Proton Magic Angle Spinning NMR Research Articles

Impact of donor health on corneal biochemistry – an unexpected caveat from a pilot study

To test the possibility that some chronic systemic maladies not directly related to the function of the eye may significantly and permanently disturb corneal metabolism. Contents of selected low molecular weight metabolites were compared among corneas collected from donors who died suddenly of an accident or non-poisoning suicide, or met a sudden non-accidental death from unidentified causes, or died of a chronic cardiovascular disease or of idiopathic liver cirrhosis (N=4 for each group). Corneal buttons were halved; one half was snap-frozen and stored at -80°C, and the other half was stored at +4°C in Eusol-C for 8 days and then was snap-frozen and stored at -80°C until analyzed. Metabolite contents were assessed using high-resolution magic angle spinning proton NMR spectroscopy. Significant between-group differences in corneal biochemical profiles were identified. Most of them were reduced or nullified by the Eusol-C storage, suggesting their link to differences in in vivo corneal environment. The corneas from donors with liver cirrhosis or cardiovascular diseases differed considerably from the remaining ones, both before and after the Eusol-C storage. Various chronic systemic diseases that are not directly related to the function of the eye markedly affect corneal biochemistry. Some of the alterations are likely related to a permanent aberration in corneal metabolism. A study is warranted in larger donor groups on the effect of idiopathic liver cirrhosis and cardiovascular diseases on corneal metabolism and/or a retrospective analysis of the long-term outcome of keratoplasty and other grafting procedures employing materials from these donor groups.

Temperature-Dependent Phase Behavior of the Synaptosomal Membranes from Mammalian and Marine Invertebrate Synaptosomes

Given reports of raft domains in rodent brain synaptosomes, we probed for their conservation in a poikilotherm, the Woods Hole squid Loligo. Because rafts are described as liquid-ordered phase-separated domains, we expected phase transition temperatures above body temperature if raft function is important to neuronal activity. We tested this hypothesis by comparing synaptosomes, intact nerve endings, from animals that live at two very different temperatures: mouse (body temperature-37°C) and squid (body temperature-20°C). We measured the temperature-dependence of the lipid phase of intact synaptosomes in the absence of exogenous probes by using line-width and spinning sideband intensities of lipid hydrocarbon chain resonances, using proton magic angle spinning NMR spectra as a function of temperature between 0 and 40°C. We also determined the phase behavior of total synaptosomal lipids by repeating the NMR protocol on extracted lipids. To our surprise, there was no detectable signal for liquid-ordered lipid phases at the body temperature of either species. However, there was a difference in the temperature of the onset of order as synaptosomes were cooled below body temperature: the phase state of mouse synaptosomal membranes changes drastically below 24°C, whereas this change occurs below 8°C for the squid synaptosomal membranes. We then measured the composition of synaptosomes in terms of total lipid heads and tails, and the main difference arises from high concentrations of omega-3 poly-unsaturated fatty acids and cholesterol in the squid. Fluorescence microscopy images of squid synaptosomes stained with lipid dyes confirmed the formation of domains below, but not above the phase transition temperatures obtained from NMR measurements. Thus although the membranes of synaptosomes contain lipids that can phase-separate, these lipids remain in the liquid-disordered state at the usual physiological temperatures for squid and mouse.

A highly ordered mesostructured material containing regularly distributed phenols: preparation and characterization at a molecular level through ultra-fast magic angle spinning proton NMR spectroscopy

Highly ordered organic-inorganic mesostructured material containing regularly distributed phenols is synthesized by combining a direct synthesis of the functional material and a protection-deprotection strategy and characterized at a molecular level through ultra-fast magic angle spinning proton NMR spectroscopy.

