Prospects for in vivo NMR methods in xenobiotic research in plants

The application of non-invasive nuclear magnetic resonance (NMR) methods in xenobiotic research is reviewed in relation to: (i) the characterisation of the effects of xenobiotics on the metabolism of plants and plant cell suspensions; (ii) the direct detection of xenobiotics and their degradation products in vivo; and (iii) the spatial localisation of xenobiotics and their derivatives at the subcellular and tissue levels. Novel information has been generated by in vivo NMR studies of both agrochemicals and heavy metals, but a lack of generality in the methods makes it difficult to extrapolate from one successful application to the next. In vivo NMR spectroscopy is shown to be informative when a xenobiotic perturbs metabolic pathways that are accessible to the technique, and it is useful for probing the partitioning of paramagnetic metal ions between the cytoplasm and the vacuole. The successful application of 19F NMR to the analysis of plant tissue extracts also suggests that in vivo 19F NMR spectroscopy may have a role in biotransformation studies of fluorinated xenobiotics. In contrast NMR imaging techniques have been little used for xenobiotic research in plants, and while the method has been shown to be capable of monitoring the uptake and translocation of paramagnetic ions in plants, the potential use of high resolution 1H and 19F NMR imaging for mapping agrochemicals in tissues is still in its infancy.     

Noninvasive In Vivo Demonstration of 2-Fluoro-2-Deoxy-d-Glucose Metabolism Beyond the Hexokinase Reaction in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy

The metabolism of 2-fluoro-2-deoxy-D-glucose (FDG) in vivo was observed noninvasively in rat brain using 19F nuclear magnetic resonance (NMR) spectroscopy following an intravenous injection of FDG (400 mg/kg). At 3 h after infusion, four resonances with discrete chemical shifts were resolved. Chemical shift analysis of these resonances suggested the chemical identity of two of the resonances to be FDG and/or FDG-6-phosphate and 2-fluoro-2-deoxy-delta-phosphogluconolactone and/or 2-fluoro-2-deoxy-6-phosphogluconate. The chemical identities of the other two resonances remain to be elucidated. The present study indicates that the metabolism of FDG in vivo is more extensive than is previously recognized and demonstrates the feasibility of using 19F NMR spectroscopy to follow the 19F-containing metabolites of FDG in vivo.     

MG-63 human osteosarcoma cells grown in monolayer and as three-dimensional tumor spheroids present a different metabolic profile: a (1)H NMR study

High resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy was used to determine if the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as small (about 50-80 microm in diameter), three-dimensional tumor spheroids with no hypoxic center has different metabolic characteristics. Consequently, the (1)H NMR spectra were obtained from both types of cultures and then compared. The results indicate that the type of cellular spatial array determines specific changes in MG-63 cells. In particular, small but significant differences in lactate and alanine indicating a perturbation in energy metabolism were observed in the two cell models. In addition, although variations in CH(2) and CH(3) groups were also seen, it is not possible at this time to establish if lipid metabolism is truly different in cells and spheroids.     

High-resolution mono- and multidimensional magic angle spinning 1H nuclear magnetic resonance of membrane peptides in nondeuterated lipid membranes and H2O.

High-speed (14 kHz) solid-state magic angle spinning (MAS) 1H NMR has been applied to several membrane peptides incorporated into nondeuterated dilauroyl or dimyristoylphosphatidylcholine membranes suspended in H2O. It is shown that solvent suppression methods derived from solution NMR, such as presaturation or jump-return, can be used to reduce water resonance, even at relatively high water content. In addition, regioselective excitation of 1H peptide resonances promotes an efficient suppression of lipid resonances, even in cases where these are initially two orders of magnitude more intense. As a consequence, 1H MAS spectra of the peptide low-field region are obtained without interference from water and lipid signals. These display resonances from amide and other exchangeable 1H as well as from aromatic nonexchangeable 1H. The spectral resolution depends on the specific types of resonance and membrane peptide. For small amphiphilic or hydrophobic oligopeptides, resolution of most individual amide resonance is achieved, whereas for the transmembrane peptide gramicidin A, an unresolved amide spectrum is obtained. Partial resolution of aromatic 1H occurs in all cases. Multidimensional 1H-MAS spectra of membrane peptides can also be obtained by using water suppression and regioselective excitation. For gramicidin A, F2-regioselective 2D nuclear Overhauser effect spectroscopy (NOESY) spectra are dominated by intermolecular through-space connectivities between peptide aromatic or formyl 1H and lipid 1H. These appear to be compatible with the known structure and topography of the gramicidin pore. On the other hand, for the amphiphilic peptide leucine-enkephalin, F2-regioselective NOESY spectra mostly display cross-peaks originating from though-space proximities of amide or aromatic 1H with themselves and with aliphatic 1H. F3-regioselective 3D NOESY-NOESY spectra can be used to obtain through-space correlations within aliphatic 1H. Such intrapeptide proximities should allow determination of the conformation of the peptide in membranes. It is suggested that high-speed MAS multidimensional 1H NMR of peptides in nondeuterated membranes and in H2O can be used for studies of both peptide structure and lipid-peptide interactions.     

