C isotopomer analysis of glutamate by heteronuclear multiple quantum coherence-total correlation spectroscopy (HMQC-TOCSY)

¹³C has become an important tracer isotope for studies of intermediary metabolism. Information about relative flux through pathways is encoded by the distribution of ¹³C isotopomers in an intermediate pool such as glutamate. This information is commonly decoded either by mass spectrometry or by measuring relative multiplet areas in a ¹³C NMR spectrum. We demonstrate here that groups of glutamate ¹³C isotopomers may be quantified by indirect detection of protons in a 2D HMQC-TOCSY NMR spectrum and that fitting of these data to a metabolic model provides an identical measure of the ¹³C fractional enrichment of acetyl-CoA and relative anaplerotic flux to that given by direct ¹³C NMR analysis. The sensitivity gain provided by HMQC-TOCSY spectroscopy will allow an extension of ¹³C isotopomer analysis to tissue samples not amenable to direct ¹³C detection (∼10 mg soleus muscle) and to tissue metabolites other than glutamate that are typically present at lower concentrations.     

TCA cycle kinetics in the rat heart by analysis of (13)C isotopomers using indirect (1)H

This study was designed to test the hypothesis that indirect (1)H[(13)C] detection of tricarboxylic acid (TCA) cycle intermediates using heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) nuclear magnetic resonance (NMR) spectroscopy provides additional (13)C isotopomer information that better describes the kinetic exchanges that occur between intracellular compartments than direct (13)C NMR detection. NMR data were collected on extracts of rat hearts perfused at various times with combinations of [2-(13)C]acetate, propionate, the transaminase inhibitor aminooxyacetate, and (13)C multiplet areas derived from spectra of tissue glutamate were fit to a standard kinetic model of the TCA cycle. Although the two NMR methods detect different populations of (13)C isotopomers, similar values were found for TCA cycle and exchange fluxes by analyzing the two data sets. Perfusion of hearts with unlabeled propionate in addition to [2-(13)C]acetate resulted in an increase in the pool size of all four-carbon TCA cycle intermediates. This allowed the addition of isotopomer data from aspartate and malate in addition to the more abundant glutamate. This study illustrates that metabolic inhibitors can provide new insights into metabolic transport processes in intact tissues.     

Determination of full 13C isotopomer distributions for metabolic flux analysis using heteronuclear spin echo difference NMR spectroscopy

13C-isotopomer labeling experiments play an increasingly important role in the analysis of intracellular metabolic fluxes for genetic engineering purposes. 13C NMR spectroscopy is a key technique in the experimental determination of isotopomer distributions. However, only subsets of isotopomers can be quantitated using this technique due to redundancies in the scalar coupling patterns and due to invisibility of the 12C isotope in NMR. Therefore, we developed and describe in this paper a 1H NMR spectroscopy method that allows to determine the complete isotopomer distribution in metabolites having a backbone consisting of up to at least four carbons. The proposed pulse sequences employ up to three alternately applied frequency-selective inversion pulses in the 13C channel. In a first application study, the complete isotopomer distribution of aspartate isolated from [1-13C]ethanol-grown Ashbya gossypii was determined. A tentative model of the central metabolism of this organism was constructed and used for metabolic flux analysis. The aspartate isotopomer NMR data played a key role in the successful determination of the flux through the peroxisomal glyoxylate pathway. The new NMR method can be highly instrumental in generating the data upon which isotopomer labeling experiments for flux analysis, that are becoming increasingly important, are based.     

