Glucose transport in human erythrocytes measured using 13C NMR spin transfer

We present the results of a new NMR-based procedure for measuring the fast transmembrane exchange of D-[1-13C]glucose in human erythrocytes. The method relies on different rates of exchange between the alpha- and beta-anomers of glucose inside and outside the cells; the rate outside the cells is greatly increased by the addition of mutarotase to the suspension. Theory is developed to describe nuclear-spin transfer in the present system and is used to analyse the data to yield estimates of transmembrane-exchange rate constants and their statistical uncertainties. For a total glucose concentration of 25.5 mmol/l at 40 degrees C the first order efflux rate constants for the alpha- and beta-anomers were 1.20 +/- 0.40 s-1 and 0.71 +/- 0.30 s-1, respectively.     

Ferrocyanide carbon-13 NMR line broadening as a probe of electron transfer reactions

The electron transfer reaction between ferrocyanide ion and the blue copper protein, stellacyanin, has been investigated by means of 13C NMR line broadening of the inorganic oxidant. The temperature dependence of the ferrocyanide line broadening gives an activation energy for the electron transfer reaction of 17 +/- 3 kJ. The apparent rate constant decreases with increasing concentration of K4Fe(CN)6, a result which can be explained either by formation of a strong precursor ferrocyanide--stellacyanin [Cu(II)] complex or by increased formation of KFe(CN)3-6 ion pairs. The direct electron transfer between ferrocyanide and ferricyanide has also been studied by 13C NMR line broadening of the former species. The ferricyanide concentration dependence of the exchange line broadening yields a value for the apparent second-order rate constant at 25 degrees C of k = 1.65 . 10(3) M-1 . s-1, in agreement with previously reported values derived from 14N NMR and isotope exchange studies. This rate constant shows a linear dependence on the K+ concentration, independent of ionic strength, a result which confirms the importance of ion pair species such as KFe(CN)3-6 and KFe(CN)2-6 in the direct electron transfer mechanism. The general applications of the method are discussed, including the considerations which suggest that a wide range of electron transfer rates, from about 1 s-1 to 4 . 10(3) s-1, are, in principle, accessible to this technique. The potential utility of ferrocyanide 13C spin--lattice relaxation time measurements is decreasing the lower limit of this range is also discussed.     

Carbon-13 NMR spectra of carbon-13 enriched chlorophylls a and b

The proton decoupled ¹³C NMR (CMR) spectra of chlorophylls a and b enriched to 90% ¹³C have been obtained at 25.2 MHz and, despite the complexity of the spectra, many of the assignments of the ¹³C resonances have been made.     

The soluble components of chromaffin granules a carbon-13 NMR survey

Carbon-13 NMR spectra of the reconcentrated chromaffin granule lysate have been obtained at 50 MHz and 62.9 MHz. The spectrum contains a number of assignable resonances in addition to those of the main soluble components (catecholamines, adenine nucleotides and chromogranin). Guanine and uridine nucleotides are present at levels of 0.13 and 0.08 mol/mol adenine nucleotides, respectively. Concentrations of cytidine nucleotides and NAD⁺ are below the detection limit (0.02 mol/mol adenine nucleotides). An unidentified low molecular weight species, thought to be an adenine-containing oligonucleotide, is also present. Ascorbic acid was observed at a concentration of 0.14 mol/mol adenine nucleotides, but both dopamine and dehydroascorbic acid were below the detection limit. Protein resonances agree well with the reported amino acid composition of chromogranin A, with the exception of tryptophan and glutamine which have not previously been measured. The concentrations of these residues are estimated to be 12 ± 3 and 39 ± 5 residues per 77 000 dalton unit of chromogranin A. Substantial intensity due to unsaturated fatty acid side-chains in solubilized lipid is seen in the olefinic carbon region and in the methylene region, suggesting the presence of lipoprotein. Unassigned carbohydrate resonances are also present, but are largely obscured by sucrose in the isolation medium.     

