Contrast agents for nuclear magnetic resonance imaging

Nuclear magnetic resonance imaging relies upon differences in relaxation times for much of its ability to resolve anatomical structures and to detect changes in tissue. The natural differences can be changed by the administration of paramagnetic substances, such as metal complexes and stable organic free radicals, and ferromagnetic materials, such as small particles of magnetite. Detailed studies of the chemistry and biophysics of such substances in the body are required if they are to become safe and effective contrast agents for use in medical NMR imaging.     

Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

Cytochrome bd is a terminal quinol:O₂ oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b₅₅₈, b₅₉₅, and d. The role of heme b₅₉₅ remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b₅₉₅ causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b₅₉₅ and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b₅₉₅, and not that of heme b₅₅₈, controls the pathway(s) by which CO migrates between heme d and the medium.     

Dissociation constants of phenothiazine drugs incorporated in phosphatidylcholine bilayer of small unilamellar vesicles as determined by carbon-13 nuclear magnetic resonance spectrometric titration

The dissociation constants (pK_ms) of the phenothiazine drugs promazine, chlorpromazine, and triflupromazine, incorporated in the phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUV), were investigated by a ¹³C nuclear magnetic resonance (NMR) titration method employing their N-¹³CH₃ (ionizable group) labelled derivatives. Use of the labelled drugs enabled direct observations of the ionization equilibrium of the N-dimethyl group. A second derivative spectrophotometric study proved that 95-98% of the phenothiazine species in the sample solutions (200 μM phenothiazine in the presence of 27 mM PC SUV) were incorporated into the PC bilayer, which simplified the calculation of pK_m values by allowing that the phenothiazines in the aqueous phase could be neglected. The pK_m values were calculated from the chemical shift dependence of the N-dimethyl ¹³C NMR signal on the pH value of sample solutions. The pK_m values obtained were smaller than those measured in aqueous solutions by about one unit. The existence of cholesterol (30 mol%) in the PC bilayer showed little effect on the pK_m values, suggesting that cholesterol in the bilayer does not largely affect the interfacial region where the N-dimethyl group of the incorporated phenothiazines is located. The results offered clear evidence for the pK_m decrease and provided their precise values.     

Characterization of the AT180 epitope of phosphorylated Tau protein by a combined nuclear magnetic resonance and fluorescence spectroscopy approach

We present here the characterization of the epitope recognized by the AT180 monoclonal antibody currently used to define an Alzheimer’s disease (AD)-related pathological form of the phosphorylated Tau protein. Some ambiguity remains as to the exact phospho-residue(s) recognized by this monoclonal: pThr231 or both pThr231 and pSer235. To answer this question, we have used a combination of nuclear magnetic resonance (NMR) and fluorescence spectroscopy to characterize in a qualitative and quantitative manner the phospho-residue(s) essential for the epitope recognition. Data from the first step of NMR experiments are used to map the residues bound by the antibodies, which were found to be limited to a few residues. A fluorophore is then chemically attached to a cystein residue introduced close-by the mapped epitope, at arginine 221, by mutagenesis of the recombinant protein. The second step of Förster resonance energy transfer (FRET) between the AT180 antibody tryptophanes and the phospho-Tau protein fluorophore allows to calculate a dissociation constant Kd of 30 nM. We show that the sole pThr231 is necessary for the AT180 recognition of phospho-Tau and that phosphorylation of Ser235 does not interfere with the binding.     

Phosphorus-31 nuclear magnetic resonance spectroscopy of human retinoblastoma cells: correlations with metabolic indices

The ³¹P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by ³¹P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1,2 and 3 days post feeding showed a 40% decrese in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.     

Primary metabolism in N2-fixing Alnus incana-Frankia symbiotic root nodules studied with 15N and 31P nuclear magnetic resonance spectroscopy

