Natural Abundance 13C NMR Research Articles

13C-NMR analysis of the positional distribution of fatty acids in plant glycolipids

High-resolution, natural abundance 13C-NMR spectroscopy was used to analyze the positional distribution of fatty acids in the predominant plant glycoglycerolipids. Fatty acid profiles attributed spectroscopically to the sn-1 and sn-2 positions were in very good agreement with the enzymatically measured distribution using a lipase from Rhizopus arrhizus. The 13C-NMR spectroscopy analysis makes use of small shift differences of the aryl carbonyl carbons. As in other lipids, in glycolipids sn-1-bound acyl carbonyls are also shifted further downfield than the sn-2-bound carbonyls. Furthermore, in both positions the carbonyl shift depends on the distance to the nearest double bond resulting in identical series of incremental sequence shifts to higher fields from saturated to Δ9, Δ7 and Δ6 double bonds, respectively. These deductions were enabled by analysis of galactolipids isolated from plants belonging to the Apiaceae and Boraginaceae, which contain high proportions of all- cis- δ7,10,13-hexadecatrienoic and all- cis- Δ6.9,12,15-octadecatetraenoic acid, respectively, in the sn-2 position.

Characterization of glycogen and amino acid pool of Entamoeba histolytica by 13C-NMR spectroscopy.

Trophozoite extracts of axenic Entamoeba histolytica were investigated by natural-abundance 13C-NMR spectroscopy. The extracts were found to contain a high level of glycogen (30 mM glucose equivalents), which had a compact structure as suggested by alpha (1-->6) branch points every 5-6 glucose residues. As other major metabolites, we identified putrescine (9.5 mM) and the following free amino acids: tyrosine and phenylalanine (1 mM), glycine, lysine and methionine (2 mM), isoleucine (5 mM), proline and valine (6-7 mM), leucine (11 mM) and glutamate (22 mM). Glutamate and proline may serve, together with putrescine, as intracellular osmolytes.

Identification and localization of two distinct microenvironments for the diacylglycerol component of lipophorin particles by 13C NMR

13C nuclear magnetic resonance spectroscopy of lipoproteins, isolated from the insect Manduca sexta, has been employed to probe the microenvironment of diacylglycerol (DG), their major neutral lipid component. Natural abundance 13C NMR spectra of high density lipophorin exhibited several well-separated resonances derived from its lipid moiety, including those for the carbonyl carbon atoms of phospholipid and DG fatty acyl chains in the region of 175-180 ppm. To verify the assignment of the DG acyl chain carbonyl carbon resonances, di[1-13C]oleoylglycerol high density lipophorin was isolated after instilling a bolus of tri[1-13C]oleoylglycerol into the midgut of larvae fed a fat-free diet. 13C NMR spectra of the isolated lipoprotein revealed a specific and dramatic enrichment of resonances at 175.5 ppm. Expansion of this region revealed two resonances separated by 0.08 ppm. These were assigned as 1,2- and 1,3- isomers of DG, the latter presumably arising from spontaneous acyl chain migration of 1,2-DG following lipoprotein isolation. On the basis of compositional and structural analysis of this lipoprotein, it is postulated that these DG species are localized predominantly in the hydrophobic core of the particle. By contrast, natural abundance 13C NMR spectra of the DG-rich, low density lipophorin (LDLp) subspecies revealed two additional resonances, separated by 0.2 ppm, that were tentatively assigned as 1,2- and 1,3-DG present at the surface of the particle. The verify this assignment, experiments employing phospholipase C, to convert lipophorin surface associated phospholipid into DG, were performed.(ABSTRACT TRUNCATED AT 250 WORDS)

The Conformations of 1-Thiacyclooctan-3-One Dynamic Nuclear Magnetic Resonance and Force-Field Calculation

The 1H and natural-abundance 13C-NMR spectra of 1-thiacyclooctan-3-one (1) have been measured from 25 to -100°C. Coalescence is observed in the 1H-NMR spectra of (1) at about -80°C, and attributed to ring inversion in a boat-chair conformation, which is the predominant conformation of (1). The free energy of activation (DG¹) for this process is 9.2±0.2 kcal/mol. The 13C-NMR spectra of (1) is temperature independent.

