Deuterium Signal Research Articles

10Be evidence for the Matuyama–Brunhes geomagnetic reversal in the EPICA Dome C ice core

An ice core drilled at Dome C, Antarctica, is the oldest ice core so far retrieved. On the basis of ice flow modelling and a comparison between the deuterium signal in the ice with climate records from marine sediment cores, the ice at a depth of 3,190 m in the Dome C core is believed to have been deposited around 800,000 years ago, offering a rare opportunity to study climatic and environmental conditions over this time period. However, an independent determination of this age is important because the deuterium profile below a depth of 3,190 m depth does not show the expected correlation with the marine record. Here we present evidence for enhanced 10Be deposition in the ice at 3,160-3,170 m, which we interpret as a result of the low dipole field strength during the Matuyama-Brunhes geomagnetic reversal, which occurred about 780,000 years ago. If correct, this provides a crucial tie point between ice cores, marine cores and a radiometric timescale.

Assignment of Absolute Configuration of Natural Abundance Deuterium Signals Associated with (R)- and (S)-Enantioisotopomers in a Fatty Acid Aligned in a Chiral Liquid Crystal: Enantioselective Synthesis and NMR Analysis

Previous experimental natural abundance deuterium (NAD) NMR results have shown an odd/even-related alternation in the ((2)H/(1)H) ratio of the methylene groups of fatty acids (ChemBioChem 2001, 2, 425) and, by NAD NMR in CLC, a marked difference between enantiotopic deuterons for each methylenic site (Anal. Chem. 2004, 76, 2827). However, to date, the assignment of the absolute configuration for each deuterium has not been possible. To investigate further the origin of these effects, the assignment of NAD quadrupolar doublets observed in chiral oriented solvent is required. Here we describe the assignment of R- and S-isomers resulting from the isotopic substitution in positions 4 and 5 in the aliphatic chain of 1,1'-bis(thiophenyl)hexane 1 (BTPH) derived from natural linoleic acid of plant origin. This was achieved using an optimized synthetic strategy to obtain separately four regio- and stereoselectively deuterated enantiomers of BTPH. By reference to the deuterium spectra of these isotopically labeled reference compounds, we demonstrate that, on both 4 and 5 positions of BTPH, the isotopic enantiomers of S configuration are depleted relative to those of R configuration. This finding effectively explains the observed low ((2)H/(1)H) ratio in NAD of some ethylenic sites of unsaturated fatty acids.

Familial Mutants of α-Synuclein with Increased Neurotoxicity Have a Destabilized Conformation

A30P and A53T mutations of the presynaptic protein alpha-synuclein are associated with familial forms of Parkinson disease. NMR spectroscopy demonstrates that Parkinsonism-linked mutations greatly perturb specific tertiary interactions essential for the native state of alpha-synuclein. However, alpha-synuclein is not completely unfolded but exhibits structural fluctuations on the time scale of secondary structure formation and loses its native conformation gradually when protein stability decreases. The redistribution of the ensemble of alpha-synuclein conformers may underlie toxic gain-of-function by fostering self-association and altered binding affinity to ligands and receptors.

Platelet Factor 4 and Interleukin-8 CXC Chemokine Heterodimer Formation Modulates Function at the Quaternary Structural Level

The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.

Exploring the Analytical Potential of NMR Spectroscopy in Chiral Anisotropic Media for the Study of the Natural Abundance Deuterium Distribution in Organic Molecules

The deuterium/hydrogen (D/H)(i) ratio measurement by quantitative (2)H NMR spectroscopy is a method of choice for the analysis of kinetic isotopic effects associated with enzyme-catalyzed reactions during a biosynthetic pathway. However, the efficiency of the current isotropic (2)H-[(1)H] NMR can be limited by the rather small chemical shift dispersion of deuterium nuclei. In addition, this method does not allow the enantiotopic deuterons in prochiral molecules to be spectrally discriminated, hence precluding the quantification of isotopic fractionation on methylene prostereogenic sites. In this work, we explore another analytical strategy able to circumvent these disadvantages. This approach is based on the use of natural abundance (2)H 2D NMR experiments on solutes embedded in polypeptidic, chiral liquid crystalline solvent. Thus, we show that NMR in these oriented phases is a powerful way to separate deuterium signals on the basis of the quadrupolar interactions, providing a promising alternative to overcrowded (2)H NMR spectra obtained in liquid state. To illustrate our purpose, we have experimentally investigated the case of 1,1'-bis(phenylthio)hexane derived by cleavage from methyl linoleate of safflower. The (2)H NMR results in chiral liquid crystals are presented and discussed. We show, for the first time, that (D/H)(pro-R) and (D/H)(pro-S) can be measured at the same methylene position of a fatty acid chain.