The primary nitrogen metabolism of the N₂-fixing root nodule symbiosis Alnus incana (L.)–Frankia was investigated by ³¹P and ¹⁵N nuclear magnetic resonance (NMR) spectroscopy. Perfusion of root nodules in a pulse–chase approach with ¹⁵N- or ¹⁴N-labeled NH₄⁺ revealed the presence of the amino acids alanine (Ala), -amino butyric acid, glutamine (Gln), glutamic acid (Glu), citrulline (Cit) and arginine (Arg). Labeling kinetics of the Gln amide-N and -amino acids suggested that the glutamine synthetase (GS; EC 6.3.1.2)–glutamate synthase (GOGAT; EC 1.4.1.13) pathway was active. Inhibition of the GS-catalyzed reaction by methionine sulphoximine abolished incorporation of ¹⁵N. Cit was labeled in all three N positions but most rapidly in the position, consistent with carbamoyl phosphate as the precursor to which Gln could be the amino donor catalyzed by carbamoyl phosphate synthase (CPS; EC 6.3.5.5). Ala biosynthesis occurred consistent with a flux of N in the sequence Gln–Glu–Ala. ³¹P NMR spectroscopy in vivo and of extracts revealed several metabolites and was used in connection with the ¹⁵N pulse–chase experiment to assess general metabolic status. Stable concentrations of ATP and UDP-glucose during extended perfusions showed that the overall root nodule metabolism appeared undisturbed throughout the experiments. The metabolic pathways suggested by the NMR results were confirmed by high activities of the enzymes GS, NADH-GOGAT and ornithine carbamoyltransferase (OCT; EC 2.1.3.3). We conclude that the primary pathway of NH₄⁺ assimilation in A. incana root nodules occurs through the GS–GOGAT pathway. Biosynthesis of Cit through GS–CPS–OCT is important and is a link between the first amino acid Gln and this final transport and storage form of nitrogen.Abbreviations AlaDH l-Alanine dehydrogenase - Cit Citrulline - CPS Carbamoyl phosphate synthase - GABA -Amino butyric acid - GOGAT Glutamate synthase - GS Glutamine synthetase - MDH Malate dehydrogenase - MSO Methionine sulphoximine - NMR Nuclear magnetic resonance - OCT Ornithine carbamoyltransferase - PEPC Phosphoenolpyruvate decarboxylase - UDPGlc Uridine 5-diphosphoglucose     

Estimation of binding constants for the substrate and activator of Rhodobacter sphaeroides adenosine 5'-diphosphate-glucose pyrophosphorylase using affinity capillary electrophoresis

Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5'-triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5'-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using affinity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buffer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a approximately 15-fold decrease in apparent affinity for F6P compared to that of WT while ACE indicated an affinity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent affinity for ATP in the presence of F6P. The ACE results were in good agreement with the activity assay data, confirming the increased affinity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study different binding sites/ligand interactions in allosteric enzymes.     

39K nuclear magnetic resonance and a mathematical model of K+ transport in human erythrocytes

³⁹K nuclear magnetic resonance was used to measure the efflux of K⁺ from suspensions of human erythrocytes [red blood cells (RBCs)], that occurred in response to the calcium ionophore, A23187 and calcium ions; the latter activate the Gárdos channel. Signals from the intra- and extracellular populations of ³⁹K⁺ were selected on the basis of their longitudinal relaxation times, T ₁, by using an inversion- recovery pulse sequence with the mixing time, τ₁, chosen to null one or other of the signals. Changes in RBC volume consequent upon efflux of the ions also changed the T ₁ values so a new theory was implemented to obviate a potential artefact in the data analysis. The velocity of the K⁺ efflux mediated by the Gárdos channel was 1.19±0.40 mmol (L RBC)⁻¹ min⁻¹ at 37°C.     

Characterisation of germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy

Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time (T₂) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T₂ data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.     

Nuclear magnetic resonance studies of Ba1 bacterium and some model systems

Lithium NMR relaxation times of some model systems and E. coli cells in high LiCl concentration were measured. The lithium NMR relaxation times were compared to the relaxation times in the holotolerant bacterium Ba₁ (Goldberg, M., Risk, M. and Gilboa, H. (1983) Biochim. Biophys. Acta 763, 35–40). Complementary studies of the water protons NMR relaxation times were carried out. It is suggested that the lithium in the H.S. Ba₁ bacterium is occulated in small pores of the cell envelope.     

Kinetic analysis of plasminogen activator inhibitor type-2: urokinase complex formation and subsequent internalisation by carcinoma cell lines

The overexpression of urokinase (uPA), which plays a key role in tumour invasion and metastasis, is an established prognostic marker and potential therapeutic target. Plasminogen activator inhibitor type 2 (PAI-2), an efficient and specific inhibitor of uPA, has been shown to selectively deliver potent cytotoxins to tumour cells. However, a direct quantitative analysis of both the inhibition kinetics and subsequent fate of PAI-2 upon interaction with cell-surface uPA has not been previously undertaken. In this study, we analysed specific PAI-2 binding to receptor-bound uPA on human breast and prostate cancer cell lines to directly measure inhibition kinetics. Cell-surface uPA:PAI-2 complex formation, which is reflective of complete uPA inhibition, was found to be very efficient (inactivation constant [K(I)] = 60-80 pM, depending on cell line used) and rapid (inactivation rate constant [k(inact)] = 0.32-0.47 min(-1) at 37 degrees C, depending on cell line used). To directly quantify and visualise cellular internalisation and localisation, we developed a novel assay based on the use of PAI-2 labelled with Alexa(488) fluorochrome and a polyclonal antibody to quench Alexa(488) fluorescence. The efficient and rapid formation of uPA:PAI-2 complexes was thus shown to be associated with specific and rapid internalisation of PAI-2, which could be localised within endosomes and lysosomes. PAI-2 was subsequently degraded, presumably within lysosomes. This study is the first to provide definitive evidence for uPA/uPAR-mediated PAI-2 endocytosis.     

