Molecular ordering of interfacially localized tryptophan analogs in ester- and ether-lipid bilayers studied by 2H-NMR.

Perdeuterated indole-d6 and N-methylated indole-d6 were solubilized in lamellar liquid crystalline phases composed of either 1,2-diacyl-glycero-3-phosphocholine (14:0)/water or 1,2-dialkyl-glycero-3-phosphocholine(14:0/water. The molecular ordering of the tryptophan analogs was determined from deuteron quadrupole splittings observed in 2H-NMR spectra on macroscopically aligned lipid bilayers. NMR spectra were recorded with the bilayers oriented perpendicular to or parallel with the external magnetic field, and the values of the splittings differed by a factor of 2 between these distinct orientations, indicating fast rotational motion of the molecules about an axis parallel to the bilayer normal. In all cases the splittings were found to decrease with increasing temperature. Relatively large splittings were observed in all systems, demonstrating that the tryptophans partition into a highly anisotropic environment. Solubilization most likely occurs at the lipid/water interface, as indicated by 1H-NMR chemical shift studies. The 2H-NMR spectra obtained for each analog were found to be rather similar in ester and ether lipids, but with smaller splittings in the ether lipid under similar conditions. The difference was slightly less for the indole molecule. Furthermore, in both lipid systems the positions of the splittings from indole were different from those of N-methyl indole. The results suggest that 1) the tryptophan analogs are solubilized in the interfacial region of the lipid bilayer, 2) the behavior may be modulated by hydrogen bonding in the case of indole, and 3) hydrogen bonding with the lipid carbonyl groups is not likely to play a major role in the solubilization of single indole molecules in the ester lipid bilayer interface.     

In vivo measurements of intra- and extracellular Na+ and water in the brain and muscle by nuclear magnetic resonance spectroscopy with shift reagent.

The introduction of new paramagnetic shift reagents in the nuclear magnetic resonance (NMR) method has made it possible to distinguish intra- and extracellular ions in tissues or organs in vitro. We measured the intra- and extracellular 23Na and 1H in vivo in the gerbil brain and skeletal muscle by NMR spectroscopy employing the shift reagent, dysprosium triethylenetetraminehexaacetate (Dy[TTHA]3-). Without Dy(TTHA)3-, the 23Na and 1H signals were seen only as single peaks, but gradual intravenous infusion of Dy(TTHA)3- separated these signals into two peaks, respectively. The unshifted peaks reflected the intracellular 23Na and 1H signals, while the shifted peaks reflected the extracellular signals. In the brain spectra, an additional small peak, which represented intravascular signals, was detected and its intensity increased after injection of papaverine hydrochloride. The present method is advantageous over the microelectrode technique because of its nondestructiveness and its capability for obtaining intra- and extracellular volume information from measurements of the 1H spectra, the peaks of which reflect the intra- and extracellular water amounts. The intracellular Na+ increase associating with increased cellular volume after ouabain in the muscle was clearly visualized by this method. The technique is clearly of use for physiological and pathophysiological studies of organs.     

Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance.

31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses. The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted. Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water: (a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water; (c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP. The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2.     

Bicelle samples for solid-state NMR of membrane proteins

Magnetically aligned bicelles are an excellent medium for structure determination of isotopically labeled membrane proteins by solid-state NMR spectroscopy. Bicelles are a mixture of long- and short-chain phospholipids that form bilayers in an aqueous medium and align spontaneously in a high magnetic field, for example that of an NMR spectrometer with a 1H resonance frequency between 400 and 900 MHz. Importantly, membrane proteins have been shown to be fully functional in these fully hydrated, planar bilayers under physiological conditions of pH and temperature. We describe a protocol for preparing stable protein-containing bicelles samples that yield high-resolution solid-state NMR spectra. Depending on the details of the protein and its behavior in the lipids, the time for sample preparation can vary from a few hours to several days.     

Effects of organic solutes on the Raman spectra of barnacle muscle fibers

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023-1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the alpha-helix conformation (1645-1650 cm-1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660-1670 cm-1 region where the amide I bands of either beta-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm-1 is assigned to a change of orientation of alpha-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.     

