Direct observation of glycogenesis and glucagon-stimulated glycogenolysis in the rat liver in vivo by high-field carbon-13 surface coil NMR

High-field 13C surface coil nuclear magnetic resonance has been employed to investigate glucose and glycogen metabolism in rat liver in vivo. Natural abundance and isotopically enriched proton-decoupled 13C NMR experiments were conducted at 90.56 MHz on a standard commercial spectrometer utilizing a laboratory-built high-sensitivity double-resonance coaxial coil probe. At variance with a previous preliminary report, natural abundance spectra of the liver in vivo from a rat fed ad libitum reveal resonances of substantial intensity from hepatic glycogen with approximately 10 min of signal averaging. The response of hepatic glycogen levels to an intravenous injection of the hormone glucagon was continuously monitored through the glycogen C-1 carbon resonance intensity; this revealed an average 60% depletion of hepatic glycogen stores in vivo within approximately 1 h. In a complementary study utilizing fasted rats, 100 mg of D-[1-13C]glucose (90% enriched) was administered via a peripheral vein injection and continuously monitored by 13C NMR with 3-min time resolution as it was incorporated into hepatic glycogen. The C-1 carbon resonances of hepatic glucose and glycogen are well-resolved in vivo enabling the time course for the relative change in concentration for both metabolites to be established simultaneously. The 13C label incorporated into the glycogen pool reaches a steady-state level in approximately 40 min.     

Metabolic pathways for ketone body production. 13C NMR spectroscopy of rat liver in vivo using 13C-multilabeled fatty acids

The hormonal regulation of ketogenesis in the liver of living rat has been studied noninvasively with 13C nuclear magnetic resonance. The protocol involved the use of a surface coil that was placed on the skin of the rat, directly over the normal location of the liver. Signals from superficial tissue were suppressed with a 180 degrees pulse at the center of the coil. A resolution of 0.6 ppm was obtained in the 13C NMR spectra at 20.1 MHz, which was equal to or better than that observed in experiments where the liver was surgically exposed and surrounded with radiofrequency coil. The spatial selection for the liver was better than 90%, with extrahepatic adipose tissue contributing only a very small amount of signal. The metabolic activities of the liver were investigated by infusion of 13C-labeled butyrate in the jugular vein of the anesthetized rat. The rate of butyrate infusion was chosen to be close to the maximum oxidative capacity of the rat liver, and the 13C signal intensities were enhanced by using doubly labeled [1,3-13C]butyrate as a substrate. Different 13C NMR spectra and hence different metabolites were observed depending on the hormonal state of the animal. In the fasted rat, the most intense 13C signal came from the end product of the Krebs cycle, namely, HCO3, with additional resonances from glutamine and glutamate. Weak resonances of the ketone bodies 3-hydroxybutyrate and acetoacetate could also be detected and allowed an evaluation of the "redox state" of the in vivo liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational flexibility of luteinizing hormone-releasing hormone in aqueous solution. A carbon-13 spin-lattice relaxation time study.

The carbon-13 nuclear magnetic resonance (13C NMR) spectra of luteinizing hormone-releasing hormone (LH-RH) and lower homologous peptides have been assigned in aqueous solutions at various pH values. 13C spin-lattice relaxation times (T1) have been measured for all proton-bearing carbons at 25.2 and 67.9 MHz. From the T1 data the rates of overall molecular motion and intramolecular motion of side chains have been estimated. LH-RH is a flexible molecule in solution, having segmental motion along the backbone as well as in the nonaromatic side chains.     

Natural abundance carbon-13 nuclear magnetic resonance studies of histone and DNA dynamics in nucleosome cores

Natural abundance carbon-13 nuclear magnetic resonance spectra (67.9 MHz) were obtained for native nucleosome cores: cores dissociated in 2 M NaCl and 2 M NaCl, 6 M urea; and cores degraded with DNase I plus proteinase K. Phosphorus-31 NMR spectra of native and dissociated cores and core length DNA were also obtained at 60.7 MHz. The 31P resonance and spin-lattice relaxation time (T1) of DNA were only slightly affected by packaging in nucleosome cores, in agreement with other reports, but 13C resonances of DNA were essentially unobservable. The loss of DNA spectral intensity suggests that rapid internal motions of DNA sugar carbons in protein-free DNA previously demonstrated by 13C NMR methods are partly restricted in nucleosomes. The 13C spectrum of native cores contains many narrow intense resonances assigned to lysine side chain and alpha-carbons, glycine alpha-carbons, alanine alpha- and beta- carbons, and arginine side chain carbons. Several weaker resonances were also assigned. The narrow line widths, short T1 values, and non-minimal nuclear Overhauser enhancements of these resonances, including alpha- and beta-carbons, show that some terminal chain segments of histones in nucleosomes are as mobile as small random coil polypeptides. The mobile segments include about 9% of all histone residues and 25% of all lysines, but only 10% of all arginines. The compositions of these segments indicate that mobile regions are located in amino- or carboxyl-terminal sequences of two or more histones. In addition, high mobility was observed for side chain carbons of 45-50% of all lysines (delta and epsilon carbons) and about 25% of all arginines (zeta carbon) in histones (including those in mobile segments), suggesting that basic residues in terminal histone sequences are not strongly involved in nucleosome structure and may instead help stabilize higher order chromatin structure.     