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.     

Acute biochemical effects of La(NO3)3 on liver and kidney tissues by magic-angle spinning 1H nuclear magnetic resonance spectroscopy and pattern recognition

High-resolution magic-angle spinning (MAS) 1H nuclear magnetic resonance (NMR) spectroscopic and pattern recognition (PR) based methods have been applied to studies on the acute biochemical effects of La(NO3)3 on rats. Male Wistar rats were treated with various doses of La(NO3)3 (2, 10, and 50 mg/kg body weight), and MAS 1H NMR spectra of their intact liver and kidney tissues were analyzed using principal components analysis to extract metabolic information. The biochemical effects of La(NO3)3 were characterized by the increase of triglyceride and bile acid and the decrease of glycogen in liver tissue, together with a slight elevation of triglyceride level in kidney tissue. The target lesion of La(NO3)3 to liver was found by MAS NMR-PR methods. This study illustrated the power of the combination of MAS 1H NMR and pattern recognition for the analysis of biochemical effects of rare earths.     

Oxygenated complex of cytochrome bd from Escherichia coli: stability and photolability

Cytochrome bd is one of the two terminal ubiquinol oxidases in the respiratory chain of Escherichia coli catalyzing reduction of O2 to H2O. The enzyme is expressed under low oxygen tension; due to high affinity for O2 it is isolated mainly as a stable oxygenated complex. Direct measurement of O2 binding to heme d in the one-electron reduced isolated enzyme gives K(d(O2)) of approximately 280 nM. It is possible to photolyse the heme d oxy-complex by illumination of the enzyme for several minutes under microaerobic conditions; the light-induced difference absorption spectrum is virtually identical to the inverted spectrum of O2 binding to heme d.     

Metabolomic analysis of Strychnos nux-vomica, Strychnos icaja and Strychnos ignatii extracts by 1H nuclear magnetic resonance spectrometry and multivariate analysis techniques

1H Nuclear magnetic resonance spectrometry and multivariate analysis techniques were applied for the metabolic profiling of three Strychnos species: Strychnos nux-vomica (seeds, stem bark, root bark), Strychnos ignatii (seeds), and Strychnos icaja (leaves, stem bark, root bark, collar bark). The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between all samples, using the three first components. The key compounds responsible for the discrimination were brucine, loganin, fatty acids, and Strychnos icaja alkaloids such as icajine and sungucine. The method was then applied to the classification of several "false angostura" samples. These samples were, as expected, identified as S. nux-vomica by PCA, but could not be clearly discriminated as root bark or stem bark samples after further statistical analysis.     

A non-invasive measurement of phloem and xylem water flow in castor bean seedlings by nuclear magnetic resonance microimaging

A flow-sensitive nuclear magnetic resonance (NMR) microimaging technique was applied to measure directly the in-vivo water flow in 6-d-old castor bean seedlings. The achieved in-plane resolution of the technique allowed discrimination between xylem and phloem water flow. Both the xylem- and the phloem-average flow velocities in the intact seedling could be quantified. Furthermore, the total conductive cross-sectional area of the xylem vessels and the phloem sieve elements could be determined using the non-invasive and non-destructive NMR microimaging technique. Hence, it was possible to calculate the in-vivo volume flow rates for both xylem and phloem water flow. Our non-destructive technique showed that previously used methods to measure phloem water flow affected the flow rate itself. In the intact seedlings we found values of 16.6 l·h^–1, two fold lower than those previously estimated from phloem exudation rates. Finally, our results demonstrate for the first time that water is internally circulated between phloem and xylem, and that water flow within the xylem is maintained by this internally circulated water, even in the absence of any significant transpiration or evaporation.Abbreviation NMR nuclear magnetic resonance     