Vitamin A Deficiency in Rats Induces Anatomic and Metabolic Changes Comparable with Those of Neurodegenerative Disorders

Anatomic and metabolic changes in central nervous system induced by 14 wk of vitamin A deprivation (VAD) were monitored and quantified in rats. In vivo brain magnetic resonance imaging (4.7T) was performed at 5, 7, 9, 11, and 14 wk of each diet after weaning in the following: 1) VAD group; 2) control pair-fed group; and 3) control group that consumed the diet ad libitum (1.15 μg retinol/g diet). After 14 wk, high-resolution magic angle spinning proton NMR spectroscopy (11.7T) was performed on small samples of cortex, hippocampus, and striatum. Serum retinol concentrations remained stable and cerebral volume (CV) increased as a linear function of body weight in the ad libitum group (R2 = 0.78; P = 0.047) and pair-fed controls (R2 = 0.78; P = 0.046). In VAD rats, retinol decreased from the onset of deprivation (2.2 ± 0.14 μmol/L) to reach 0.3 ± 0.13 μmol/L at wk 5, followed by a stopping of body weight gain from wk 7. In VAD rats, the CV decreased from wk 5 and reached a value 11% lower than that of the control group (P < 0.001) at wk 14 and was correlated with retinol status (R2 = 0.99; P = 0.002). The VAD hippocampal volume decreased beginning at wk 9 and was 22% lower than that of the control group at wk 14 (P < 0.001). Compared with the control, VAD led to lower N acetyl aspartate:creatine+phosphocreatine (Cr) in cortex (−36%), striatum (−22%), and hippocampus (−19%) and higher myoinositol:Cr in cortex (+127%) and striatum (+150%). VAD induced anatomic and metabolic changes comparable to those associated with neurodegenerative disorders. By wk 7 of deprivation, the slowing in cerebral growth that correlated with the retinol level could be considered as a predictive marker of brain disorders, confirmed by metabolic data from VAD rats after 14 wk.

Progressive ordering with decreasing temperature of the phospholipids of influenza virus

Using linewidth and spinning sideband intensities of lipid hydrocarbon chain resonances in proton magic angle spinning NMR spectra, we detected the temperature-dependent phase state of naturally occurring lipids of intact influenza virus without exogenous probes. Increasingly, below 41 degrees C ordered and disordered lipid domains coexisted for the viral envelope and extracts thereof. At 22 degrees C much lipid was in a gel phase, the fraction of which reversibly increased with cholesterol depletion. Diffusion measurements and fluorescence microscopy independently confirmed the existence of gel-phase domains. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. Above the physiological temperatures of influenza infection, the physical properties of viral envelope lipids, regardless of protein content, were indistinguishable from those of the disordered fraction. Viral fusion appears to be uncorrelated to ordered lipid content. Lipid ordering may contribute to viral stability at lower temperatures, which has recently been found to be critical for airborne transmission.

Characterization of the Liquid-Ordered State by Proton MAS NMR

We investigated if magic angle spinning (MAS) 1H NMR can be used as a tool for detection of liquid-ordered domains (rafts) in membranes. In experiments with the lipids SOPC, DOPC, DPPC, and cholesterol we demonstrated that 1H MAS NMR spectra of liquid-ordered domains (l o) are distinctly different from liquid-disordered (l d) and solid-ordered (s o) membrane regions. At a MAS frequency of 10 kHz the methylene proton resonance of hydrocarbon chains in the l d phase has a linewidth of ∼50 Hz. The corresponding linewidth is ∼1 kHz for the l o phase and several kHz for the s o phase. According to results of 1H NMR dipolar echo spectroscopy, the broadening of MAS resonances in the l o phase results from an increase in effective strength of intramolecular proton dipolar interactions between adjacent methylene groups, most likely because of a lower probability of gauche/ trans isomerization in l o. In spectra recorded as a function of temperature, the onset of l o domain (raft) formation is seen as a sudden onset of line broadening. Formation of small domains yielded homogenously broadened resonance lines, whereas large l o domains (diameter >0.3 μm) in an l d environment resulted in superposition of the narrow resonances of the l d phase and the much broader resonances of l o. 1H MAS NMR may be applied to detection of rafts in cell membranes.

High-Resolution MAS 1H NMR Spectroscopic Analysis of Rabbit Cornea After Treatment with Dexamethasone and Exposure to UV-B Radiation

Metabolic changes in rabbit cornea after combined long-term steroid treatment and UV-B exposure were investigated. Corneas were exposed to UV-B radiation (2.05 J/cm2) after 36 days topical pretreatment with either 0.1% dexamethasone or saline. Twenty-four hours after UV-B exposure, corneas were excised and aqueous humour aspirated. Intact corneal tissues were analyzed by magic angle spinning proton NMR spectroscopy and pattern recognition methods. UV-B decreased corneal ascorbate (63% reduction), taurine (62%), and choline (63%), whereas glucose was elevated. Dexamethasone pretreatment further depleted corneal taurine and ascorbate, decreased aqueous ascorbate (85%), and accumulated glucose in cornea and aqueous humour. The results reflect antioxidative mechanisms and osmoregulation.