Intramembrane Polarity by Electron Spin Echo Spectroscopy of Labeled Lipids

The association of water (D 2O) with phospholipid membranes was studied by using pulsed-electron spin resonance techniques. We measured the deuterium electron spin echo modulation of spin-labeled phospholipids by D 2O in membranes of dipalmitoyl phosphatidylcholine with and without 50 mol% of cholesterol. The Fourier transform of the relaxation-corrected two-pulse echo decay curve reveals peaks, at one and two times the deuterium NMR frequency, that arise from the dipolar hyperfine interaction of the deuterium nucleus with the unpaired electron spin of the nitroxide-labeled lipid. For phosphatidylcholine spin-labeled at different positions down the sn-2 chain, the amplitude of the deuterium signal decreases toward the center of the membrane, and is reduced to zero from the C-12 atom position onward. At chain positions C-5 and C-7 closer to the phospholipid headgroups, the amplitude of the deuterium signal is greater in the presence of cholesterol than in its absence. These results are in good agreement with more indirect measurements of the transmembrane polarity profile that are based on the 14N-hyperfine splittings in the conventional continuous-wave electron spin resonance spectrum.

NMR water mobility in xanthan and locust bean gum mixtures: possible explanation of microbial response

Molecular and structural mobility of xanthan and locust bean gum mixtures (with and without mannitol) were studied in relation to microbial stability. Molecular mobility was measured by solid state 1H and 2H NMR and by 2H high resolution NMR while differential scanning calorimetry and dynamic mechanical analysis were used to investigate structural mobility. The NMR mobile signal was found to increase significantly with increasing moisture content and, at above 30% moisture content, the solid component disappeared with observed structural collapse. Cell survival decreased with moisture content increasing from 6 to 15% corresponding with an increasing mobile proton and deuterium signals (i.e. increased mobility). Presence of mannitol protected the cells from death at increasing moisture content and a relationship with a lower mobile 1H signal (as compared with the control) seems to be present. A strong mannitol–water interaction, leading to a decreased mobility, is suggested to play a role. No evidence of a glassy to rubbery transition was observed from DSC and DMA analyses, suggesting that structural relaxation did not play a significant role in NMR solid-liquid transition, i.e. NMR molecular mobility.

1H and 2H NMR mobility in cellulose

Mobility of water in cellulose was studied by solid-state 1H and high-resolution 2H NMR as a function of moisture content within the unfreezable moisture range (0–19% dry basis). Measurements of relative mobilities were based on relative intensities, transverse and longitudinal relaxation times and lineshape analysis. At 2–16% moisture content (dry basis), water molecules reoriented anisotropically, suggesting an interaction with cellulose fibers. At moisture content below the monolayer value (2.8%, dry basis), 90% of the protons were immobile and no liquid deuterium signal was detected. A sharp increase in liquid or mobile 1H intensity (accompanied by a decreased LW) and increases in 2H NMR T 1 and T 2 relaxation times were observed as moisture increased above 9% (dry basis). At this moisture content the molecular mobility approached the fast exchange regime. The data confirmed earlier reports that unfreezable water could be highly mobile and not in a rigid state. No glass transition was observed by DSC. However, NMR showed a significant mobility transition as the material transformed from a slow exchange (retarded mobility) to a fast exchange regime.

The dynamic disorder of azulene: A single crystal deuterium nuclear magnetic resonance study

Deuterium nuclear magnetic resonance measurements on single crystals of azulene, specifically deuterated in the 1 and 3 positions, are reported. The quadrupole coupling tensor of these deuterons was determined by rotation experiments, yielding Qzz=182.2 kHz and η=0.056, with Qxx, the intermediate component (magnitude wise), oriented perpendicular to the molecular plane. The deuterium signals are inhomogeneously broadened and their widths are strongly anisotropic. This is quantitatively interpreted in terms of alignment disorder, induced by polar (up–down) disorder already known to be present in azulene from earlier x-ray measurements. It is shown that the alignment disorder is due to a planar distribution in the orientation of the molecules about the short axis, with a root mean square deviation of ±1°. The linewidths are strongly temperature dependent and reduce from about 8.9 kHz (maximum width) at room temperature to 1.6 kHz at 67 °C. This effect is interpreted in terms of molecular “up–down” flips, which average out both the polar and the alignment disorder. The rate of this process is found to be 104 s−1 at 40 °C, with an activation energy of 65 kJ/mol. Magnetization transfer experiments were performed by selectively inverting the magnetization of one of the deuterium doublets, followed by monitoring the subsequent approach to equilibrium of the whole spectrum. The results show the presence of additional dynamic processes in the ultraslow motion regime. These include molecular π flips about their long axes, as well as jumps between different sites in the lattice. The rate of both processes is about 0.084 s−1 at 57 °C. The intersite jumps are predominantly of the flip type, which interchange crystallographic symmetry related deuterons. From the rate of this process, a self-diffusion constant of 0.35×10−22 m2 s−1, at 57 °C, is estimated.