Nuclear Magnetic Resonance Studies on Intracellular Sodium in Human Erythrocytes and Frog Muscle

The nuclear magnetic resonance (NMR) spectrum of sodium was determined in muscle and erythrocytes using conventional continuous wave techniques. NMR spectra of fresh intact muscle revealed a single line with a width of about 38 Hz equivalent in intensity to about 53% of the total muscle sodium, in general agreement with previous work. Prolonged washing with sodium-free solutions led to a marked loss of both total and NMR-detectable sodium. The NMR-visible sodium remaining in the muscle was somewhat larger than the fraction calculated to remain extracellular and, presumably, was intracellular. The original sodium signal is thus interpreted as arising from both extracellular sodium and the narrow line portion of the signal from intracellular sodium. NMR spectra of sodium were also obtained for human erythrocytes under conditions preserving the sodium transport system. The intensity of the sodium signal in fresh cells was 98% of that present in the same samples after complete hemolysis of the cells. The NMR sodium present in intact cells was 92% of the sodium recovered by flame photometric determination of sodium from ashed samples. It is concluded that no NMR-“invisible” sodium occurs in human erythrocytes and that the presence of such sodium is not necessary for the normal functioning of the sodium transport system in erythrocytes.     

Proton magnetic resonance assay of total and taurine-conjugated bile acids in bile.

Biliary bile acids, coexisting with phospholipid and cholesterol, are partly conjugated with taurine. In the present report we show that total and taurine-conjugated bile acids in bile can be simultaneously and quantitatively measured by high-resolution (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy. We used a 7.05-Tesla NMR spectrometer to obtain the (1)H-NMR spectra of model and biological biles. Only addition of trimethylsilyl-3-propionic acid sodium salt-D(4) (TSP) to each sample as an internal standard was required in preparation for (1)H-NMR measurement. In (1)H-NMR spectra of rat bile, peaks of C-18 methyl protons of bile acids and of C-25 methylene protons on the taurine moiety of taurine-conjugated bile acids were detected at 0.7 ppm and 3.1 ppm, respectively. Peak areas, of C-18 and C-25 peaks, increased in proportion to the concentrations of bile acids or taurine-conjugated bile acids, even in the presence of phospholipid and cholesterol. The accuracy of NMR measurement of total and taurine-conjugated bile acids was confirmed by comparing the results of NMR with those of enzyme-fluorimetry.The results clearly demonstrate that (1)H-NMR spectroscopy can be applied to the quantitative determination of total and taurine-conjugated bile acids in bile without troublesome preparative steps.     

Two-dimensional proton magnetic resonance of human muscle extracts

In continuation of our previous work on one-dimensional (1D) proton nuclear magnetic resonance (1H-NMR) of normal and diseased human muscle extracts we recorded the two-dimensional (2D) J-correlated proton magnetic resonance spectra of these extracts. Significant differences between normal and diseased muscle extracts, not observed in the 1D 1H-NMR spectra, were seen from their 2D connectivity contour patterns. Taurine was not present in cerebral palsy muscle extract while both normal and scoliosis muscles contained this metabolite. Only the normal muscle had carnitine. Carnosine was present in all muscles. alpha-Ketoglutarate was found only in the diseased muscle extracts. While the amino acids lysine, cysteine and glutamine were common to normal and diseased muscles, threonine was seen only in the diseased muscles. Additional small differences were detected in the 2D patterns of human muscle extracts.     

NMR Evidence for Complexing of Na+ in Muscle, Kidney, and Brain, and by Actomyosin. The Relation of Cellular Complexing of Na+ to Water Structure and to Transport Kinetics