INCA 2.0: A tool for integrated,dynamic modeling of NMR- and MS-based isotopomer measurements and rigorous metabolic flux analysis

Metabolic flux analysis (MFA) combines experimental measurements and computational modeling to determine biochemical reaction rates in live biological systems. Advancements in analytical instrumentation, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), have facilitated chemical separation and quantification of isotopically enriched metabolites. However, no software packages have been previously described that can integrate isotopomer measurements from both MS and NMR analytical platforms and have the flexibility to estimate metabolic fluxes from either isotopic steady-state or dynamic labeling experiments. By applying physiologically relevant cardiac and hepatic metabolic models to assess NMR isotopomer measurements, we herein test and validate new modeling capabilities of our enhanced flux analysis software tool, INCA 2.0. We demonstrate that INCA 2.0 can simulate and regress steady-state ¹³C NMR datasets from perfused hearts with an accuracy comparable to other established flux assessment tools. Furthermore, by simulating the infusion of three different ¹³C acetate tracers, we show that MFA based on dynamic ¹³C NMR measurements can more precisely resolve cardiac fluxes compared to isotopically steady-state flux analysis. Finally, we show that estimation of hepatic fluxes using combined ¹³C NMR and MS datasets improves the precision of estimated fluxes by up to 50%. Overall, our results illustrate how the recently added NMR data modeling capabilities of INCA 2.0 can enable entirely new experimental designs that lead to improved flux resolution and can be applied to a wide range of biological systems and measurement time courses.     

Isotopomer analysis using GC-MS

Knowledge of the complete isotopomer distribution represents the ultimate amount of information on the labeling pattern of a metabolite. One technique for measuring the isotopomer distributions is the analysis of the multiplet intensities arising from the 13C-13C couplings in NMR spectroscopy. While this technique has proven to be very valuable in the elucidation of labeling patterns of C2 and C3 units of various amino acids, fragments larger than C3 are very difficult to measure. Another technique, GC-MS, offers a unique possibility of analyzing fragments larger than C3 and GC-MS is therefore able to give information which is complementary to the information that can be obtained from NMR spectroscopy. In this work we have developed fast, simple, and robust GC-MS methods that can be used to gain information on the labeling patterns of the amino acids in a crude biomass hydrolysate. It is shown that a combination of information obtained from these analyses and information from the NMR spectroscopy is able to yield a much more complete picture of the isotopomer distributions of the amino acids than any of the two techniques alone. The GC-MS method was used for analyzing the labeling patterns of amino acids from a batch cultivation of Penicillium chrysogenum grown on fully labeled glucose. The data from this analysis showed no signs of any significant carbon isotope effects, and the measurements can therefore be used without corrections for metabolic flux analysis.     

Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY

Isotopomer analysis is a very powerful technique for determining site enrichment with stable isotopes. Such information helps determine the relative flux through metabolic pathways. We have developed ¹H NMR detection methods to isotopomer analysis of human rhabdomyosarcoma cells grown in the presence of uniformly ¹³C-labeled glucose. We show that TOCSY can be used both to identify the isotopomer distributions in a substantial number of key compounds and to determine the site-specific enrichment with good precision. Effects of differential relaxation have been specifically addressed. We have identified and quantified isotopomer distributions in Ala, Lactate, (glycolysis markers), nucleotide riboses (pentose phosphate markers), Asp, Glu and Gln (citric acid cycle and anaplerosis markers) as well as in nucleotide pyrimidine rings. Due to the high sensitivity of proton experiments, a reasonable throughput was achieved using a cold probe on only 3–5 mg dry cell weight. This methodology can be applied to biological system using different labeled precursors to examine their metabolic phenotypes and their response to external perturbations. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simultaneous tracers and a unified model of positional and mass isotopomers for quantification of metabolic flux in liver