Comparison of warfarin sensitivity between rat and bird species

Scattering coumarin derivative rodenticides in broad areas have caused primary- and secondary-poisoning incidents in non-target wild birds. In this study, we compared factors determining warfarin sensitivity between bird species and rats based on vitamin K 2,3-epoxide reductase (VKOR) kinetics, VKOR inhibition by warfarin and warfarin metabolism assays. In VKOR characterization, chickens and ostriches showed significantly lower enzymatic efficiencies than rats (one-sixth and one-third, respectively), suggesting bird species depend more on a non-VKOR vitamin K source. On the other hand, the inhibition constants (K_i) of VKOR for warfarin were significantly different between chickens and ostriches (11.3 ± 2.5 μM and 0.64 ± 0.39 μM, respectively). Interestingly, the ostrich K_i was similar to the values for rats (0.28 ± 0.09 μM). The K_i results reveal a surprising possibility that VKOR in some bird species are easily inhibited by warfarin. Warfarin metabolism assays also showed a large inter-species difference in bird species. Chickens and ostriches showed higher metabolic activity than that of rats, while mallards and owls showed only a slight ability to metabolize warfarin. In this study, we clarified the wide inter-species difference that exists among birds in xenobiotic metabolism and sensitivity to a rodenticide.     

High-resolution nuclear magnetic resonance spectroscopy studies of polysaccharides crosslinked by sodium trimetaphosphate: a proposal for the reaction mechanism

An NMR spectroscopy study ((31)P, (1)H, (13)C) of the postulated crosslinking mechanism of sodium trimetaphosphate (STMP) on polysaccharides is reported using methyl alpha-D-glucopyranoside as a model. In a first step, reaction of STMP with Glc-OMe gives grafted sodium tripolyphosphate (STPP(g)). On the one hand, STTP(g) can react with a second alcohol functionality to give a crosslinked monophosphate. On the other hand, a monophosphate (grafted phosphate) could be obtained by alkaline degradation of STPP(g). NMR spectroscopy allows to detect the various species formed and to obtain the crosslinking density of STMP-polysaccharides hydrogels.     

Determination of dissociation constants of (4RS)-[4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid] in water and biological fluids by means of nuclear magnetic resonance spectroscopy and the DISCO software package

The pK(A) values of (4RS)-[4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid] (BOPTA), a polyprotic molecule whose gadolinium complex is an important magnetic resonance imaging contrast agent for clinical use, have been determined in water, in physiologic solution (PS), in serum (S), and in cerebrospinal fluid (CSF), by means of 13C nuclear magnetic resonance spectroscopy data processed by a dedicated software package called DISCO. The aim of this study was to supply the BOPTA pK(A) values in media very similar to the in vivo environment and, consequently, to get a picture of the in vivo behavior of its Gd complex, whose thermodynamic stability is directly linked to the pK(A) values. The pK(A) values appeared to be almost equal both in D(2)O and in PS, while pK(1) and pK(5) values in CSF differ a little. In S, only pK(2) and pK(3) were calculated due to the narrow pH range used for data collection. However, these pK(A) values were found equal to those in the other media. These results represent the first direct spectroscopic evidence of a substantial invariability of BOPTA behavior in different media and they justify the extrapolation to biological fluids of the data obtained in water. The values also confirmed the high-quality performance of DISCO in calculating pK(A) values of polyprotic molecules in complex media.     

Metabolic-flux analysis of continuously cultured hybridoma cells using (13)CO(2) mass spectrometry in combination with (13)C-lactate nuclear magnetic resonance spectroscopy and metabolite balancing

Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO(2), and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste products derived from glucose. Continuously cultured hybridoma cells are grown in medium containing either 1-(13)C-, 2-(13)C-, or 6-(13)C-glucose. The uptake and production rates of amino acids, glucose, ammonia, O(2), and CO(2) as well as the cellular composition are measured. In addition, the (13)C distribution of the lactate produced and alanine produced by the hybridomas is determined by (1)H-NMR spectroscopy, and the (13)CO(2)/(12)CO(2) ratio is measured by on-line mass spectrometry. These data are used to calculate the intracellular fluxes of the glycolysis, the pentose phosphate pathway, the TCA cycle, and fluxes involved in amino acid metabolism. It is shown that: (i) approximately 20% of the glucose consumed is channeled through the pentose shunt; (ii) the glycolysis pathway contributes the most to lactate production, and most of the CO(2) is produced by the TCA cycle; (iii) the pyruvate-carboxylase flux is negligibly small; and (iv) the malic-enzyme flux is estimated to be 10% of the glucose uptake rate. Based on these flux data suggestions are made to engineer a more efficient glucose metabolism in mammalian cells.