Detection of hydrogen isotope atoms by mass spectrometry combined with resonant ionization

We examined the application of mass spectrometric methods using resonant ionization by a tunable laser and proposed its use for analyzing hydrogen isotopes. We conducted resonance ionization mass spectrometry (RIMS) to detect gas-phase hydrogen isotope atoms. The ionization efficiency was increased by more than 1000 times that obtained with conventional methods using nonresonant ionization. Resonant laser ablation mass spectrometry (RLAMS) was applied for deuterium detection in solid samples. A graphite substrate implanted with deuterium was used for ordinary laser ablation mass spectrometry (LAMS) and RLAMS. The deuterium signal was observed very clearly by RLAMS, in contrast to LAMS. Mass spectrometry combined with resonance ionization was very useful for hydrogen isotope detection, because components with equal mass numbers were resolved and the method demonstrated higher ionization efficiency.

Solid-State Deuterium NMR of Imidazole Ligands in Cytochrome c Peroxidase

We have measured hyperfine shifted NMR signals associated with deuterated imidazole bound to the high-spin Fe3+ state of cytochrome c peroxidase using deuterium magic angle spinning solid-state NMR. These experiments were performed on a mutant of cytochrome c peroxidase, CcP(H175G), for which replacement of the proximal histidine with glycine produced a cavity that can bind a variety of substituted imidazoles including imidazole or 2-methylimidazole. The mutant with imidazole bound is inactive; specifically its reaction with H2O2 is blocked. We observed deuterium NMR signals from the methyl-d3 group of the perdeuterated 2-MeIm sample bound to H175G CcP. The signal displayed an upfield chemical shift and exhibited non-Curie temperature dependence, indicating the existence of low-lying excited electronic states. Upon introducing a nondeuterated competitive ligand, imidazole, a decrease in the intensity of this signal was detected, consistent with the assignment of the deuterium signal to the bound 2-methyli...

Reconstructing annual and seasonal climatic responses from volcanic events since A.D. 1270 as recorded in the deuterium signal from the Greenland Ice Sheet Project 2 ice core

A 200 m ice core at the GISP 2 site (Summit, Greenland) provides a 720 year record of volcanic eruptions, identified by pronounced increases in SO42− in relation to the background concentration. Deuterium/hydrogen isotope ratios (δD) measured in the same core provide a proxy climate record (temperature) used to examine the response of the climate system in Greenland to these eruptions. A group of 34 known volcanic events and a subgroup of the six largest volcanic events are isolated and examined. For both groups, significant negative (cooler) isotopic excursions occur beginning the year the volcanic event is detected in the ice core. Cooling is significant for 1 to 2 years. The maximum isotopic excursion averages 4‰ (∼0.7°C) for all 34 events and 10‰ (∼1.8°C) for the subgroup of largest events. For individual events the response in the isotope signal to volcanic events is highly variable but occurs at most 1 year before SO42− is detected in the core. Examination of subannual isotope data suggests that peak winter isotope values are lowered more than peak summer values. We hypothesize that this result is flawed by posted‐positional alteration of the isotopic profile by vapor diffusion. To test this hypothesis, we use numerical deconvolution to account for the smoothing effects of vapor diffusion in the firn. Analysis of the deconvolved data shows greater summer than winter cooling, a result more consistent with instrumental temperature data.

Action of Oxidants on Water Sorption, 2H Nuclear Magnetic Resonance Mobility, and Glass Transition Behavior of Gluten

ABSTRACTOxidation of vital wheat gluten with potassium bromate and ascorbic acid significantly extends or broadens glassy‐rubber transition to a higher final temperature range or moisture content. Thermomechanical and deuterium nuclear magnetic resonance (NMR) data show that the increased stiffness due to oxidation could be detected from the thermomechanical and deuterium NMR mobility level, indicating increased rigid fractions. The oxidation also resulted in increased water sorption, but no significant change in “freezable” water, and a much decreased mobile deuterium NMR signal. Room temperature sorption of water resulted in a glassy‐rubbery transition over a ≈10–20% mc range for the control. For the oxidized sample, it started at ≈10%mc, but the transition was gradual and extended into much higher moisture ranges that corresponded to a more rigid fraction of deuterium (NMR) signal. This suggests that the oxidative interactions led to a more rigid gluten fraction, extending the transition to a higher temperature range, perhaps resulting in a more elastic dough.