Rates of decay in wood measured by carbon dioxide production

The applicability of two methods of respirometry to measurement of the carbon dioxide output of naturally decaying branches and wood of standing trees was studied. The Warburg respirometer was judged unsuitable for general use on decaying wood. A conductivity respirometer was found satisfactory. Carbon dioxide production was essentially unaffected by fragmentation suggesting that the measurements obtained are likely to be a valuable indication of decay in the intact tree or branch. The carbon dioxide production of samples was fairly stable when conditions were kept constant but responded promptly to increased or decreased moisture. Wood from branches infested with Polyporus tulipiferae in which moisture was increased from approximately 20%–50% (fresh weight basis) increased its carbon dioxide output over a 4-day period by some seven times. Comparable wood in which the moisture content was reduced from 45 to 20 % showed an almost linear reduction in rate over a similar period to about one-sixth the original rate. Rate of decay in stained and unstained zones of living trees showed no consistent effect of the stain. However, rates of decay in heart-rot of poplar caused by Fomes igniarius were only one-third those reported by Verrall (1937) for decay in culture.     

Fluoride transmembrane exchange in human erythrocytes measured with 19F NMR magnetization transfer

¹⁹F NMR spectra of sodium fluoride in suspensions of human erythrocytes were seen to yield separate resonances for the F^- populations inside and outside the cells. Selective saturation of the magnetization of the intracellular population gave rise to transfer of that saturation to the extracellular population. The extent of magnetization transfer was high and it was blocked by the capnophorin (band 3) anion exchange inhibitor 4,4-dini-trostilbene-2,2-disulfonic acid (DNDS). A series of magnetization-inversion transfer experiments was carried out for the range of intracellular fluoride concentrations of 11 mM to 136 mM and analysed using one-dimensional overdetermined exchange analysis. This yielded an estimate of the equilibrium exchange Michaelis constant and maximal velocity of 27 ± 3 mM and 180 ± 5 × 10^-16 mol cell^-1 s^-1, respectively. There was no alteration of exchange flux of fluoride at an intracellular concentration of 49 mM in the presence of 50 mM glucose; thus suggesting no interaction between glucose and anions in capnophorin-mediated exchange of solutes.     

Chemical properties and stereoisomerism of heterogeneous long chain bases in lysosphingolipids by positive ion fast atom bombardment mass spectrometry and carbon-13 NMR spectroscopy

Positive ion fast atom bombardment (FAB) mass spectrometry of galactopsychosine and glucopsychosine was capable of showing not only the pseudo molecular ion peaks, but also various fragment ion peaks such as protonated sphingosine and its fragment ions. The percent distribution of sphingosine and dihydrosphingosine in each lysosphingolipid was determined by GLC of the trimethyl-silylated derivatives of long chain bases after methanolysis and was comparable to the relative intensities of ion peaks derived from the sphingosine and dihydrosphingosine groups. The FAB mass spectra showed that during the fast atom bombardment the sphingosine more preferentially gave rise to one and/or two fragment ions by loss of one and/or two molecules of water than the dihydrosphingosine did. The stereoisomerism of sphingosylphosphorylcholine containing mainly L-threo-sphingosine could be reconfirmed by carbon-13 NMR spectroscopy. Furthermore, although the carbon-13 NMR signals of sphingosine C-1, C-2, C-3, C-4, and C-5 showed significant chemical shift differences between D-erythro and L-threo-sphingosines of lysosphingolipids, it was concluded that the signal position of sphingosine C-3 was the most important for the determination of D-erythro and L-threo configuration in the long chain base moieties of lysosphingolipids.     

One-dimensional 13C NMR and HPLC-1H NMR techniques for observing carbon-13 and deuterium labelling in biosynthetic studies