Carbon-13 and proton nuclear magnetic resonance of unsonicated model and mitochondrial membranes

Proton nuclear magnetic resonance (PMR) spectra at 270 MHz of aqueous dispersions of nonsonicated egg lecithin, dipalmitoyl lecithin, egg lecithin-cholesterol (1 : 1) and dipalmitoyl lecithin-cholesterol (1 : 1), together with PMR spectra of mitochondrial membranes and their extracted lipids, have been obtained.Carbon-13 nuclear magnetic resonance (CMR) spectra at 25.2 MHz of egg lecithin, egg lecithin-cholesterol (1 : 1) and sphingomyelin, together with CMR spectra of chloroplast and mitochondrial membranes, and erythrocyte ghosts, have also been obtained. The results obtained using CMR appear very promising for further study of intact membrane structure.It is suggested, on the basis of CMR evidence, that the proteins in mitochondrial membranes may be considerably less mobile than the lipids.     

Carbon-13 nuclear magnetic resonance studies of myocardial glycogen metabolism in live guinea pigs

Myocardial glycogen metabolism was studied in live guinea pigs by 13C NMR at 20.19 MHz. Open-chest surgery was used to expose the heart, which was then positioned within a solenoidal radio frequency coil for NMR measurements. The time course of myocardial glycogen synthesis during 1-h infusions of 0.5 g of D-[1-13C]glucose (and insulin) into the jugular vein was investigated. The possible turnover of the 13C-labeled glycogen was also studied in vivo by following the labeled glucose infusion with a similar infusion of unlabeled glucose. The degree of 13C enrichment of the C-1 glycogen carbons during these infusions was measured in heart extracts by 1H NMR at 360 MHz. High-quality proton-decoupled 13C NMR spectra of the labeled C-1 carbons of myocardial glycogen in vivo were obtained in 1 min of data accumulation. This time resolution allowed measurement of the time course of glycogenolysis of the 13C-labeled glycogen during anoxia by 13C NMR in vivo. With the solenoidal coil used for 13C NMR, the spin-lattice relaxation time of the labeled C-1 carbons of myocardial glycogen could be measured in vivo. For a comparison, spin-lattice relaxation times of heart glycogen were measured in vitro at 90.55 MHz. Natural abundance 13C NMR studies of the quantitative hydrolysis of extracted heart glycogen in vitro at 90.55 MHz showed that virtually all the carbons in heart glycogen contribute to the 13C NMR signals. The same result was obtained in 13C NMR studies of glycogen hydrolysis in excised guinea pig heart.     

Nuclear magnetic resonance investigation of the helix in random coil transformation in poly-alpha-amino acids. I

High-resolution nuclear magnetic resonance spectra at 100 MHz and 220 MHz have been obtained on two samples of poly-L -alanine of differing molecular weights (2500 and 42 500) in the chloroform–trifluoroacetic acid system under various conditions of solvent composition, temperature, and polypeptide concentration. Separate helix and random coil peaks are observed for the α-CH and peptide NH backbone proton resonances, thereby permitting the determination of helix content. This observation of separate peaks demonstrates that the lifetimes of the helix and random coil portions of poly-L -alanine have lower limits of about 10^?1 sec. It is suggested that solvent–peptide versus peptide–peptide hydrogen bond competition, coupled with a destabilizing effect of the trifluoroacetic acid on the helix, is responsible for the helix–random coil transformation.     