Carbon‐13 NMR measurments of polybutadiene local dynamics in dilute solution: Further evidence for non‐Kramers' behavior

AbstractThe local segmental dynamics of cis‐polybutadiene in dilute solutions have been investigated using natural abundance 13C NMR spectroscopy. The time integral of the CH orientation autocorrelation function 〈σ〉 is extracted from T1 measurements in five solvents covering a viscosity range of two decades. The hydrodynamic Kramers' equation, which is generally assumed to be appropriate for local polymer dynamics in dilute solution, cannot describe these results. 〈σ〉 is found to depend upon the solvent viscosity η raised to the 0.33 power whether η is varied by changing the temperature or the solvent. This apparent power law behavior has been observed for other polymers in dilute solution and can be rationalized if the solvent is viewed as viscoelastic. This view is supported by experimental results which show that local polymer dynamics can occur as much as an order of magnitude faster than solvent reorientation. Trends for a mixed microstructure polybutadiene are similar to those of cis‐polybutadiene. © 1994 John Wiley & Sons, Inc.

Solution structure by 1H and dynamics by natural abundance 13C NMR of a receptor recognising peptide derived from a C-terminal fragment of neuropeptide Y

A peptide consisting of 20 amino acid residues, derived from a C-terminal fragment of neuropeptide Y (NPY) and showing high affinity to NPY receptors, was synthesised. Its sequence is PAADLARYRHYINLITRQRY-NH2, and the solution structure was calculated from NMR-derived distance and torsion angle restraints, obtained at 15 degrees C in a solvent mixture of water and 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, by using DIANA and restrained energy minimisation. The structure was found to consist of a well-defined alpha-helix in the centre, with a few residues at the termini having less well defined conformations. The spin-lattice and spin-spin relaxation rates of alpha-carbons have been determined on 13C at natural abundance. From 1D experiments the global rotational correlation time was determined and from 2D experiments the dynamics of each individual residue was obtained. The results demonstrate that the C alpha-H alpha vectors in the alpha-helix essentially follow the global motion. Towards the termini, contributions from local dynamics increase. This tendency is correlated to the increasing uncertainty of the structure towards the peptide ends. An effective molecular volume was calculated from the temperature dependence of the global rotational correlation time. This is well compatible with a monomeric peptide, solvated by water and 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of peptide dimers was ruled out as being inconsistent with the relaxation data.

Turnover of human muscle glycogen with low-intensity exercise

To determine whether glycogen turnover occurs during prolonged low-intensity exercise, five subjects performed plantar flexion of the right leg at 15% MVC for 5 h. At rest and during the initial 2.5 h of exercise gastrocnemius glycogen was monitored in both legs with natural abundance 13C NMR. At 2.5 h exercise, a step-up infusion of 99% enriched 1-13C glucose was begun and maintained over the next 1.5 h of continued exercise to monitor 1-13C glucose incorporation into the exercising muscle's glycogen pool. Exercise was continued for an hour following the infusion, and NMR scans were performed throughout the session. During the first 2 h of exercise, glycogen 1-13C signal amplitudes dropped approximately 30% and remained there at 2.5 h, indicating that glycogen concentration had leveled. Following infusion, glycogen signal amplitudes rose to 123% of resting values, remaining there during an hour of subsequent exercise. There was no change of glycogen 1-13C signal in the nonexercising leg. Venous glucose levels remained stable until the infusion was begun and then rose < 7% (5.57-5.96 mmol.l-1) during the infusion. Venous insulin and C-peptide levels did not change during the infusion. We conclude that the human gastrocnemius can degrade and synthesize glycogen simultaneously during prolonged low-intensity exercise.