The nuclear magnetic resonance (NMR) spectrum of Na⁺ is suitable for qualitative and quantitative analysis of Na⁺ in tissues. The width of the NMR spectrum is dependent upon the environment surrounding the individual Na⁺ ion. NMR spectra of fresh muscle compared with spectra of the same samples after ashing show that approximately 70% of total muscle Na⁺ gives no detectable NMR spectrum. This is probably due to complexation of Na⁺ with macromolecules, which causes the NMR spectrum to be broadened beyond detection. A similar effect has been observed when Na⁺ interacts with ion exchange resin. NMR also indicates that about 60% of Na⁺ of kidney and brain is complexed. Destruction of cell structure of muscle by homogenization little alters the per cent complexing of Na⁺. NMR studies show that Na⁺ is complexed by actomyosin, which may be the molecular site of complexation of some Na⁺ in muscle. The same studies indicate that the solubility of Na⁺ in the interstitial water of actomyosin gel is markedly reduced compared with its solubility in liquid water, which suggests that the water in the gel is organized into an icelike state by the nearby actomyosin molecules. If a major fraction of intracellular Na⁺ exists in a complexed state, then major revisions in most theoretical treatments of equilibria, diffusion, and transport of cellular Na⁺ become appropriate.     

Laser Raman investigation of intact single muscle fibers on the state of water in muscle tissue

Laser Raman spectroscopy has been used to investigate the state of water in intact single muscle fibers of the giant barnacle (Balanus nulilus). The spectra in the region of the O-H (or O-²H) stretching modes of water in unfrozen fibers show that there is no appreciable difference between the shape and relative intensity of the Raman bands due to the water molecules located inside a muscle fiber and those of the corresponding bands in the spectrum of pure water. The presence of significant amounts of “structured” intracellular water, greater than approx. 5% of the total water content, in these fibers is thus excluded. The Raman spectra of frozen fibers have also been recorded in order to evaluate the amount of intracellular water which remains unfrozen at temperatures below the normal freezing point of water. We have been able to reproduce these spectra by assuming that the spectrum of a frozen fiber is the sum of the individual spectra of water and ice. To calculate the amount of unfrozen water from these curve fittings, it was also necessary to determine the intensities of the water and ice Raman bands relative to one another. We have found the I(ice)/I(water) ratio is 1.07 ± 0.01 for H₂O and 1.05 ± 0.03 for ²H₂O With these figures, we have calculated that for a fiber with a normal water content of 80%, 20% of the water molecules remain in the supercooled state at ?5°C, which corresponds to 1 g of water per of fiber dry weight. This amount of bound water was also found to be independent of the water content of the fibers.     

Proton magnetic resonance spectroscopy of leech muscle and nervous system

1. Proton nuclear magnetic resonance spectroscopy (1H NMR) was used to measure the major intracellular metabolites in perchloric acid extracts of the Macrobdella decora muscle and nervous systems and the Oryctolagus cuniculus cerebrum. 2. Acetate, alanine, choline, glutamate, inositol, and lactate were assigned in the spectrum of leech ventral cord, leech muscle, and rabbit cerebrum. 3. Hirudonine and propionate were clearly observed only in the spectrum of leech muscle. 4. Creatine, N-acetyl aspartate, gamma aminobutyric acid, aspartate, and taurine, distinctive components of spectra of the mammalian cerebrum, were not seen in the invertebrate spectra. 5. 1H NMR spectroscopy provides a simple and rapid means of characterizing the major organic metabolites found in leech muscle and nervous tissues.     

The depletion of protein signals in metabonomics analysis with the WET-CPMG pulse sequence

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool capable of providing a comprehensive metabolic profile of biofluids such as urine, plasma, and serum. Unfortunately, when measuring serum and plasma, the high protein concentration can obscure the signals originating from low molecular weight metabolites. We evaluated the use of different parameters within the Carr-Purcell-Meiboom-Gill (CPMG) pulse train of fast spin-echoes to remove the macromolecular signal contribution in one-dimensional proton (1H) NMR spectra. Experimental parameters such as the refocusing delay in the CPMG pulse train, pulse miscalibration, and recycle time were examined to assess the ability to remove the protein signals from the spectrum without causing a deleterious effect on the signals originating from free, low molecular weight metabolites. The 1H-NMR spectra of a variety of serum samples spiked with 2'-deoxyadenosine were acquired using various acquisition parameters. Our results show that the delay used in the CPMG spin-echo and the combination of the acquisition pulse flip angle and recycle time are the two major factors affecting the observed metabolite signal amplitudes in the resulting 1H-NMR spectrum.     

The use of neocarrabiose oligosaccharides with different length and sulphate substitution as model compounds for 1H-NMR spectroscopy.

