Gheorghe D. Mateescu

Characterization of H3PO4-equilibrated Nafion® 117 membranes using 1H and 31P NMR spectroscopy

Abstract 1H and 31P NMR spectroscopic measurements were used to examine the behavior of H3PO4 sorbed in Nafion® 117 membranes. The 31P NMR signal for phosphoric acid sorbed in Nafion was found to have the same chemical shift as that of 100% phosphoric acid in bulk liquid phase. This, however, was broadened compared to that in the bulk acid (FWHM = 121 Hz versus 8 Hz for bulk 100% H3PO4), suggesting lower mobility, possibly due to hydrogen-bonding-type interactions. In the 1H-NMR spectrum for H3PO4-equilibrated Nafion, the peak was shifted downfield compared to a relatively dry membrane (~ 2 H2O per −SO3H), consistent with a higher mole fraction of acidic protons. The presence of a single peak also indicates an exchange of protons between the sulfonic acid groups, phosphoric acid and residual water. The latter may in fact mediate the exchange between −SO3H and H3PO4. The peak was relatively broad (FWHM = 240 Hz) compared to those in “dry” (FWHM = 100 Hz) and water-equilibrated Nafion (FWHM = 80 Hz), indicating lower mobility and/or the effect of finite proton exchange rates.

Functional oxygen-17 magnetic resonance imaging and localized spectroscopy.

Functional oxygen-17 magnetic resonance (MR) imaging and localized spectroscopy is defined as the ensemble of MR measurements aiming at in vivo, noninvasive characterization of oxygen transport and utilization. After a brief description of the present status of in vivo 17O-MR, preliminary results are reported on oxygen delivery and consumption in cell suspensions of Saccharomyces cerevisiae. It is shown that parallel 31P-MR at high magnetic fields has an important corroborative value.

Assessing mitochondrial respiration in isolated hearts using 17O MRS

The application of 17O MRI and MRS for the evaluation of cardiac mitochondrial function has been limited because of the challenge of detecting metabolic H217O in the vast background of naturally abundant H217O. In this study, we have developed a direct 17O MRS approach to examine the feasibility and sensitivity of detecting metabolically produced H217O in isolated rat hearts perfused with 17O2‐enriched Krebs–Henseleit buffer containing normal (1.5 mm) and high (2.5 mm) calcium (Ca2+) concentrations to induce high workload. Consistent with increased workload at high Ca2+ concentration, the measured myocardial oxygen consumption rate (MVO2) increased by 82%. Dynamic 17O MRS showed an accelerated increase in the H217O signal at high Ca2+ concentration, suggesting increased mitochondrial production of H217O in concordance with the increased workload. A compartment model was developed to describe the kinetics of H217O production as a function of MVO2. The myocardial 17O2 consumption rate (MV17O2) was determined by least‐squares fitting of the model to the NMR‐measured H217O concentration. Consistent with the measured MVO2, the model‐determined MV17O2 showed a 92% increase at high Ca2+ concentration. The increase in metabolic activity at high workload allowed the balance between ATP production and utilization to be maintained, leading to a similar phosphocreatine to ATP ratio. These results demonstrate that dynamic 17O MRS can provide a valuable tool for the detection of an altered metabolic rate associated with a change in cardiac workload. Copyright

[94]13C NMR spectroscopy of the chromophore of rhodopsin

Publisher Summary This chapter discusses the C nuclear magnetic resonance (nmr) spectroscopy of the chromophore of rhodopsin. C nmr spectroscopy has provided valuable information on the structure and function of water-soluble proteins. Rhodopsin is one membrane protein for which C nmr has proved feasible, as these drawbacks may be remedied by the introduction of a C enriched retinylidene chromophore and detergent solubilization. Solubilization by octyl glucoside enables high concentrations of rhodopsin to be obtained, as the lipids can be removed by chromatography and the detergent is not concentrated by ultrafiltration because of its high carboxymethyl cellulose (CMC). Octyl glucoside does not contribute resonances in the alkene region of the C nmr spectrum—that is, approximately 120–130 ppm. The rhodopsin/detergent micelle size appears to be relatively small, which leads to a short rotational correlation time and therefore narrow linewidths. The low ratio obtained for enriched rhodopsin indicates that free opsin is removed from regenerated rhodopsin during affinity chromatography, thereby maximizing the ratio.

In vivo assessment of oxygen consumption via Deuterium Magnetic Resonance.

We present a novel approach to simultaneously measure, in vivo, noninvasively, glucose and oxygen consumption via Deuterium Magnetic Resonance (DMR). Mice are administered deuteriated glucose by intravenous injection. The rate of formation of nascent (deuteriated) mitochondrial water is then measured via DMR. The rate of glucose metabolism and oxygen utilization is assessed by tracking their separate peaks in DMR spectra during dynamic scanning. Further studies will aim to validate these results by comparison with in vivo (17)O-MRI (mitochondrial function), (13)C-MRI and (19)FDG-PET (glucose metabolism) and ex vivo 1H- and 2H-MR, as well as mass spectrometry.

