¹³C NMR metabolomics: applications at natural abundance.

(13)C NMR has many advantages for a metabolomics study, including a large spectral dispersion, narrow singlets at natural abundance, and a direct measure of the backbone structures of metabolites. However, it has not had widespread use because of its relatively low sensitivity compounded by low natural abundance. Here we demonstrate the utility of high-quality (13)C NMR spectra obtained using a custom (13)C-optimized probe on metabolomic mixtures. A workflow was developed to use statistical correlations between replicate 1D (13)C and (1)H spectra, leading to composite spin systems that can be used to search publicly available databases for compound identification. This was developed using synthetic mixtures and then applied to two biological samples, Drosophila melanogaster extracts and mouse serum. Using the synthetic mixtures we were able to obtain useful (13)C-(13)C statistical correlations from metabolites with as little as 60 nmol of material. The lower limit of (13)C NMR detection under our experimental conditions is approximately 40 nmol, slightly lower than the requirement for statistical analysis. The (13)C and (1)H data together led to 15 matches in the database compared to just 7 using (1)H alone, and the (13)C correlated peak lists had far fewer false positives than the (1)H generated lists. In addition, the (13)C 1D data provided improved metabolite identification and separation of biologically distinct groups using multivariate statistical analysis in the D. melanogaster extracts and mouse serum

Development of LC-13C NMR

This study involves the development of C-13 nuclear resonance as an on-line detector for liquid chromatography (LC-C-13 NMR) for the chemical characterization of aviation fuels. The initial focus of this study was the development of a high sensitivity flow C-13 NMR probe. Since C-13 NMR sensitivity is of paramount concern, considerable effort during the first year was directed at new NMR probe designs. In particular, various toroid coil designs were examined. In addition, corresponding shim coils for correcting the main magnetic field (B sub 0) homogeneity were examined. Based on these initial probe design studies, an LC-C-13 NMR probe was built and flow C-13 NMR data was obtained for a limited number of samples

A large geometric distortion in the first photointermediate of rhodopsin, determined by double-quantum solid-state NMR

Double-quantum magic-angle-spinning NMR experiments were performed on 11,12-C-13(2)-retinylidene-rhodopsin under illumination at low temperature, in order to characterize torsional angle changes at the C11-C12 photoisomerization site. The sample was illuminated in the NMR rotor at low temperature (similar to 120 K) in order to trap the primary photointermediate, bathorhodopsin. The NMR data are consistent with a strong torsional twist of the HCCH moiety at the isomerization site. Although the HCCH torsional twist was determined to be at least 40A degrees, it was not possible to quantify it more closely. The presence of a strong twist is in agreement with previous Raman observations. The energetic implications of this geometric distortion are discussed

Complicated Conformational Exchange of New Pyridoxine Derivative. Dynamic¹³C NMR Characterization

© 2017, Springer Science+Business Media, LLC. New pyridoxine derivative with multiple chemical exchanges was studied by 13 C (at 298 K, 188 K) and 2D NMR ( 1 H- 13 C HSQC, 1 H- 13 C HMBC) spectroscopy in acetone-d 6 solution. Complete 13 C NMR data (table with chemical shifts at different temperatures) is presented. Intramolecular mobility of the molecule was analyzed based on the results of 13 C NMR experiments in combination with the data obtained from previous study of this compound by dynamic 1 H NMR spectroscopy

Spin Dynamics at the Mott Transition and in the Metallic State of the Cs_{3}C_{60} Superconducting Phases

We present here ^{13}C and ^{133}Cs NMR spin lattice relaxation T_{1} data in the A15 and fcc-Cs_{3}C_{60} phases for increasing hydrostatic pressure through the transition at p_{c} from a Mott insulator to a superconductor. We evidence that for p>> p_{c} the (T_{1}T)^{-1} data above T_{c} display metallic like Korringa constant values which match quantitatively previous data taken on other A_{3}C_{60} compounds. However below the pressure for which T_{c} goes through a maximum, (T_{1}T)^{-1} is markedly increased with respect to the Korringa values expected in a simple BCS scenario. This points out the importance of electronic correlations near the Mott transition. For p > p_{c} singular T dependences of (T_{1}T)^{-1} are detected for T >> T_{c}. It will be shown that they can be ascribed to a large variation with temperature of the Mott transition pressure p_{c} towards a liquid-gas like critical point, as found at high T for usual Mott transitions.Comment: 6 pages, 6 figures, submitted to EP

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells.

A network model for the determination of tumor metabolic fluxes from (13)C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-(13)C2]glucose under normoxic conditions at 37 °C and monitored by (13)C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism

Biochemical quantification of total brain glycogen concentration in rats under different glycemic states.