H-1, C-13, and F-19 nuclear magnetic resonance assignments of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate.

The molecular structure of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate was analyzed by 1H, 13C, and 19F nuclear magnetic resonance (NMR) techniques; two-dimensional NMR was used to assigned 1H and 13C resonances. The 1H NMR spectrum in deuterated chloroform shows three sharp singlets (delta = 0.74, 0.79, and 2.00 ppm) integrating for three protons each, an isolated triplet at 4.55 ppm integrating for one proton, and overlapping multiplets between 0.72 and 2.12 ppm integrating for 31 protons. The 13C spectrum shows 18 resonances between 10 and 55 ppm, and three additional resonances at 82.9, 124.0, and 171.5 ppm. The 19F[1H] spectrum shows two sets of doublets (observed 2J = 150 Hz) at 5.00 and -4.80 ppm. Multiplets arising from 19F-13C J-coupling provide the starting assignment for all resonances by means of 1H homonuclear correlation (COSY) and 1H-13C heteronuclear correlation spectroscopy.     

Dual 19F/1H MR gene reporter molecules for in vivo detection of β-galactosidase

Increased emphasis on personalized medicine and novel therapies requires the development of noninvasive strategies for assessing biochemistry in vivo. The detection of enzyme activity and gene expression in vivo is potentially important for the characterization of diseases and gene therapy. Magnetic resonance imaging (MRI) is a particularly promising tool, since it is noninvasive and has no associated radioactivity, yet penetrates deep tissue. We now demonstrate a novel class of dual (1)H/(19)F nuclear magnetic resonance (NMR) lacZ gene reporter molecule to specifically reveal enzyme activity in human tumor xenografts growing in mice. We report the design, synthesis, and characterization of six novel molecules and evaluation of the most effective reporter in mice in vivo. Substrates show a single (19)F NMR signal and exposure to β-galactosidase induces a large (19)F NMR chemical shift response. In the presence of ferric ions, the liberated aglycone generates intense proton MRI T(2) contrast. The dual modality approach allows both the detection of substrate and the imaging of product enhancing the confidence in enzyme detection.     

Interactions of diastereomeric tripeptides of lysyl-5-fluorotryptophyllysine with DNA. 1. Optical and 19F NMR studies of native DNA complexes

Lysyl-5-fluoro-L-tryptophyllysine and lysyl-5-fluoro-D-tryptophyllysine were synthesized, and their interactions with double-stranded DNA were investigated as a model for protein-nucleic acid interactions. The binding to DNA was studied by monitoring various 19F NMR parameters, the fluorescence, and the optical absorbance in thermal denaturation. The 19F resonance of the L-Trp peptide shifts upfield in the presence of DNA, and that of the D-Trp peptide shifts downfield with DNA present. The influence of ionic strength on the binding of each peptide to DNA and the fluorescence quenching titration of each with DNA indicate that electrostatic bonding (approximately 2 per peptide-DNA complex) dominates the binding in each case and accounts for the similar binding constants determined from the fluorescence quenching, i.e., 7.7 X 10(4) M-1 for the L-Trp complex and 6.2 X 10(-1) for the D-Trp complex. The 19F NMR chemical shift, line width, 19F[1H] nuclear Overhauser effect, and spin-lattice relaxation time (T1) changes all indicate that the aromatic moiety of the L-Trp complex, but not that of the D-Trp complex, is stacked between the bases of DNA. The relative increases in DNA melting temperature caused by binding of the tripeptide diastereomers are also consistent with stacking in the case of the L-Trp peptide. The magnitude of the changes and the susceptibility of the 19F NMR chemical shift to altering the solvent isotope (H2O vs. D2O) suggest that the L-Trp ring is not intercalated in the classical sense but is partially inserted between the bases of one strand of the double helix.     