Quantitative assessment of brain glucose metabolic rates using in vivo deuterium magnetic resonance spectroscopy

Quantitative assessment of cerebral glucose consumption rate (CMRglc) and tricarboxylic acid cycle flux (VTCA) is crucial for understanding neuroenergetics under physiopathological conditions. In this study, we report a novel in vivo Deuterium (2H) MRS (DMRS) approach for simultaneously measuring and quantifying CMRglc and VTCA in rat brains at 16.4 Tesla. Following a brief infusion of deuterated glucose, dynamic changes of isotope-labeled glucose, glutamate/glutamine (Glx) and water contents in the brain can be robustly monitored from their well-resolved 2H resonances. Dynamic DMRS glucose and Glx data were employed to determine CMRglc and VTCA concurrently. To test the sensitivity of this method in response to altered glucose metabolism, two brain conditions with different anesthetics were investigated. Increased CMRglc (0.46 vs. 0.28 µmol/g/min) and VTCA (0.96 vs. 0.6 µmol/g/min) were found in rats under morphine as compared to deeper anesthesia using 2% isoflurane. This study demonstrates the feasibility and new utility of the in vivo DMRS approach to assess cerebral glucose metabolic rates at high/ultrahigh field. It provides an alternative MRS tool for in vivo study of metabolic coupling relationship between aerobic and anaerobic glucose metabolisms in brain under physiopathological states.

In Vivo 17O Magnetic Resonance Spectroscopy

After the first reports on the feasibility of 17O magnetic resonance imaging (MRI) and localized spectroscopy (MRS)1–3 significant progress has been recorded in applications to vertebrates and invertebrates. Water diffusion in gels and time release preparations have also been reported.4 Of particular importance are the experiments leading to the determination, in vivo, of the rate of oxygen consumption.5–9 These investigations reached a new dimension with the introduction of interleave 17O/31P measurements which are yielding valuable information on the degree of uncoupling of the oxidative phosphorylation (OXPHOS) by various physical and chemical agents.10–11 The significance of such information derives from the fact that degenerative diseases (Alzheimer, Parkinson, ischemic heart, late onset diabetes, etc) are associated to OXPHOS perturbations induced by genetic mitochondrial and nuclear DNA mutations.12 Given the increasing interest in the elucidation of the protective mechanism of hypothermia, as compared to that of barbiturates, in traumatic or ischemic brain damage13–16 we initiated a feasibility study for the determination of the Q 10factor.

Solution and Solid State C-13 and N-15 NMR Studies of Visual Pigments and Related Systems: Rhodopsin and Bacteriorhodopsin1a

This paper constitutes a chronological review of C-13 and N-15 solution and solid state NMR investigations of bovine rhodopsin and the purple membrane of Halobacterium halobium. Model retinylideneimines and their protonated species yielded rather informative variation in C-13 chemical shifts, as a function of structure and conformation, Retinals, specifically enriched with C-13 at key atom positions, were incorporated in rhodopsin and bacteriorhodopsin during the bleaching-regeneration process. The labeled pigments exhibited C-13 signals which were interpreted in terms of the state of protonation and conformation of the Schiff base linkage between the chromophore and opsin (15-anti in rhodopsin and light adapted bacteriorhodopsin) and chromophore-protein interactions. Peaks arising from both all-trans and 13-cis-retinylidene moieties were observed in dark adapted bacteriorhodopsin. Upon protonation, the Schiff base nitrogen of model compounds displayed dramatic (up to 146 ppm) high field N-15 shifts (an order of magnitude larger than C-13). Hydrogen bonding also led to important (up to 50 ppm) nitrogen shielding. H. halobium grown on a medium containing ɛ15N-lysine incorporated, with high yield, the labeled amino acid in all of the seven lysine sites of bacteriorhodopsin. Of these, one forms the shift could be observed only in lyophilized preparations. It appears that the degree of protonation of the Schiff base that the degree of protonation of the Schiff base linkage depends on the degree of hydation of the linkage depends on the degree of hydration of the specimen. Double labeling (13C=15N) is expected to provide valuable information concerning the structure and conformation of this moiety. The six lysine residues not involved in retinal binding yielded, in a micellar (octyl-β-glucoside) solution, N-15 signals of various linewidths, spread over a narrow (2–4 ppm) range. This could be interpreted in terms of lysine residue interactions within the protein. Preliminary results indicate the feasibility of in vivo measurements which avoid alterations inherent to extraction, purification, and/or lyophilization procedures.