Phosphorus NMR and Its Application to Metabolomics (original) (raw)
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Combining solid-state and solution-state 31P NMR to study in vivo phosphorus metabolism
Proceedings of the National Academy of Sciences, 1985
Otherwise unavailable information concerning the distribution of phosphorylated compounds in biological systems is obtained by a combined solid-state/solution-state NMR approach, illustrated here for oocytes from Rana pipiens. General methodology is developed, and further extensions are proposed. The following conclusions pertain to the specific system under examination. (i) Nucleoside phosphates can be observed by magic-angle sample spinning of the lyophilized material. (ii) The solid-state NMR technique of dipolar decoupling provides no additional resolution of the phospholipid and phosphoprotein components of the yolk. However, cellular death produces sufficient pH changes to cause the phospholipid and protein phosphate peaks to become resolvable. The concentration of nucleoside phosphates also decreases. (iii) The phospholipid and phosphoprotein components are shown by computer simulation to be present in a ratio of 40:60, respectively. (iv) The amounts of inorganic phosphate, n...
Physiological Genomics, 2012
Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope18O-assisted31P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The18O labeling procedure is based on the incorporation of one18O into Pifrom [18O]H2O with each act of ATP hydrolysis and the distribution of18O-labeled phosphoryls among phosphate-carrying molecules. This enables simultaneous recording of ATP synthesis and utilization, phosphotransfer fluxes through adenylate kinase, creatine kinase, and glycolytic pathways, as well as mitochondrial substrate shuttle, urea and Krebs cycle activity, glycogen turnover, and intracellular energetic communication. Application of expanded18O-labeling procedures has revealed significant differences in the dynamics of G-6-P[18O] (glycolysis), G-3-P[18O] (substrate shuttle), a...
Determination of renal molar concentrations of phosphorus-containing metabolitesin vivo using31P NMR
Magnetic Resonance in Medicine, 1987
A technique to determine absolute metabolite concentrations of the kidney in vivo using 31P NMR is described. The technique is based on the use of methylphosphonic acid (MPA), which gives rise to a well-resolved peak upfield from in vivo phosphorous metabolite resonances, as an "internal standard." The method involves acquisition of a fully relaxed kidney spectrum with an implanted coil followed by intravenous infusion of MPA (4 ml of 150 mM) for a period of 30 min. The animal is then sacrificed to insure a steady state level of renal MPA and another spectrum is obtained. From these two spectra the ratio of intensities of MPA to P-ATP was derived. In the method used here, no significant contribution from tissues outside the kidney was observed. In addition, a relatively homogenous distribution of MPA throughout the kidney was achieved. The amount of MPA per gram wet weight of kidney was also obtained through NMR methods by placing the excised organ in a phosphate-calibrated solenoidal coil. The calibration spectra along with the ratio of intensities for MPA/@-ATP were used to calculate the number of micromoles of ATP per gram wet weight of kidney. Infusion of a higher concentration of MPA (1.25 M) produced a visible MPA resonance in other organs besides the kidney. Thus, MPA could be useful in determining phosphate metabolite concentrations in other tissues. o 1987 Academic Press, Inc.
NMR-based stable isotope resolved metabolomics in systems biochemistry
Journal of Biomolecular NMR, 2011
An important goal of metabolomics is to characterize the changes in metabolic networks in cells or various tissues of an organism in response to external perturbations or pathologies. The profiling of metabolites and their steady state concentrations does not directly provide information regarding the architecture and fluxes through metabolic networks. This requires tracer approaches. NMR is especially powerful as it can be used not only to identify and quantify metabolites in an unfractionated mixture such as biofluids or crude cell/tissue extracts, but also determine the positional isotopomer distributions of metabolites derived from a precursor enriched in stable isotopes such as 13 C and 15 N via metabolic transformations. In this article we demonstrate the application of a variety of 2-D NMR editing experiments to define the positional isotopomers of compounds present in polar and non-polar extracts of human lung cancer cells grown in either [U-13 C]-glucose or [U-13 C, 15 N]-glutamine as source tracers. The information provided by such experiments enabled unambiguous reconstruction of metabolic pathways, which is the foundation for further metabolic flux modeling.
Analytical Biochemistry, 1999
31 P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved 31 P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum (1 H-31 P HSQC) and 31 P correlation spectroscopy (31 P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts. © 1999 Academic Press 31 P NMR spectroscopy has been shown to be a useful tool for the study of cell metabolism. This is mainly due to the fact that a variety of important metabolites are phosphorylated and that the 31 P nucleus has a 100% natural abundance, thereby eliminating the need of
Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. As 18O can be derived from heavy water (H218O), it is comparably cheap and particularly suited for labelling of phosphorylated compounds, provided the introduction is straight-forward and phosphate neutral loss in the ion source can be avoided. Here, a unifying synthetic concept for 18O-labelled phosphates is presented, based on a family of modified 18O2‑phosphoramidite reagents. This flexible toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including - but not limited to - nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. 18O-enrichment ratios >95% and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the 18O labelled inositol phosphates and pyr...