All (13)C NMR studies of brain glycogen to date relied on observing the incorporation of (13)C label into glycogen, and thus interpretation was potentially affected by changes in (13)C label turnover rates. The goal of this study was to quantify total brain glycogen concentration under conditions of hypoglycemia or normoglycemia using biochemical methods. Rats were sacrificed using a focused microwave fixation device. The results showed that metabolism of brain glycogen was Glc- and insulin-sensitive and that insulin-induced hypoglycemia promoted a gradual glycogenolysis. Moreover, we show that there are very mild effects of isoflurane and alpha-chloralose anesthesia on brain glycogen concentration. Altogether these results show that total brain glycogen serves as a substantial source of glucosyl units during insulin-induced moderate hypoglycemia and therefore may be neuroprotective. Finally we also conclude that previous interpretation of (13)C NMR spectroscopy data accurately reflected the changes in total brain glycogen content

Conformationally selective multidimensional chemical shift ranges in proteins from a PACSY database purged using intrinsic quality criteria

We have determined refined multidimensional chemical shift ranges for intra-residue correlations ([superscript 13]C–[superscript 13]C, [superscript 15]N–[superscript 13]C, etc.) in proteins, which can be used to gain type-assignment and/or secondary-structure information from experimental NMR spectra. The chemical-shift ranges are the result of a statistical analysis of the PACSY database of >3000 proteins with 3D structures (1,200,207 [superscript 13]C chemical shifts and >3 million chemical shifts in total); these data were originally derived from the Biological Magnetic Resonance Data Bank. Using relatively simple non-parametric statistics to find peak maxima in the distributions of helix, sheet, coil and turn chemical shifts, and without the use of limited “hand-picked” data sets, we show that ~94 % of the [superscript 13]C NMR data and almost all [superscript 15]N data are quite accurately referenced and assigned, with smaller standard deviations (0.2 and 0.8 ppm, respectively) than recognized previously. On the other hand, approximately 6 % of the [superscript 13]C chemical shift data in the PACSY database are shown to be clearly misreferenced, mostly by ca. −2.4 ppm. The removal of the misreferenced data and other outliers by this purging by intrinsic quality criteria (PIQC) allows for reliable identification of secondary maxima in the two-dimensional chemical-shift distributions already pre-separated by secondary structure. We demonstrate that some of these correspond to specific regions in the Ramachandran plot, including left-handed helix dihedral angles, reflect unusual hydrogen bonding, or are due to the influence of a following proline residue. With appropriate smoothing, significantly more tightly defined chemical shift ranges are obtained for each amino acid type in the different secondary structures. These chemical shift ranges, which may be defined at any statistical threshold, can be used for amino-acid type assignment and secondary-structure analysis of chemical shifts from intra-residue cross peaks by inspection or by using a provided command-line Python script (PLUQin), which should be useful in protein structure determination. The refined chemical shift distributions are utilized in a simple quality test (SQAT) that should be applied to new protein NMR data before deposition in a databank, and they could benefit many other chemical-shift based tools.National Institutes of Health (U.S.) (Grant GM066976

NMR evidence for a strong modulation of the Bose-Einstein Condensate in BaCuSi2_2O6_6

We present a $^{63,65}$Cu and $^{29}$Si NMR study of the quasi-2D coupled spin 1/2 dimer compound BaCuSi$_2$O$_6$ in the magnetic field range 13-26 T and at temperatures as low as 50 mK. NMR data in the gapped phase reveal that below 90 K different intra-dimer exchange couplings and different gaps ($\Delta_{\rm{B}}/\Delta_{\rm{A}}$ = 1.16) exist in every second plane along the c-axis, in addition to a planar incommensurate (IC) modulation. $^{29}$Si spectra in the field induced magnetic ordered phase reveal that close to the quantum critical point at $H_{\rm{c1}}$ = 23.35 T the average boson density $\bar{n}$ of the Bose-Einstein condensate is strongly modulated along the c-axis with a density ratio for every second plane $\bar{n}_{\rm{A}}/\bar{n}_{\rm{B}} \simeq 5$. An IC modulation of the local density is also present in each plane. This adds new constraints for the understanding of the 2D value $\phi$ = 1 of the critical exponent describing the phase boundary

AMENTOFLAVON DARI DAUN NYAMPLUNG (Calophyllum inophyllum Linn.)

Satu senyawa biflavonoid yaitu amentoflavon (1) berhasil diisolasi dari daun tumbuhan nyamplung (Calophyllum inophyllum Linn). Ekstraksi menggunakan metode maserasi dan pemurnian dengan kromatografi kolom dengan fasa diam Si-gel dan sephadex LH-20. Elusidasi struktur menggunakan metode spektroskopi UV, IR, 1 H dan 13 C NMR beserta HMQC dan HMBC. Hasil yang diperoleh dianalisis dan dibandingkan dengan data referensi. Amentoflavone (1), a biflavonoid was isolated from leaves of Calophyllum inophyllum Linn. Isolation and purification of the compound used maceration and chromatography methods using Si-gell and sephadex LH-20. The structure was determined by UV, IR, 1 H and 13 C NMR spectroscopy includes HMQC/HMBC analysis and comparison with references