Validation of 13C NMR measurements of liver glycogen in vivo

The natural abundance 13C NMR intensity of the glycogen C1 resonance was measured in the surgically exposed liver of rabbits in vivo (n = 17) by integration from 98 to 104 ppm and compared double blindedly to the subsequent biochemical measurement. Coil loading was measured each time from a reference sphere at the coil center and the NMR intensity was normalized accordingly. For quantification, the normalized NMR intensity was calibrated using aqueous glycogen solutions ranging from 110 to 1100 mumol glucosyl units/g (n = 14). An in vivo range from 110 to 800 mumol glucosyl units/g wet weight was measured with a highly linear correlation with concentration (r = 0.85, P < 0.001). The in vivo NMR concentration was 0.95 +/- 0.05 (mean +/- standard error, n = 17) of the concomitant enzymatic measurement of glycogen content. We conclude that the 13C NMR signal of liver glycogen C1 is essentially 100% visible in vivo and that natural abundance 13C NMR spectroscopy can provide reliable noninvasive estimates of in vivo glycogen content over the physiological range of liver glycogen concentrations when using adequate localization and integration procedures.

13C-NMR relaxation in three DNA oligonucleotide duplexes: model-free analysis of internal and overall motion.

Natural-abundance 13C-NMR spectra of [d(TCGCG)] (1), [d(CGCGCG)]2 (2), and [d(GGTATACC)]2 (3) were measured at 90.6 MHz to obtain 13C-1H NOEs and T1 relaxation times; relaxation data were also measured at 125.7 MHz for 1 and 2 and at 62.9 MHz for 1. Analysis of the relaxation data was performed in the context of the "model-free" approach of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559], leading to the following conclusions: (i) Optimized values for the overall correlation times of 0.9 ns for 1 and 1.4 ns for 2 are close to those predicted by light-scattering results on similar molecules [Eimer et al. (1990) Biochemistry 29, 799-811]. (ii) For the nonterminal residues, the "order parameter", S2, is around 0.8 for the protonated base carbons and 0.6 for the sugar carbons, indicating less spatial restriction on the sugar carbons (in the model-free approach, the order parameter is 1 for a rigid body and 0 for a system with completely unrestricted internal motion). (iii) The order parameters for the terminal residues vary over a wide range with the smallest values around 0.2-0.3 for the HO-13C5' and the 13C3'-OH; rational trends are seen in the variation of S2 with chain position in the terminal residues. (iv) The analysis shows that the order parameters are accurate within 15%. (v) The "effective internal correlation time", tau e, is very short for the sugar carbons (30-300 ps) and less well-defined, but probably also short, for the bases. (vi) The analysis indicates that most of the relaxation in DNA is accounted for by S2 and the tau e is so short that a good approximation to any relaxation property, P (e.g., T1, T2, 13C-1H NOE, 1H-1H cross-relaxation rate), is P = S2Prigid, where Prigid is the value for the property in a system without internal motion (the analysis assumes the same isotropic overall motion for both the rigid and flexible bodies).

13C-NMR of the deoxyribose sugars in four DNA oligonucleotide duplexes: assignment and structural features.

Natural-abundance 13C-NMR spectra have been obtained for four self-complementary DNA oligonucleotides: [d(TAGCGCTA)]2, [d(GGTATACC)]2, [d(CG)3]2, and [d(TCGCG)]2; this paper focuses on the deoxyribose resonances. Assignments were made by a combination of the two-dimensional proton-detected heteronuclear correlation experiment and comparison of 1D spectra, accounting for 31P coupling, base composition, and similarities in chemical shift versus temperature profiles (delta vs T). Large shielding and deshielding of the sugar resonances (between 2.0 and -1.9 ppm) are observed upon thermal dissociation of the duplex. The shapes of the delta vs T profiles correlate strongly with the purine/pyrimidine nature of the base attached at C1' in these duplexes that have a substantial fraction of residues within alternating purine-pyrimidine sequences. The correlation is primarily associated with changes in the equilibrium distribution of furanose pseudorotational states that may arise in part from the relief of interstrand purine-purine steric clashes.

Determination of orientational order parameters in liquid crystals from temperature-dependent 13C NMR experiments

We have shown that natural abundance 13C NMR of solutes in liquid crystals may be used to obtain order parameters from a series of temperature-dependent measurements even with limited information on the carbon shift tensors. A procedure for simultaneously analysing data at the various temperatures is proposed. We have obtained the ordering matrix as a function of temperature for various biaxial (D2h) solutes: pyrene, perylene, naphthalene, anthracene and for one effectively uniaxial probe (triphenylene), in the nematic mixture ZLI–1167. We compare these results with deuterium data where possible and we also provide estimates of the chemical shift tensors for perylene and anthracene, where solid-state data are, as yet, unavailable.

Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy

Natural abundance 13C NMR spectroscopy has identified sucrose and trehalose as the principle compatible solutes accumulated by non-halophilic purple and green sulphur bacteria respectively, in response to osmotic stress. Synthesis of glycine betaine as a compatible solute was rare in non-halophilic phototrophic sulphur bacteria and appears to be limited almost exclusively to halotolerant isolates, although all isolates tested were able to accumulate exogenous glycine betaine from the growth medium in response to osmotic stress. These data support the hypothesis that the degree of halotolerance of a microorganism may be due, at least in part, to the metabolic effects of the compatible solute(s) accumulated.

Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy

Natural abundance 13C NMR spectroscopy has identified sucrose and trehalose as the principle compatible solutes accumulated by non-halophilic purple and green sulphur bacteria respectively, in response to osmotic stress. Synthesis of glycine betaine as a compatible solute was rare in non-halophilic phototrophic sulphur bacteria and appears to be limited almost exclusively to halotolerant isolates, although all isolates tested were able to accumulate exogenous glycine betaine from the growth medium in response to osmotic stress. These data support the hypothesis that the degree of halotolerance of a microorganism may be due, at least in part, to the metabolic effects of the compatible solute(s) accumulated.

Detecting fatty acids of dietary origin in normal and cancerous human breast tissue by 13C nuclear magnetic resonance spectroscopy.

Natural abundance 13C NMR was used to determine relative amounts of fatty acid subclasses present in fibroadipose tissue from the human breast in healthy and cancer patients and in breast carcinoma tissue. Resonances corresponding to the carbon atoms of triacylglycerides were obtained when adipose tissue constituted more than 10% of the carcinoma. Resonances corresponding to phospholipids and proteins were also observed when the percentage of adipose tissue was lower. No significant difference between the levels of unsaturated fatty acids in adipose tissue from cancer and non-cancer patients was found. However, significant differences in the levels of monounsaturated and saturated fatty acids of carcinoma compared to non-cancerous tissue was found, as was a nearly significant difference for the levels of polyunsaturated fatty acids in these two tissue types. These findings suggest an alteration of cellular lipid composition in neoplastic mammary tissue.

The water relations of growth and polyhydroxy alcohol production by ascomycetous yeasts

Summary: The response of 31 ascomycetous yeasts to a reduction in water activity (aW) adjusted with D-glucose or NaCl was investigated. The growth of most yeasts was more tolerant to glucose than to NaCl at equivalent aW. Zygosaccharomyces rouxii was the most osmotolerant yeast examined. Natural abundance 13C-NMR spectroscopy and HPLC analyses of eight yeasts indicated that glycerol and arabitol or mannitol were accumulated intracellularly in response to aW. reduction. Pichia sorbitophila, Candida cacaoi, Candida magnoliae and Zygosaccharomyces bisporus responded to reduced aW by a decrease in specific growth rate and cell volume, and an accumulation of glycerol. The other polyol accumulated did not increase in concentration with aW reduction to the same degree as glycerol. A polyol concentration ratio (intra/extracellular) as high as 800-fold was attained across the membrane. Greater amounts of polyols were produced at equivalent aW values when adjusted with glucose than with NaCl. The ability to accumulate high concentrations of polyols appears to be the most important criterion in determining osmotolerance.

Localized 13C NMR spectroscopy of myo-inositol in the human brain in vivo.

Natural abundance 13C NMR spectra obtained from 144-cm3 volumes in the human brain contained well-resolved resonances of myo-inositol after 60 min of data accumulation. A mean concentration of 7.2 +/- 0.5 mumol/g (+/- SE, n = 7) was calculated from the comparison with phantoms. 13C NMR spectroscopy thus provides a complementary role in the quantitation of metabolites also observed in the crowded 1H spectrum.

Multinuclear NMR spectroscopy of the cellular slime mold Polysphondylium pallidum. Monitoring of the encystment and excystment processes.

