP performance over an array of probes without altering DNP functionality. We present three MAS probes (two supporting 3.2 mm rotors and one supporting 9.5 mm rotors) interfacing with the single cryostat. Mechanical details, transmission line radio frequency design, and performance of the cryostat and three probes are described.