For several decades isotope labelling techniques have been the indispensable tools used to unravel pathways of secondary product biosynthesis. NMR spectroscopy, together with mass spectrometry, is the most effective measuring technique used in the analysis of metabolites enriched with stable isotopes. ²H and ¹³C are the NMR-detectable nuclides which have been most frequently employed in plant secondary metabolite synthesis. Examples from the biosynthesis of phenylpropanoids, phenylphenalenones, and glucosinolates are used when discussing some aspects of one-dimensional NMR analysis of metabolites selectively labelled with ²H and ¹³C. Besides direct NMR detection of ¹³C-enriched metabolites, special emphasis is placed on indirect detection of ¹³C and ²H, especially by HPLC-¹H NMR coupling, to analyse the isotopomer pattern of compounds in low concentration. The examples discussed in this paper were obtained from studies with Anigozanthos preissii (root cultures) (Haemodoraceae) and Eruca sativa (Brassicaceae).     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Structure and dynamics of porcine submaxillary mucin as determined by natural abundance carbon-13 NMR spectroscopy

Nearly all of the resonances in the 13C NMR spectrum of porcine submaxillary mucin glycoprotein (PSM) have been assigned to the peptide core carbons and to the carbons in the eight different oligosaccharide side chains that arise from the incomplete biosynthesis of the sialylated A blood group pentasaccharide (alpha-GalNAc(1-3) [alpha-Fuc(1-2)]-beta-Gal(1-3) [alpha-NeuNGl(2-6)]- alpha-GalNAc-O-Ser/Thr). By use of these assignments, a nearly complete structural analysis of intact PSM has been performed without resorting to degradative chemical methods. Considerable structural variability in the carbohydrate side chains was observed between mucins obtained from different animals, while no variability was observed between glands in a single animal. The dynamics of the PSM core and carbohydrate side chains were examined by using the carbon-13 nuclear magnetic resonance relaxation times and nuclear Overhauser enhancements of each assigned carbon resonance. The peptide core of PSM exhibits internal segmental flexibility that is virtually identical with that of ovine submaxillary mucin (OSM), whose carbohydrate side chain consists of the alpha-NeuNAc(2-6)alpha-GalNAc disaccharide. The longer oligosaccharide side chains of PSM, therefore, have no significant effect on peptide core mobility compared to the shorter side chains of native OSM or asialo-OSM. Although the dynamics of the shorter carbohydrate side chains shared by both OSM and PSM appear to be identical, the A and H blood group structures in PSM have reduced mobilities, indicating that the glycosidic linkages of the terminal sugars in these determinants are relatively inflexible. These results differ from most reports of glycoprotein dynamics, which typically find the terminal carbohydrate residues to be undergoing rapid internal rotation about their terminal glycosidic bonds. The results reported here are consistent with previous studies on the conformations of the A and H determinants derived from model oligosaccharides and further indicate that the conformations of these determinants are unchanged when covalently bound to the mucin peptide core. In spite of their carbohydrate side-chain heterogeneity, mucins appear to be ideal glycoproteins for the study of O-linked oligosaccharide conformation and dynamics and for the study of the effects of glycosylation on polypeptide conformation and dynamics.     

A complete proton and carbon-13 NMR analysis of fluocinonide, a fluorinated corticosteroid

This paper presents a complete analysis of the proton and carbon-13 NMR spectra of 21-acetoxy-6 alpha,9-difluoro-11 beta-hydroxy-16 alpha,17-(1-methylethylidene) bis-(oxy) pregna-1,4-diene-3,20-dione, a potent anti-inflammatory fluorosteroid. The 300 MHz proton spectrum was analyzed using a combination of the two-dimensional homonuclear chemical shift correlation (COSY) technique and one-dimensional NOE difference spectra. Exact coupling constants and chemical shifts were obtained by spectral simulation and iteration. The carbon-13 spectrum was assigned from the proton spectrum via a two-dimensional heteronuclear chemical shift experiment, and long-range fluorine-proton couplings were confirmed by a fully coupled heteronuclear COSY-type experiment.     

Imazalil-cyclomaltoheptaose (beta-cyclodextrin) inclusion complex: preparation by supercritical carbon dioxide and 13C CPMAS and 1H NMR characterization

An inclusion complex between imazalil (IMZ), a selected fungicide, and cyclomaltoheptaose (beta-cyclodextrin, betaCD) was obtained using supercritical fluid carbon dioxide. The best preparation conditions were determined, and the inclusion complex was investigated by means of 1H NMR spectroscopy in aqueous solution and 13C CPMAS NMR spectroscopy in the solid state. Information on the geometry of the betaCD/IMZ complex was obtained from ROESY spectroscopy, while the dynamics of the inclusion complex in the kilohertz range was obtained from the proton spin-lattice relaxation times in the rotating frame, T(1rho) (1H).     