Radioimmunotherapy of human lymphoma in athymic, nude mice as monitored by 31P nuclear magnetic resonance

Human B cell lymphoma (Raji) growing in athymic, nude mice has been successfully treated with a single pulse dose of 131I-labeled monoclonal antibody (Lym-1) specific for this tumor. Sequential in vivo measurements of phosphate metabolites in the tumors by 31P surface coil nuclear magnetic resonance showed a significant initial decrease of phosphocreatine following radioimmunotherapy. Diminution of relative ATP to Pi peak area ratio suggesting tissue damage occurred within 3-4 days. The contribution from metabolites resonating at ca 3.8 ppm (putative sugar phosphate region) increased. There was no significant change in pH either as a function of tumor volume or treatment. The sequence of alterations of nuclear magnetic resonance spectra from tumors of treated mice were strikingly different from sequential nuclear magnetic resonance spectra obtained from tumors of control mice. These observations lead us to conclude that 31P surface coil nuclear magnetic resonance is a promising non-invasive method for assessing and predicting the efficacy of radioimmunotherapy. Further spatial discrimination of the region of tissue observed by the surface coil nuclear magnetic resonance experiment is under exploration in an effort to increase the utility of these methods.     

Structural elucidation of 4'-epiadriamycin by nuclear magnetic resonance spectroscopy and comparison with adriamycin and daunomycin using quantum mechanical and restrained molecular dynamics approach

The structural and electronic properties of 4′-epiadriamycin, adriamycin, and daunomycin have been studied using density functional theory (DFT) employing B3LYP exchange correlation. The chemical shifts of ¹H and ¹³C resonances in nuclear magnetic resonance spectra have been calculated using Gauge-Invariant Atomic Orbital (GIAO) method as implemented in Gaussian 98 and compared with experimental spectra recorded at 500 MHz. ¹³C resonances of drugs have been assigned for the first time. A restrained molecular dynamics approach was used to get the optimized solution structure of drugs using inter-proton distance constraints obtained from 2D NOESY spectra. The glycosidic angle C7-O7-C1′-C2′ is found to show considerable flexibility by adopting 156°-161° (I), 142°-143° (II), and 38°-78° (III) conformations, of which the biological relevant structure appears to be the conformer II. The observed different conformations of the three drugs are correlated to the differential anticancer activity and the available biochemical evidence exhibited by these drugs.     

Qualitative aspects of hydrogen-deuterium exchange in the 1H, 13C, and 15N nuclear magnetic resonance spectra of viomycin in aqueous solution.

The 1H, 13C, and 15N high field nuclear magnetic resonance spectra of the cyclic peptide viomycin have been fully assigned using homo- and heteronuclear double resonance experiments and pH effects. In addition it is shown how the two- and three-bond H-D isotope effects upon carbonyl resonances may assist in their assignment. The resistance to exchange with solvent water of the amide proton involved in the transannular hydrogen bond is observed directly in the 1H spectra, via the isotope effect on a carbonyl resonance in the 13C spectra, and via the one-bond 1H couppling in the 15N spectra.     

A carbon-13 magnetic resonance study of the helix-coil transition in polyuridylic acid

The temperature-dependent conformations of poly(U) in 0.5M CsC1 have been studied by carbon-13 nuclear magnetic resonance. The transition from random coil to an ordered structure results in broadening of lines in the ¹³C spectra, due to intramolecular ¹H–¹³C dipolar interactions and restricted motions in the ordered state. Changes in the chemical shifts suggest that the bases are interacting below the transition temperature. The random coil form shows conformation preferences for internal rotation about C4′–C5′, C5′–O5′, and C3′–O 3′ bonds. The statistical randomness of the coil arises mainly because of flexibility about O–P bonds. The results are analyzed in conjunction with theoretical calculations and light-scattering data.     

Comparison of membrane organization in mitochondria from yeast and rat liver by nuclear magnetic resonance spectroscopy

Summary Proton magnetic resonance (PMR) and carbon-13 magnetic resonance (CMR) spectra of intact, unsonicated yeast and rat liver motochondria show differences which may be correlated with the composition of the membranes. High resolution PMR and CMR signals in intact yeast mitochondria have been assigned to regions of fluid lipid-lipid interaction on the basis of spectra of extracted lipid and protein, and the temperature dependence of NMR signals from the intact membrane. PMR spectra suggest that about 20% of total yeast phospholipid is in regions where both intramolecular fatty acid chain mobility and lateral diffusion of entire phospholipid molecules are possible. No such regions appear to exist in rat liver mitochondria. For both yeast and rat liver mitochondria, comparison of PMR and CMR spectra suggests that about 50% of phospholipid appears to be in regions where intramolecular fatty acid chain motion is considerable, but lateral diffusion is restricted. The remaining phospholipid appears to have little inter- or intramolecular mobility. Since NMR observation of lipid extracts from membranes indicates that phospholipid-sterol interactions do not account for the spectra of intact mitochondria, these effects are interpreted in terms of extensive lipid-protein interactions.     