Anionic Polyelectrolyte Binding to Mixed Cationic−Zwitterionic Surfactant Micelles: A Molecular Perspective from 2H NMR Spectroscopy

Complex formation between an anionic polyelectrolyte poly(sodium styrenesulfonate) (PSSS) and mixed zwitterionic−cationic surfactant micelles was monitored using deuterium NMR spectroscopy of the specifically deuterium-labeled surfactants hexadecylphosphocholine (HDPC-γ-d6) and cetyltrimethylammonium bromide (CTAB-γ-d9). The deuterium signal from both surfactants decreased in direct proportion to the amount of added PSSS, reflecting the phase separation of the charge-neutralized PSSS−surfactant complex. The insoluble complex was preferentially enriched with CTAB over HDPC. The PSSS/CTAB charge ratio was the primary determinant of the degree of complex formation. Deuterium NMR T1 and T2 relaxation time measurements indicated that soluble PSSS−surfactant complexes exist prior to their phase separation. Within these soluble complexes motions of the cationic surfactant CTAB, but not the zwitterionic surfactant HDPC, were hindered. Quadrupolar echo deuterium NMR spectra of the phase-separated complexes showed that the flocculated material is inhomogeneous in composition and that surfactant mobility within the insoluble complex varies according to the CTAB/HDPC ratio.

Deuterium MR Spectroscopy at 4.7 T

Deuterium MR spectroscopy was used for the determination of tissue blood flow (TBF). The tracer D2O was injected into the tissue of interest, and tracer washout was followed using a 4.7 T spectroscopy/imaging unit. Normal subcutaneous tissue in rats was studied, as well as tissue influenced by vasoactive agents (papaverine and adrenaline). The vasoactive agents introduced changes of 40% in TBF, compared with normal tissue. Normal tissue measurements were repeated using various D2O injection volumes (5-400 microliters). The injection volume 5 microliters gave TBF 11.7 +/- 2.0 ml/100 g.min (mean +/- 1 SD). This value was 40% higher than corresponding values observed at larger injection volumes (200-400 microliters). This injection volume effect is probably partly due to a capillary dilution caused by tracer administration, and partly related to the non-physiological deuterium signal decrease observed in dead rats. Blood flow measurements in human colon tumours implanted in nude mice showed a rather poor reproducibility, not improved by the use of a multiple site injection technique.

Deuterium MR Spectroscopy at 4.7 T

Deuterium MR spectroscopy was used for the determination of tissue blood flow (TBF). The tracer D2O was injected into the tissue of interest, and tracer washout was followed using a 4.7 T spectroscopy/imaging unit. Normal subcutaneous tissue in rats was studied, as well as tissue influenced by vasoactive agents (papaverine and adrenaline). The vasoactive agents introduced changes of 40% in TBF, compared with normal tissue. Normal tissue measurements were repeated using various D2O injection volumes (5–400 μl). The injection volume 5 μl gave TBF 11.7 ± 2.0 ml/100 g · min (mean ± 1 SD). This value was 40% higher than corresponding values observed at larger injection volumes (200–400 μl). This injection volume effect is probably partly due to a capillary dilution caused by tracer administration, and partly related to the non-physiological deuterium signal decrease observed in dead rats. Blood flow measurements in human colon tumours implanted in nude mice showed a rather poor reproducibility, not improved by...

2H NMR study of the self-assembly of an azo dye-cyclomaltooctaose (γ-cyclodextrin) complex

Orange II and γ-cyclodextrin form 2:1 and 2:2 complexes. These complexes self-associate, and microscopic viewing indicates the formation of a fibroid aggregate. The deuteron resonance (in 2H NMR spectroscopy) from deuteron exchange and solvation between the aggregate and deuterium oxide exhibits quadrupole splitting (Δν) in the region of 0-1.2 KHz. The orientation behavior obtained from this splitting suggests the formation of a liquid-crystaloid substance. (1) The aggregate aligned by means of the super conductive and permanent magnetic fields shows a different NMR pattern and turbidity. (2) Dilution of the 0.12 M aggregate with deuterium oxide proceeds discontinuously via the nonoriented pattern to the oriented-pattern. (3) With an increase in temperature, the 2H NMR spin-spin splitting values decrease continuously. A phase diagram that plots melting points in variable concentrations reveals separate solution and aggregate layers. (4) When the 0.06 M aggregate is diluted by deuterated solvents (water, methanol, ethanol, propanol, dimethyl sulfoxide, and acetone), increases in the amount added and the length of solvent molecules cause a regular reduction of Δν. The deuterium signal of the methyl group in dimethyl sulfoxide- d 6 exhibits a small Δν, suggesting that this solvent is included in the orientation system.