1H nuclear magnetic resonance double resonance study of oxytocin in aqueous solution.

Peptide NH resonances in the 250 MHZ 1H nuclear magnetic resonance (NMR) spectrum of oxytocin in H2O were assigned to specific amino acid residues by the "underwater decoupling" technique (i.e., decoupling from corresponding CalphaH resonances, which are buried beneath the intense water peak). These experiments confirm previous assignments of A. I. Brewster an V. J. Hruby ((1973), Proc. Natl. Acad. Sci. U.S.A. 70, 3806) and A. F. Bradbury et al. ((1974), FEBS Lett. 42, 179). Three methods of assigning NH resonances of peptides--solvent titration, underwater decoupling, and isotopic labeling--are compared. As the solvet composition is gradually changed from dimethyl sulfoxide to H2O, oxytocin undergoes a conformational change at 70-90 mol % of H2O. Exposure to solvent of specific hydrogens of oxytocin in H2O was studied by monitoring intensity changes of solute resonances when the solvent peak was saturated. Positive nuclear Overhauser effects (NOE's) of 14 +/- 5 were observed for the Tyr ortho CH and meta CH resonances, respectively. Comparative studies with deamino-oxytocin indicate that these effects result predominantly from intermolecular dipoledipole interaction between aromatic side chain CH protons and protons of the solvent. The NOE's therefore indicate intimate contact between water and the aromatic CH hydrogens of the Tyr side chain. The extent of saturation transferred by proton exchange between water and NH group varies with Ph in a manner which appears to reflect the acid-base catalysis of the protolysis reaction. There is no indication that any NH protons are substantially shiedled from the solvent.     

Synthesis of [2-3H-ethyl]S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine and its use in covalent-binding studies.

Metabolism of S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) yields chlorofluorothioacetyl fluoride, which reacts with cellular proteins to form stable lysine adducts. Little is known about the subcellular localization of these protein adducts or about their role in CTFC-induced nephrotoxicity. A method for the synthesis of CTFC and other cysteine S-conjugates labeled with 3H at the S-alkyl or S-alkenyl position would be useful in studies of S-conjugate metabolism and toxicity. Reaction of L-cysteine, chlorotrifluoroethene, 1,8-diazabicyclo[5.4.0]undec-7-ene, and 3H-labeled water followed by repeated crystallization yielded radiochemically pure [3H]CTFC (235 mg, 20% yield; sp act 1.07 x 10(9) Bq/mmol), which was identical to CTFC by TLC, 1H NMR, and 19F NMR. 3H NMR revealed a doublet of triplets at 6.5 ppm with geminal and vicinal T-F couplings of 51.5 and 6.0 Hz, respectively, consistent with the proposed structure. When 2H-labeled water was used, [2H]CTFC was formed, and its structure was confirmed by 1H and 19F NMR, FAB-MS, and TLC. Analysis of renal and hepatic subcellular fractions of rats given 1, 10, or 100 mumol/kg [3H]CTFC showed a dose-dependent binding of 3H-containing metabolites to liver and kidney proteins.     

Isolation and survey of novel fluoroacetate-degrading bacteria belonging to the phylum Synergistetes

Microbial dehalogenation of chlorinated compounds in anaerobic environments is well known, but the degradation of fluorinated compounds under similar conditions has rarely been described. Here, we report on the isolation of a bovine rumen bacterium that metabolizes fluoroacetate under anaerobic conditions, the mode of degradation and its presence in gut ecosystems. The bacterium was identified using 16S rRNA gene sequence analysis as belonging to the phylum Synergistetes and was designated strain MFA1. Growth was stimulated by amino acids with greater quantities of amino acids metabolized in the presence of fluoroacetate, but sugars were not fermented. Acetate, formate, propionate, isobutryate, isovalerate, ornithine and H(2) were end products of amino acid metabolism. Acetate was the primary end product of fluoroacetate dehalogenation, and the amount produced correlated with the stoichiometric release of fluoride which was confirmed using fluorine nuclear magnetic resonance ((19) F NMR) spectroscopy. Hydrogen and formate produced in situ were consumed during dehalogenation. The growth characteristics of strain MFA1 indicated that the bacterium may gain energy via reductive dehalogenation. This is the first study to identify a bacterium that can anaerobically dehalogenate fluoroacetate. Nested 16S rRNA gene-specific PCR assays detected the bacterium at low numbers in the gut of several herbivore species.     