Proton magic-angle spinning–NMR investigation of surfactant aqueous suspensions

In this Note we present the advantages of 1H magic-angle spinning nuclear magnetic resonance (MAS-NMR) for the investigation of surfactant suspensions via transverse relaxation rate ( R 2) measurements. 1H-relaxation rates can be determined by the classical CPMG method from high-resolution spectra obtained either under conditions of liquid-state NMR for monomers and small spherical micelles or by using MAS-NMR for larger aggregates. For a mixture of alkyl dioxyethylene sulfate and alkylbetaine (80:20, w/w), up to a percentage of surfactant in water of 20%, we found that R 2 increased, in accordance with an increased micellar size and very likely the formation of an H I phase. However, above 25%, R 2 decreased. This result suggests a change from a hexagonal to a lamellar phase that would be difficult to observe by proton NMR without magic-angle spinning because the lines would be very broad, or by light scattering because of sample opacity. This NMR approach seems to have been overlooked by the community of surfactant physical chemists. It can be complementary to other analytical techniques and presents the advantage of not requiring isotopic labeling.

High-resolution magic angle spinning proton NMR analysis of human prostate tissue with slow spinning rates.

The development of high-resolution magic angle spinning (HR-MAS) NMR spectroscopy for intact tissue analysis and the correlations between the measured tissue metabolites and disease pathologies have inspired investigations of slow-spinning methodologies to maximize the protection of tissue pathology structures from HR-MAS centrifuging damage. Spinning sidebands produced by slow-rate spinning must be suppressed to prevent their complicating the spectral region of metabolites. Twenty-two human prostatectomy samples were analyzed on a 14.1T spectrometer, with HR-MAS spinning rates of 600 Hz, 700 Hz, and 3.0 kHz, a repetition time of 5 sec, and employing various rotor-synchronized suppression methods, including DANTE, WATERGATE, TOSS, and PASS pulse sequences. Among them, DANTE, as the simplest scheme, has shown the most potential in suppression of tissue water signals and spinning sidebands, as well as in quantifying metabolic concentrations.

Proton magic angle spinning NMR reveals new features in photodynamically treated bacteria.

The bacterium Propionibacterium acnes is light-sensitive due to porphyrin-induced photosensitization. The light sensitivity increases with incubation of 5-aminolevulinic acid, ALA. For the first time, 1H magic angle spinning NMR spectroscopy is used to describe the photoinduced changes in the bacterium after ALA incubation. Successful photosensitization was performed with light-emitting diodes in the blue and red regions (430 and 654 nm, respectively). The irradiation setup, suitable for irradiation of bacterium suspensions in petri dishes is described. For NMR studies blue light diodes with about 90 micromol/m2s were chosen. After blue light irradiation, the endogenous glycine betaine, proline, glutamate and choline levels in P. acnes decreased with increasing irradiation time. For sublethal light doses (50% survival fraction), the endogenous glycine betaine level decreased 80% on average. The corresponding percentages for proline, choline and glutamate were about 40, 25 and 10, respectively. It is hypothesized that the irradiation, inducing porphyrin photosensitization amplified by ALA incubation, leads to elimination of the osmolyte glycine betaine and possibly also proline by so-called regulatory volume decrease (RVD) mechanisms. These mechanisms are known to be active in several prokaryotic and eukaryotic cells when exposed to hypotonic stress. They are also known to be present in several eukaryotic cells during photodynamic therapy (PDT) exposure leading to hypotonoc stress. The findings contribute to the knowledge of the inactivation mechanisms of P. acnes in photosensitization, and could therefore be of interest in the efforts to use PDT as treatment of the acne disease.