Quantitation of model digestive mixtures by 13C NMR.

13C nuclear magnetic resonance (NMR) spectra were obtained at 50.3 and 100.5 MHz for methanolic and aqueous mixtures of sodium taurocholate, 1-monocapryloyl-rac-glycerol, and caprylic acid. Distortionless Enhancement by Polarization Transfer (DEPT) was used to improve spectral sensitivity and resolution, and to generate calibration curves for quantitative determinations of each lipid in methanol. Alternatively, the heights for nonoverlapping peaks in a 13C NMR spectrum acquired with inverse-gated decoupling provide reliable quantitative estimates for each component of the mixture, particularly when the data are obtained in methanol. These experiments also demonstrate the feasibility of detailed NMR structural investigations in model systems for glyceride digestion.     

500 MHz NMR characterization of synthetic bombesin and related peptides in DMSO-d6 by two-dimensional techniques

The proton NMR characterization of bombesin has been carried out at 500 MHz in DMSO-d6 using two-dimensional homo- and 1H-13C hetero-correlated techniques. All resonances in the NMR spectra have been assigned and several coupling constants have been measured. The backbone J alpha CH-NH coupling constants have constant values that vary between 7.8 and 8.2 Hz and indicate an unfolded structure in DMSO-d6. Discrepancies with data recently obtained at 300 MHz [(1987) Eur. J. Biochem. 168, 193-199] are discussed.     

Dihydrofolate reductase. 1H resonance assignments and coenzyme-induced conformational changes

Lactobacillus casei dihydrofolate reductase has been studied in solution by one and two-dimensional 1H nuclear magnetic resonance (n.m.r.) spectroscopy at 500 MHz. By using a combination of n.m.r. methods in conjunction with the crystal structure of the enzyme-methotrexate-NADPH complex, resonances have been assigned for 32 of the 162 residues of the enzyme. These are widely distributed throughout the structure of the protein, and include all the histidine and tyrosine residues, as well as several valine, leucine, isoleucine and phenylalanine residues. The assignments have been made for the enzyme-methotrexate and enzyme-methotrexate-NADP+ complexes as well as the enzyme-methotrexate-NADPH complex. Comparison of assigned resonances in the spectra of the three complexes has permitted a preliminary assessment of structural differences between them. The beta-sheet "core" of the protein is unaffected by coenzyme binding, but two regions of the structure that undergo coenzyme-induced conformation changes have been identified. These are the loop comprising residues 13 to 23, and alpha-helix C (residues 42 to 49).     

Complete structure of the polysaccharide from Streptococcus sanguis J22

The cell wall polysaccharides of certain oral streptococci such as Streptococcus sanguis strains 34 and J22, although immunologically distinct, act as receptors for the fimbrial lectins of Actinomyces viscosus T14V. We report the complete covalent structure of the polysaccharide from S. sanguis J22 which is composed of a heptasaccharide subunit linked by phosphodiester bonds. The repeating subunit, which contains alpha-GalNAc, alpha-rhamnose, beta-rhamnose, beta-glucose, and beta-galactose all in the pyranoside form and beta-galactofuranose, is compared with the previously published structure of the polysaccharide from strain 34. The structure has been determined almost exclusively by high-resolution nuclear magnetic resonance methods. The 1H and 13C NMR spectra of the polysaccharides from both strains 34 and J22 have been completely assigned. The stereochemistry of pyranosides was assigned from JH-H values determined from phase-sensitive COSY spectra, and acetamido sugars were assigned by correlation of the resonances of the amide 1H with the sugar ring protons. The 13C spectra were assigned by 1H-detected multiple-quantum correlation (HMQC) spectra, and the assignments were confirmed by 1H-detected multiple-bond correlation (HMBC) spectra. The positions of the glycosidic linkages were assigned by detection of three-bond 1H-13C correlation across the glycosidic linkage in the HMBC spectra. The positions of the phosphodiester linkages were determined by splittings observed in the 13C resonances due to 31P coupling and also by 1H-detected 31P correlation spectroscopy.     