Noninvasive Demonstration of In Vivo 3-Fluoro-3-Deoxy-D-Glucose Metabolism in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy: Suitable Probe for Monitoring Cerebral Aldose Reductase Activities

The metabolism of 3-fluoro-3-deoxy-D-glucose (3-FDG) in rat brain in vivo was investigated noninvasively using ¹⁹F nuclear magnetic resonance (NMR) Spectroscopy. Following an intravenous infusion of 3-FDG, 400 mg/kg, four resonances assigned to the α and β anomers of 3-FDG, 3-fluoro-3-deoxy-D-sorbitol, and 3-fluoro-3-deoxy-D-fructose were clearly resolved in brain, a result indicating that 3-FDG is metabolized primarily into the aldose reductase sorbitol (ARS) pathway. An orally administered aldose reductase inhibitor, sorbinil, caused reduction of the flux of 3-FDG into the ARS, an observation suggesting that the method can be applied in quantitative studies of ARS path way activities. Studies of 24-h urine specimens showed that in addition to the two metabolites observed in brain, F^-was excreted into the urine. 3-FDG appears to be a suitable metabolic probe for assessing glucose metabolism in the ARS pathway by in vivo ¹⁹F NMR Spectroscopy.     

Phosphorus nuclear magnetic resonance of diverse phosvitin species

1. High resolution 31P nuclear magnetic resonance (NMR) spectra, with and without proton decoupling, of the principal egg phosphoproteins--phosvitins--of a bird (Gallus gallus), an amphibian (Xenopus laevis) and a fish (Salmo gairdneri) were obtained. 2. The spectra were evaluated with special reference to available amino acid sequences and the major NMR resonance in all three spectra was assigned to phosphoserine clusters. 3. The resolution of numerous additional phosphorus resonances provides the basis for further investigation of the particular molecular environments of phosvitin-bound phosphoryl groups and their involvement in the diverse binding modes for metal complex formation by phosvitins.     

Magnetic resonance spectroscopy: a powerful tool for drug metabolism studies.

Studies on the metabolism and disposition of drugs using nuclear magnetic resonance spectroscopy (MRS) as the analytical technique are reviewed. An overview of the main studies classed in terms of the observed magnetic nucleus (1H, 2H, 7Li, 13C, 19F, 31P, 77Se) is followed by some typical examples of the way in which 19F and 31P MRS can be profitably employed to gain more understanding about the metabolism and disposition of the anticancer fluoropyrimidines (5-fluorouracil (FU) and its prodrugs) and ifosfamide (IF). The results of three recent studies carried out in our laboratory are developed. They concern the direct quantitative monitoring of the hepatic metabolism of FU in the isolated perfused mouse liver, the elucidation of the origin of the cardiotoxicity of FU and the metabolism of IF from an analysis of biofluids of patients. Finally, the advantages and limitations of MRS for investigations on drug metabolism are discussed.     

The application of 19F nuclear magnetic resonance to investigate microbial biotransformations of organofluorine compounds

Fluorinated organic compounds, although rare in nature, are significant environmental contaminants owing to the numerous applications for which this class of compounds is employed. It is important that biodegradation of these compounds can be readily assessed in order to provide information on their fate in the environment. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy has emerged as a very useful technique to readily determine the catabolism of fluorinated aromatic compounds by microorganisms, either in whole cell or cell-free systems. The principal advantage of this technique is that fluorinated compounds can be observed directly in the culture supernatant or enzyme assay, without purification or derivatization. In this review an account of the application of 19F NMR in the study of microbial metabolism of organofluorine compounds is presented.     

Adiabatic Heteronuclear Decoupling in Rotating Solids

In magic angle spinning solid state NMR experiments the potential of heteronuclear (1)H decoupling employing a continuous train of adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. It is shown that, with a (1)H RF field strength of approximately 100 kHz that is typically available in MAS NMR probes, it is possible to achieve efficient adiabatic (1)H decoupling at low magic angle spinning frequencies. It is pointed out that in the presence of H (1) inhomogeneities it will be advantageous to employ adiabatic decoupling in MAS solid state NMR experiments.     