The high-field 1H-NMR spectra of various carrageenan oligosaccharides at room temperature are given. The assignments were faciliated by the use of proton double-quantum coherence (DQCOSY) and 1H-13C chemical shift correlation 2D NMR spectroscopy, and by comparing high-field 1H-NMR spectra of various 4-sulphated oligosaccharides of the neocarrabiose type. The effects of anomeric configuration on the 1H resonances on the same or neighbouring units are discussed. The 13C-NMR shift data are given for the tetrasaccharide of kappa-carrageenan.     

A proton nuclear magnetic resonance study of the interaction of cadmium with human erythrocytes

The binding of Cd²⁺ by molecules in the intracellular region of human erythrocytes has been studied by ¹H-NMR spectroscopy. From changes in spin-echo Fourier transform NMR spectra for both intact and hemolyzed erythrocytes to which CdCl₂ was added, direct evidence was obtained for the binding of Cd²⁺ by intracellular glutathione and hemoglobin. Time-courses were measured by ¹H-NMR for the uptake of Cd²⁺ by intact erythrocytes in saline/glucose solution and in whole blood. In both cases, the uptake, as indicated by changes in the ¹H-NMR spectrum for intracellular glutathione, plateaus after about 30 min. The effectiveness of the disodium salt of EDTA and of various thiol-chelating agents for releasing glutathione from its Cd²⁺ complexes in hemolyzed erythrocytes was also studied. EDTA was found to be more effective than thiols, and dithiols more effective than monothiols.     

Proton nuclear magnetic resonance spectroscopy of isolated rabbit fundic glands

1H-nuclear magnetic resonance spectroscopy (NMR) was adapted to isolated rabbit fundic glands and identification made of compounds responsible for several observed spectral resonances. A minimum gland concentration of 0.5 mg dry weight or 5 mg wet weight per 0.5 ml was needed for adequate signal-to-noise ratio. At physiological temperature and pH, the glands demonstrated reproducible spectra, stability for accumulation times greater than 30 min and responsiveness to histamine stimulation, as measured by oxygen consumption and aminopyrine uptake. The relatively anaerobic conditions favored use of proton compared to phosphorus NMR, since 1H-NMR allowed significantly shorter spectral accumulation times and therefore did not compromise glandular viability to the same extent as 31P-NMR. The most conspicuous resonance in the gland spectrum was assigned to the -N+(CH3)3 protons of choline and related compounds. In membrane-free lysates, several components of the signal were resolvable and assigned to choline, phosphatidylcholine, phosphocholine and L-alpha-glycerophosphocholine. Thin-layer chromatography verified that phosphatidylcholine and phosphatidylethanolamine were the major phospholipids present in gland lipid. Presumably, they represent the source of the surface-active phospholipids present in gastric juice, which may play a role in gastric cytoprotection.     

Changes in water status and water distribution in maturing lupin seeds studied by MR imaging and NMR spectroscopy

The changes in water distribution in maturing lupin (Lupinus luteus L.) seeds were visualized with magnetic resonance imaging (MRI). MRI data showed local inhomogeneities of water distribution inside the seed. At the late seed-filling stage the most intense signal was detected in the seed coat and the outer parts of cotyledons in the hilum area, but during maturation drying the decline in MR image intensity was faster in the outer part of the seed than in the central part. The changes in water status were characterized by NMR spectroscopy. Analyses of T(2) relaxation times revealed a three-component water proton system in maturing lupin seeds. Three populations of protons found during seed maturation, each with a different magnetic environment causing a different relaxation rate, were correlated with three fractions of water (structural, intracellular, and extracellular) that were observed during seed germination. This study provides evidence that lupin seeds have similar states of the different water components with regard to seed moisture content at two distinct physiological stages, seed maturation and germination. The unique feature of maturing lupin seeds is the presence of the high (1)H-NMR signal in areas corresponding to the vascular bundles. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of vascular tissue. An anti-dehydrin antibody detected three polypeptides in lupin embryos with molecular masses of 73, 43 and 28 kDa, respectively. The temporal pattern of dehydrin protein accumulation correlates well with seed desiccation.     