A bimodal Floquet analysis of phase modulated Lee–Goldburg high resolution proton magic angle spinning NMR experiments

A description of phase modulated Lee–Goldburg experiments PMLG- n, with 2 n pulses per rf cycle, on coupled proton systems under magic angle spinning is presented using bimodal Floquet theory. Level crossing conditions between Floquet states are derived and perturbation theory is used to account for spectral changes as a function of the spinning frequency and the number of pulses per PMLG- n sequence. High resolution solid-state proton spectra are shown that are obtained for n equal to 9, 5 and 3. Line broadening and the rf rotor frequency line positions are correlated to level anticrossings.

Fast magic-angle spinning proton NMR studies of polymers at surfaces and interfaces

Solid-state proton NMR with fast magic-angle sample spinning has been used to study the structure and dynamics of polymers and the water interface in porous glass composites. The composites were prepared by photopolymerization of poly(ethyl acrylate) and other acrylate formulations in a high surface-area rigid glass matrix with 40-Å interconnected pores. High resolution solid-state proton spectra were obtained for polymer films and composites with 15 kHz magic-angle sample spinning at temperatures above the polymer glass transition temperature. The solid-state proton spectra can be detected with high sensitivity and used to determine the composition of polymer and water filling the pores. These results and spin diffusion studies using 1H– 29Si 2D heteronuclear correlation and wideline separation NMR show that the polymer fills the central 30 Å of the pore, and that the remaining volume is filled with surface hydroxyl groups and water.

Membrane hydration and structure on a subnanometer scale as seen by high resolution solid state nuclear magnetic resonance: POPC and POPC/C12EO4 model membranes

The position on a subnanometer scale and the dynamics of structurally important water in model membranes was determined using a combination of proton magic-angle spinning NMR (MAS) with two-dimensional NOESY NMR techniques. Here, we report studies on phosphocholine lipid bilayers that were then modified by the addition of a nonionic surfactant that is shown to dehydrate the lipid. These studies are supplemented by 13C magic-angle spinning NMR investigations to get information on the dynamics of segmental motions of the membrane molecules. It can be shown that the hydrophilic chain of the surfactant is positioned at least partially within the hydrophobic core of the lipid bilayer. With the above NMR approach, we are able to establish molecular contacts between water and the lipid headgroup as well as with certain groups of the hydrocarbon chains and the glycerol backbone. This is possible because high resolution proton and 13C-NMR spectra of multilamellar bilayer membranes are obtained using MAS. A phase-sensitive NOESY must also be applied to distinguish positive and negative cross-peaks in the two-dimensional plot. These studies have high potential to investigate membrane proteins hydration and structural organization in a natural lipid bilayer surrounding.

Detection of water in the internal aqueous phase of W/O/W multiple emulsions using magic angle spinning proton NMR

Increased resolution in proton ( 1H) magic angle spinning (MAS) NMR spectra of emulsions is demonstrated. Relative volumes of the internal and external water phases of water-in-oil-in-water (W/O/W) type multiple emulsions were estimated by integration of water resonances separated by use of a shift reagent, while broadening due to magnetic susceptibility mismatch among phases was removed by magic angle sample spinning. Strategies are presented for reducing effects on sample integrity due to spinning and due to addition of reagents. Internal water may also be uniquely detected in the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo spectrum of a sample containing added MnCl 2 as a relaxation reagent.

Investigation of Natural Mordenites by Magic Angle Proton NMR

Abstract Magic angle spinning proton NMR techniques were used to study natural and synthetic mordenites, and faujasite. The proton NMR line width of water molecules (whose chemical shift is similar to free water) was correlated with frame structure. In the case of heat-treated natural mordenite (250°C, 18 hours) we observed two unresolved peaks whose chemical shift are different from free water. These peaks were assigned to protons around A1 and Si sites in natural mordenites.

Proton magic angle spinning nuclear magnetic resonance and temperature programmed desorption studies of ammonia on the acidity of the framework hydroxyl groups in the zeolite H-ZSM-5 and in H-boralite

The acidity of the framework hydroxyl groups of H-ZSM-5 and H-boralite is investigated by proton magic angle spinning NMR and temp. programmed desorption (TPD) of NH3. The acidity decreases from Si-OH-Al, the Broensted acidic site in H-ZSM-5, to Si-OH-B, the Broensted acidic site in H-boralite, to Si-OH, the terminating OH group of the zeolite lattice (silanol group). This sequence agrees with the NH3-TPD results.