Polysphondylium pallidum microcysts and amoebae have been investigated by 31P- and natural-abundance proton-decoupled 13C-NMR spectroscopy. Microcysts have been found to contain as prominent metabolites a phosphomonoester, inositol hexakisphosphate (1.4 mM), two phosphodiesters [glycerophosphocholine (5.5 mM) and glycerophosphoethanolamine (2.6 mM)], as well as nucleoside triphosphates (3 mM) and polyphosphates (greater than 10 mM), the polyamines 1,3-diamino-propane (3.5 mM), putrescine (16 mM) and spermidine (3 mM) and the sugar trehalose (31 mM). In vivo 31P-NMR has shown that the level of nucleoside triphosphates in microcysts was maintained metabolically and that the pH of their cytosol, deduced from the chemical shift of cytosolic Pi was 7.2. The absence of trehalose, glycerophosphocholine and glycerophosphoethanolamine in P. pallidum amoebae was the most remarkable difference from microcysts. Microcyst germination (excystment), induced by reduction of the ionic strength of the microcyst bathing medium, was monitored non-invasively by 31P- and 13C-NMR spectroscopy. The major modifications observed during excystment were the progressive disappearance of trehalose used as energy source, of glycerophosphocholine and glycerophosphoethanolamine used as membrane phospholipid precursors, and, finally, the appearance of NMR-visible polyphosphates and of cellobiose. As a mirror situation, P. pallidum amoebae responded to a high-ionic-strength stress by production of trehalose, glycerophosphocholine, and glycerophosphoethanolamine, and induction of an encystment process.

High Pressure NMR Studies of Confined Liquids

AbstractThe main goal of our novel NMR experiments on confined liquids was to determine the effects of pressure on the dynamics of liquids in the surface layer by using the twostate, fast exchange model and compare them to the pressure effects observed for bulk liquids. With this goal in mind, the deuteron NMR spin-lattice relaxation times, T1, in liquid pyridine-d5, nitrobenzene-d5, and methylcyclohexape-d confined to sol-gel porous silica glasses with pore radii in the range from 18Å to 49˚A were measured as a function of pressure up to 5 kbar at 300 K. In another set of high resolution natural abundance 13C NMR experiments, the 13C relaxation behavior of each carbon in 2- ethylhexyl benzoate model lubricant was measured as a function of pressure in porous silica glasses. In fact, the described experimental approach which allows investigation of the effects of pressure on the dynamic behavior of surface-layer liquids, may provide a new tool in studies of model liquid lubricants at extreme conditions of pressure and temperature. In addition selected results of a recent study of acetonitrile-d3 are reviewed. The 2H and 14N spin-lattice relaxation times of acetonitrile-d3 in porous silica glasses were measured in order to study the confinement effects on the anisotropic reorientation characterized by rotational diffusion constants, D⊥ and D‖, of this symmetric-top molecule.

Iron–histidine bonding interaction in deoxymyoglobin and deoxyhaemoglobin

Natural abundance 13C NMR resonances of the proximal histidyl imidazole carbons in the deoxy form of O2-binding haemoproteins have been observed and these resonances are found to serve as sensitive probes for the electonic stucture of this ubiquitous histidine, which is thought to control the iron reactivity.

Elimination of surface signals by a surface-spoiling magnetic field gradient. Theoretical optimization and application to human in vivo NMR spectroscopy

W. Chen and J. J. H. Ackerman ( J. Magn. Reson. 82, 655, 1989; NMR Biomed. 2, 267, 1989) used a superficial magnetic field gradient to eliminate surface signals when observing rat liver in vivo. We have developed a method for computing the optimal gradient coil for a given in vivo application. An analytical solution for the magnetic field created by a planar array of antiparallel current elements was derived for the calculations. The surface-signal suppression obtained by gradient coils of various sizes is presented in a synthetic plot which directly provides the electrical and geometrical parameters of the optimal coil as well as the residual signal in the deep-lying region of interest. This approach was applied to in vivo 31P and 31C spectroscopy of the human liver. Hepatic glycogen was detected by natural-abundance 13C NMR without contamination from muscle glycogen, and physiological variation during starvation could be observed.