Synthesis and characterization of a binuclear coumarin-3-carboxylate copper(II) complex

The copper(II) complex of coumarin-3-carboxylic acid (CcaH) has been prepared and characterized on the basis of elemental and thermal analysis, IR, Raman, EPR, UV-Vis reflectance and 1H-NMR spectra. A detail analysis of all spectra data is presented with particular emphasis on the elucidation of the coordination mode of the ligand and the structure of the complex. All data are consistent with a binuclear structure for the complex with four coumarin-3-carboxylates as bridges and one water ligand per copper ion. The significantly lower than the spin-only value magnetic moment of the complex and the EPR spectra at various temperature are indicative of a magnetic interaction between the two copper atoms.     

Intramyocellular lipid content in human skeletal muscle

Fat can be stored not only in adipose tissue but also in other tissues such as skeletal muscle. Fat droplets accumulated in skeletal muscle [intramyocellular lipids (IMCLs)] can be quantified by different methods, all with advantages and drawbacks. Here, we briefly review IMCL quantification methods that use biopsy specimens (biochemical quantification, electron microscopy, and histochemistry) and non-invasive alternatives (magnetic resonance spectroscopy, magnetic resonance imaging, and computed tomography). Regarding the physiological role, it has been suggested that IMCL serves as an intracellular source of energy during exercise. Indeed, IMCL content decreases during prolonged submaximal exercise, and analogously to glycogen, IMCL content is increased in the trained state. In addition, IMCL content is highest in oxidative, type 1 muscle fibers. Together, this, indeed, suggests that the IMCL content is increased in the trained state to optimally match fat oxidative capacity and that it serves as readily available fuel. However, elevation of plasma fatty acid levels or dietary fat content also increases IMCL content, suggesting that skeletal muscle also stores fat simply if the availability of fatty acids is high. Under these conditions, the uptake into skeletal muscle may have negative consequences on insulin sensitivity. Besides the evaluation of the various methods to quantify IMCLs, this perspective describes IMCLs as valuable energy stores during prolonged exercise, which, however, in the absence of regular physical activity and with overconsumption of fat, can have detrimental effects on muscular insulin sensitivity.     

Investigation of germination and aging in Moravian III barley grain by nuclear magnetic resonance.

High-resolution, solid-state 1H nuclear magnetic resonance (NMR) techniques are used for the first time to study germination in imbibed Moravian III barley grains. Whereas magic-angle spinning 1H NMR spectra reveal the water and lipid components in barley grains, combined rotation and multiple-pulse spectroscopy techniques provide 1H NMR spectra of grains that reveal the protein and carbohydrate as well as the water and lipid components. Spectra of grains are compared with spectra of model compounds to verify assignments. 1H T1 and T2 measurements using magic-angle spinning only and combined rotation and multiple-pulse spectroscopy techniques provide information about molecular mobility within the grains during inhibition. Some grains were subjected to artificial aging conditions. 1H NMR spectral comparisons are made between normal, viable grains and artificially aged grains.     

Muscle 31P-NMR in humans: estimate of bias and qualitative assessment of ATPase activity.

A mathematical model is developed whereby the longitudinal magnetization of phosphocreatine (PC), ATP, Pi, and total phosphate (PT) can be calculated on the basis of assumed chemical rate constants (kappa i) and spin lattice relaxation times of the muscle PC in equilibrium ATP in equilibrium Pi exchange system. By means of this model, some unexplained 31P nuclear magnetic resonance (NMR) spectroscopy results from the literature (e.g., a decrease of PT in a closed system) could be explained simply on the basis of the physiological variability of kappa i. Moreover, appropriate model simulations indicate that 1) 31P-NMR spectra obtained with short relaxation delays may be influenced to various extents by the metabolic and physicochemical status of the muscle; 2) the assessment of kappa i by standard NMR spectroscopy techniques may be extremely critical; 3) delta PC/delta Pi, as obtained from conventional 31P-NMR spectra, may represent a sensible index of kappa 2 (the pseudo first-order chemical exchange rate constant of the adenosinetriphosphatase reaction); 4) delta PC/delta Pi changes as detected from sequential (short relaxation delays) 31P-NMR spectra obtained in humans during metabolic transients (e.g., during transition from rest to work and vice versa) may represent an index of transient changes of kappa 2.