Temperature- and concentration-dependence of urea permeability of human erythrocytes determined by NMR

The temperature- and concentration-dependence of [13C]urea self-exchange across the human red cell membrane has been determined by NMR measurements of T1 (spin-lattice) relaxation times. T1 for intracellular label is 17 s, which is much longer than the urea exchange time across the cell membrane (about 0.5 s). T1 for urea in extracellular solution is quenched with 17 mM of impermeable Mn2+ in less than 2 ms. Hence the observed T1 (corrected for intracellular decay) is a measure of urea exchange across the cell membrane. The method is tested by showing both PCMBS and increasing concentrations of urea lengthen T1. Urea exchange permeability, defined as Purea = flux/conc, can be described by Purea = Vmax/(K1/2 + conc). Studies of temperature-dependence showed that activation energies were strongly dependent on both temperature and concentration. However, this apparently anomalous behavior was resolved into two well-behaved functions, K1/2 and Vmax, with linear Arrhenius plots and apparent 'activation energies' of 15.5 and 12.4 kcal/mol, respectively. These were used to construct an equation for calculating Purea at any concentration and temperature. Assuming a simple channel model with single binding, K1/2 becomes the dissociation equilibrium constant for the site with delta H degree = 15.5 kcal/mol and delta S degree = 51.8 cal/(mol.deg); dissociation is entropically driven.     

Measurement of oxygen consumption and carbon dioxide production rates by a tracer-bolus method

A new method for measuring oxygen consumption and carbon dioxide production is described. The method is based on the injection of a helium bolus into the inspired gas for repeated breaths; the helium can be delivered through the mouth or through a nostril (the injection system being controlled by the integrated flow signal or by the signal of a thermistor, respectively). Compared with the conventional gas-collection technique, the tracer-bolus method allows more rapid and frequent measurements. The method does not necessarily require an airtight respiratory circuit, so that it can be conveniently applied in less cooperative subjects, including children. The results of validation studies based on a comparison with the standard gas-collection technique are presented; these results indicate that the measurements by the tracer-bolus method are accurate in normal subjects. In patients with obstructive lung disease, the determinations are associated with a systematic error, which is expected, the tracer-bolus method being based on a single-compartment, uniformly ventilated and perfused lung model; the error is, however, predictable from the degree of functional impairment and can be corrected using equations based on routine pulmonary function tests.     

Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces.

Fecal suspensions from humans were incubated with 13CO2 and H2. The suspensions were from subjects who harbored 10(8) and 10(10) methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 10(10) per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and 13CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.     

Characterization of the wound-induced material in Citrus paradisi fruit peel by carbon-13 CP-MAS solid state NMR spectroscopy

Grapefruit, Citrus paradisi, were injured, inoculated with Penicillium digitatum and incubated under conditions favourable for the accumulation of defence related material. Histochemical examination revealed that tissues adjacent to inoculated injuries contained phloroglucinol-HCl (PG-HCl) reactive material. Solvent washed cell wall preparations of intact and injured-inoculated peel were further purified using a mixture of cell wall degrading enzymes. Samples from injured inoculated tissue contained PG-HCl reactive globular material in addition to the fragments of xylem and cuticle found in controls. The principal chemical moieties of the material that accumulates in grapefruit injuries during wound-healing were studied by solid state 13C cross-polarization magic angle spinning NMR. A complete assignment of the NMR signals was made. From the analysis evidence was found that cellulose and hemicellulose are the biopolymers present in the intact peel samples, in addition, relevant quantities of cutin were found in the residues of enzyme digest. The NMR difference spectrum intact- wounded peels showed resonances which were attributed to all major functional groups of the aromatic-aliphatic suberin polyester of new material produced by the wounds. Information on the latter polyester was obtained by analyzing the T(1)rho (1H) relaxation.