Proton nuclear magnetic resonance studies of 8 alpha-N-imidazolylriboflavin in its oxidized and reduced forms

The oxidized and hydroquinone forms of synthetic 8 alpha-N-imidazolylriboflavin have been investigated by proton nuclear magnetic resonance spectroscopy at 360 MHz. Proton resonances due to the imidazole ring, isoalloxazine ring, and ribityl side chain have been assigned on the basis of two-dimensional 1H-1H correlated spectra (COSY), selective decoupling, and nuclear Overhauser effect difference spectra and by comparison of computer-simulated with experimental spectra. The effect of pH on the imidazolyl resonances shows a pKa for the unsubstituted imidazole nitrogen of 6.0 +/- 0.1 for the oxidized form and a value of 7.0 +/- 0.1 for the reduced form, in good agreement with the values obtained from oxidation-reduction potential data in a previous paper [Williamson, G., & Edmondson, D. E. (1985) Biochemistry 24, 7790-7797]. Slow exchange of the flavin 8 alpha-methylene and imidazolyl C(2) protons was observed at pH 6.1 but not at pH values below 4.0 for the oxidized form of the flavin. The reduced form, but not the oxidized form, of the flavin exhibits geminal coupling of the 8 alpha-methylene protons and of the C(1') methylene protons of the ribityl side chain. The magnetic nonequivalence of the protons of these two methylene groups is suggested to result from intermolecular association of the reduced flavin in aqueous solutions at the concentrations required for the spectral experiments.     

Carbon nuclear magnetic resonance spectra of oligosaccharides isolated from human milk and ovarian cyst mucin

Natural abundance Carbon-13 nuclear magnetic resonance spectra at 20 MHz were reported for the three common human milk oligosaccharides, lacto-N-tetraose and lacto-N-fucopentaoses I and II, as well as for two related tetra- and hexasaccharide alditols isolated from the alkaline borohydride degradation products of an ovarian cyst glycoprotein. Spectral assignments made with the help of deuterium-induced shift (DIS), attached proton test (APT), and T1 data indicated some very irregular glycosylation shifts which were attributed to effects of steric crowding and non-nearest-neighbor interactions. Samples as small as 10 mumol of oligosaccharide gave acceptable 20-MHz spectra with the use of a 5-mm probe coil.     

Conformational changes of alamethicin induced by solvent and temperature. A 13C-NMR and circular-dichroism study.

13C nuclear magnetic resonance (NMR) and circular dichroism (CD) have been used for studies on the conformation of alamethicin. The 13C NMR spectrum is assigned with the aid of signals of synthetic partial sequences and selective proton decoupling. The solvent and temperature-dependence of the 13C NMR spectra, T1 measurements and the use of lanthanide-shift reagents allow the differentiation between the amino acids belonging to a rigid alpha-helical portion of the alamethicin sequence and those belonging to a more flexible part. The 13C NMR results are in agreement with results obtained from extended solvent and temperature-dependent CD studies which indicate a highly stabilized nonpolar and intrachenar alpha-helical part. The concentration-dependence of the CD spectrum of alamethicin in a nematic phase revealed aggregation phenomena which might simulate those observed in natural and synthetic membranes. After dissolving alamethicin in aqueous alcohol there is a time-dependence of the ellipticity of the Cotton effects showing a sort of memory effect on the mode of dissolution. Four different conformations can be characterized by CD spectra depending on the solvent and concentration. A model illustrating the dynamic conformations and aggregation phenomena within a membrane is proposed.     

Proton/Deuterium Magnetic Resonance Imaging of Rodents at 9.4T Using Birdcage Coils

The purpose of the present study was to fabricate a volume coil for proton/deuterium magnetic resonance imaging (MRI) in rodents at 9.4 T. Two birdcage radiofrequency (RF) coils have been designed for proton/deuterium MRI: the rungs of two concentric birdcages were azimuthally interleaved with each other for better decoupling, and the two coils were tuned to 400.3 and 61.4 MHz for ¹H/²H resonance at 9.4 T. Compared to a commercially available coil, the proposed ¹H/²H RF coil provides reasonable transmission efficiency and imaging signal-to-noise ratio (SNR); the relationships among imaging parameters such as SNR, voxel size, and deuterium oxide concentrations have been quantitatively studied, and the linear correlation results together with the spectroscopic data in vivo indicate its feasibility in deuterium metabolic imaging (DMI) in vivo. Our study indicates that using the birdcage design for MRI signal excitation combined with surface coil array for signal reception can facilitate DMI investigations more effectively towards future pre-clinical and clinical applications. As a noninvasive method by measuring nonhydrogen nuclear deuterium signals to reflect metabolite information, DMI will feature prominently in future precision medicine through the whole process of diagnosis, treatment, and prognosis. © 2021 Bioelectromagnetics Society.