Noninvasive quantitation of phosphorus metabolites in human tissue by NMR spectroscopy
1969
Quamitation of metabolite concentrations by NMR spectroscopy is complicated by the need to determine the volume from which signals are detected, and by the need to obtain the relative sensitivity of detection within this volume. The use of coils with inhomogeneous B, fields further complicates these problems. In order to quantify metabolite concentrations using 3'P NMR spectroscopy, an external reference of hexamethyl phosphoroustriamide was used. Studies were performed on phantoms, using either a surface coil or a Helmholtz head coil to confirm the accuracy of both the ISIS volume selection technique and the use of an external reference. The limitations ofthis method are related to contamination and signal loss inherent in the ISIS technique and difficulties with integration of broad overlapping peaks. The method was appiied to seven normal human subjects. The integrals for metabolite signals in normal brain and calf muscle were determined by using NMRl software. The T, values of the signals of all phosphorus metabolites in the selected volume were measured in order to correct for saturation el%cts. The concentrations for PCr, Pi, and ATP were 4.9,2.0, and 2.5 mMin brain and 36.5, 5.7, and 7.3 mM in muscle. These results are in good agreement with those reported for animals, demonstrating the validity of this quantitation technique. o 1989 Academic Press. Inc.
Rapid, reliable in vivo assays of human phosphate metabolites by nuclear magnetic resonance
Clinical Chemistry, 1989
This accurate, reliable, and fast method of assaying absolute concentrations of phosphate metabolites noninvasively in living tissue, including that of humans, combines 31P nuclear magnetic resonance (NMR) spectroscopy and 1H NMR imaging. The images are used to measure the areas of metabolite-beanng tissue in selected sections through the subject, and31 P spectra are acquired from the same section, together with a concentration reference located on the penphery. Metabolite concentrations are calculated from the ratios of areas and integrated signal intensities. Apparatus and protocol are designed to eliminate corrections due to magnetic field nonuniformities and NMR relaxation times. Mean (and SD) concentrations of adenosine triphosphate (ATP), phosphocreatine, and inorganic phosphate (P1) measured in the brains of 15 normal adult human volunteers with a 1.5-T NMR system were 3.03 (0.49), 5.18 (0.89), and 1.5 (0.7) mmol per liter of wet tissue, respectively. Acquisition times of only a few minutes should facilitate metabolic studies of patients with disorders in limbs and brain, particularly those affecting entire organs.
NMR in Biomedicine, 1999
A semi-automated, metabolite prior-knowledge-based, lineshape fitting analysis has been developed to assess the dynamic biochemical changes found in ex vivo 31 P NMR pig liver preservation studies. Due to the inherent experimental limitations of the ex vivo study and the complexity of the composite phosphorus resonances, metabolite information obtained in vitro was incorporated into the ex vivo analysis. This approach has allowed complete metabolite analysis (phosphomonoesters, inorganic phosphate, phosphodiesters and nucleotide triphosphates) in over 2000 spectra in a fraction of the time compared with more conventional analysis methods. The developed analysis will enable complete and rapid assessment of the biochemical changes in ongoing cold preservation studies of the pig liver which will result in thousands of ex vivo 31 P NMR spectra. It is also envisaged that comparative studies on human donor livers will be carried out, in which this type of analysis would be the method of choice. Moreover, this kind of analysis approach could be advantageous in many complex in vivo NMR spectroscopy applications. Copyright
Humanin vivo phosphate metabolite imaging with31P NMR
Magnetic Resonance in Medicine, 1988
Phosphorus ("P) spectroscopic images showing the distribution of high-energy phosphate metabolites in the human brain have been obtained at 1.5 T in scan times of 8.5 to 34 min at 27 and 64 cm3 spatial resolution using pulsed phase-encoding gradient magnetic fields and three-dimensional Fourier transform (3DFT) techniques. Data were acquired as free induction decays with a quadrature volume NMR detection coil of a truncated geometry designed to optimize the signal-to-noise ratio on the coil axis on the assumption that the sample noise represents the dominant noise source, and self-shielded magnetic field gradient coils to minimize eddy-current effects. The images permit comparison of metabolic data acquired simultaneously from different locations in the brain, as well as metabolite quantification by inclusion of a vial containing a standard of known "P concentration in the image array. Values for the NMR visible adenosine triphosphate in three individuals were about 3 mM of tissue. The ratio of NMR detectable phosphocreatine to ATP in brain was I. 15 k 0.17 SD in these experiments. Potential sources of random and systematic error in these and other 'IP measurements are identified. o