The search for d-glucose derivatives suitable for the study of natural hydrogen isotope fractionation

In order to apply Site-specific Natural Isotope Fractionation (SNIF) to chemical, biochemical, and environmental studies of d-glucose, there is a need for readily prepared compounds which give 2H NMR spectra with all or most signals resolved. Changing substituents of C-1 and/or C-6 of α- d-glucopyranose pentaacetate improved the dispersion of deuterium signals, the best results being achieved with 1,2,3,4-tetra- O-acetyl-6-deoxy-6-thiocyanato-α- d-glucopyranose, for which only the 2H-2 and 2H-4 signals were not resolved, and with 2,3,4-tri- O-acetyl-6-bromo-6-deoxy-α- d-glucopyranosyl bromide, for which only the 2H-6a and 2H-6b signals were not resolved. Periodate oxidation of methyl 4,6- O-benzylidene-α- d-glucopyranoside and 4,6-dichloro-4,6-dideoxy- d-galactose was also examined as a possible source of useful compounds. Products obtained from the benzylideneglucoside gave inadequate resolution and broad deuterium signals. The oxidation of 4,6-dichloro-4,6-dideoxy- d-galactose was not straightforward. The α anomer was oxidised more rapidly than the β anomer. The oxidation product, 2,4-dichloro-2,4-dideoxy-3- O-formyl- d-threose, was too labile to be isolated pure and its hydrolysis to 2,4-dichloro-2,4-dideoxy- d-threose was accompanied by a slow elimination to form 2,4-dichlorobut-2-enal.

Determination of absolute phosphate metabolite concentrations in RIF-1 tumors in vivo by 31P-1H-2H NMR spectroscopy using water as an internal intensity reference.

The absolute metabolite quantification method of Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] in which the tissue water proton signal is used as an internal intensity standard and its more recent variation in which NMR peak intensities are referenced to that of the natural abundance deuterium signal of water [Li et al., SMRM Abstr. 2, 825 (1988); Song et al., Magn. Reson. Med. 25, 45 (1992) have been implemented to obtain absolute phosphate metabolite concentrations in subcutaneous RIF-1 tumors during untreated growth and following treatment with 5-fluorouracil. The equivalence of these two hydrogen isotopes as intensity standards and the validity of their use in the determination of absolute metabolite concentrations in vivo by NMR has been demonstrated. On matched in vivo and extract tumor samples (n = 5), excellent agreement has been obtained between nucleoside triphosphate concentrations determined by NMR and those derived by HPLC analysis for the control tumors. Following 3 days of untreated growth, absolute concentrations of phosphate metabolites in RIF-1 tumors (n = 10) decreased significantly, except for the Pi concentration which did not vary. For the treated tumors (n = 10) there were no changes in metabolite concentrations except for a decrease in the PCr and, possibly, Pi concentrations. The PCr/Pi ratio in the latter tumors did not change. These observations suggest that changes in absolute metabolite concentrations may be more sensitive indices of response to therapy than changes in metabolite peak amplitude ratios, a parameter commonly used to express in vivo NMR data.

Quantitative evaluation of water content in a solid protein by deuterium NMR

A method for evaluating absolute water content in a solid protein based on deuterium NMR measurements in solution is described. By dissolving the hydrated solid protein, which has been specifically deuterium-labeled, into deuterium-depleted water and by comparing the deuterium NMR signal intensity of water (1H2HO) with that of the protein, the amount of water contained in the solid protein is evaluated quantitatively. The method requires a heat pretreatment of the protein sample in water of an enriched (e.g. 2%) deuterium composition for complete hydrogen exchange of the labile protons, and hence is applicable to a protein with a reasonably good reversibility of thermal unfolding. By utilizing this method, the absolute content of the bound water in a protein, Streptomyces subtilisin inhibitor (SSI), lyophilized for 8 h was determined to be 9.2%. The extent of hydration of solid SSI during its exposure to a deuterium-enriched water vapor could also be followed from the deuterium NMR signals in solution. In addition, solid state deuterium NMR measurements of SSI suggested that direct measurement of the natural abundance deuterium signal can give a reasonable estimate of the water content in a solid protein.