Structure and solvation of melittin in hexafluoroacetone/water

Intermolecular (1)H[(19)F] and (1)H[(1)H] nuclear Overhauser effects have been used to explore interaction of solvent components with melittin dissolved in 50% hexafluoroacetone trihydrate (HFA)/water. Standard nuclear Overhauser effect experiments and an analysis of C(alpha)H proton chemical shifts confirm that the conformation of the peptide in this solvent is alpha-helical from residues Ala4 to Thr11 and from Leu13 to Arg24. The two helical regions are not collinear; the interhelix angle (144 +/- 20 degrees ) found in this work is near that observed in the solid state and previous NMR studies. Intermolecular NOEs arising from interactions between spins of the solvent and the solute indicate that both fluoroalcohol and water molecules are strongly enough bound to the peptide that solvent-solute complexes persist for > or =2 ns. Preferential interactions of HFA with many hydrophobic side chains of the peptide are apparent while water molecules appear to be localized near hydrophilic side chains. These results indicate that interactions of both HFA and water are qualitatively different from those present when the peptide is dissolved in 35% hexafluoro-2-propanol/water, a chemically similar helix-supporting solvent system.     

High resolution deuterium NMR studies of bacterial metabolism

High resolution deuterium NMR spectra were obtained from suspensions of five bacterial strains: Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Deuterium-labeled D-glucose at C-1, C-2, and C-6 was used to monitor dynamically anaerobic metabolism. The flux of glucose through the various bacterial metabolic pathways could be determined by following the disappearance of glucose and the appearance of the major end products in the 2H NMR spectrum. The presence of both labeled and unlabeled metabolites could be detected using 1H NMR spectroscopy since the proton resonances in the labeled species are shifted upfield due to an isotopic chemical shift effect. The 1H-1H scalar coupling observed in both the 2H and 1H NMR spectra was used to assign definitively the resonances of labeled species. An increase in the intensity of natural abundance deuterium signal of water can be used to monitor pathways in which a deuteron is lost from the labeled metabolite. The steps in which label loss can occur are outlined, and the influence these processes have on the ability of 2H NMR spectroscopy to monitor metabolism are assessed.     

Benzothiazole degradation by Rhodococcus pyridinovorans strain PA: evidence of a catechol 1,2-dioxygenase activity

The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by Rhodococcus pyridinovorans strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ (1)H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional (1)H-(13)C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by (1)H and (19)F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this Rhodococcus strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.     

Biological 1H NMR spectroscopy

Proton nuclear magnetic resonance spectroscopy (1H NMR) is a powerful analytical method used to identify and quantitate chemical compounds. In recent years, it has been used to study rates of metabolism in microbes, isolated perfused tissues, intact animals, and human beings. This review highlights some of the more recent biological applications of 1H NMR in the study of metabolic pathophysiology in animals and man. 1H NMR can rapidly analyze complex mixtures of metabolites found in body fluid and biopsy specimens. In vivo 1H NMR methods can measure intracellular pH, a wide variety of metabolites, tissue perfusion, and rates of metabolism of endogenous and exogenous compounds. Using 13C labeled compounds or magnetization transfer techniques metabolic fluxes may be measured in vivo during virtually all normal and abnormal physiological conditions.     

Quantitative evaluation of NMR and MRI methods to measure sucrose concentrations in plants

Summary Developing pea (Pisum sativum L.) seeds were chosen to evaluate the performance of various nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) methods of detecting sucrose in plants. The methods included chemical shift selective imaging (CHESS), heteronuclear correlation via¹³C-¹H coupling (HMQC), and homonuclear correlation via¹H-¹H coupling (DQF). The same experiments were also performed on sucrose phantom samples to evaluate the methods in the absence of the line broadening observed in plant systems. Using the spin echo technique for multi-slice imaging, we could discern the detailed internal structure of the intact seed with a resolution of tens of microns. The proton spin-lattice relaxation time and linewidth as a function of the age of the seed were measured to optimize the efficiency of the NMR and MR experiments. The age-dependent changes in these NMR parameters are consistent with the accumulation of insoluble starch as age increases. Both the NMR and MRI results are in accord with the results of chemical analysis, which reveal that the sucrose concentration is higher in the embryo than in the seed coat, and glucose is at low concentration throughout the seed. Of the three methods for proton observation, the enhanced version of the CHESS approach (CD-CHESS) provides the best combination of sucrose detection and water suppression. Direct observation of¹³C is preferable to indirect detection using HMQC because of water signal bleed-through in samples with large (>200 Hz) linewidths.Abbreviations CD-CHESS continuous wave decoupling chemical shift selective imaging - CHESS chemical shift selective imaging - CSI chemical shift imaging - CW continuous wave - DQF homonuclear double quantum filtering - FOV field of view - FW fresh weight - GHMQC gradient version of the heteronuclear multiple quantum coherence