Computational models based on the metabolism of stable isotope tracers can yield valuable insight into the metabolic basis of disease. The complexity of these models is limited by the number of tracers and the ability to characterize tracer labeling in downstream metabolites. NMR spectroscopy is ideal for multiple tracer experiments since it precisely detects the position of tracer nuclei in molecules, but it lacks sensitivity for detecting low-concentration metabolites. GC-MS detects stable isotope mass enrichment in low-concentration metabolites, but lacks nuclei and positional specificity. We performed liver perfusions and in vivo infusions of ²H and ¹³C tracers, yielding complex glucose isotopomers that were assigned by NMR and fit to a newly developed metabolic model. Fluxes regressed from ²H and ¹³C NMR positional isotopomer enrichments served to validate GC-MS-based flux estimates obtained from the same experimental samples. NMR-derived fluxes were largely recapitulated by modeling the mass isotopomer distributions of six glucose fragment ions measured by GC-MS. Modest differences related to limited fragmentation coverage of glucose C1–C3 were identified, but fluxes such as gluconeogenesis, glycogenolysis, cataplerosis and TCA cycle flux were tightly correlated between the methods. Most importantly, modeling of GC-MS data could assign fluxes in primary mouse hepatocytes, an experiment that is impractical by ²H or ¹³C NMR.     

Metabolite and isotopomer balancing in the analysis of metabolic cycles: I. Theory

Proper analysis of label distribution in metabolic pathway intermediates is critical for correct interpretation of experimental data and strategic experimental design. While, for example, 13C nuclear magnetic resonance (NMR) spectroscopy is usually limited to the measurement of degrees of 13C enrichment, more information about metabolic fluxes can be extracted from the fine structure of NMR spectra, or molecular weight distributions of isotopomers of metabolic intermediates (measured by gas chromatography-mass spectrometry). For this purpose, rigorous accounting for the contribution of all pathways to label distribution is required, especially contributions resulting from multiple turns of metabolic cycles. In this paper we present a mathematical model developed to analyze isotopomer distributions of tricarboxylic acid cycle (TCA) intermediates following the administration of 13C (or 14C) labeled substrates. The theory presented provides the basis to analyze 13C NMR spectra and molecular weight distributions of metabolites. In a companion paper (Park et al., 1999), the theory is applied to the analysis of several cases of biological significance. Copyright 1999 John Wiley & Sons, Inc.     

Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices

Within the last decades NMR spectroscopy has undergone tremendous development and has become a powerful analytical tool for the investigation of intracellular flux distributions in biochemical networks using (13)C-labeled substrates. Not only are the experiments much easier to conduct than experiments employing radioactive tracer elements, but NMR spectroscopy also provides additional information on the labeling pattern of the metabolites. Whereas the maximum amount of information obtainable with (14)C-labeled substrates is the fractional enrichment in the individual carbon atom positions, NMR spectroscopy can also provide information on the degree of labeling at neighboring carbon atom positions by analyzing multiplet patterns in NMR spectra or using 2-dimensional NMR spectra. It is possible to quantify the mole fractions of molecules that show a specific labeling pattern, i.e., information of the isotopomer distribution in metabolite pools can be obtained. The isotopomer distribution is the maximum amount of information that in theory can be obtained from (13)C-tracer studies. The wealth of information contained in NMR spectra frequently leads to overdetermined algebraic systems. Consequently, fluxes must be estimated by nonlinear least squares analysis, in which experimental labeling data is compared with simulated steady state isotopomer distributions. Hence, mathematical models are required to compute the steady state isotopomer distribution as a function of a given set of steady state fluxes. Because 2(n) possible labeling patterns exist in a molecule of n carbon atoms, and each pattern corresponds to a separate state in the isotopomer model, these models are inherently complex. Model complexity, so far, has restricted usage of isotopomer information to relatively small metabolic networks. A general methodology for the formulation of isotopomer models is described. The model complexity of isotopomer models is reduced to that of classical metabolic models by expressing the 2(n) isotopomer mass balances of a metabolite pool in a single matrix equation. Using this approach an isotopomer model has been implemented that describes label distribution in primary carbon metabolism, i.e., in a metabolic network including the Embden-Meyerhof-Parnas and pentose phosphate pathway, the tricarboxylic acid cycle, and selected anaplerotic reaction sequences. The model calculates the steady state label distribution in all metabolite pools as a function of the steady state fluxes and is applied to demonstrate the effect of selected anaplerotic fluxes on the labeling pattern of the pathway intermediates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:831-840, 1997.     

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 comparison between NMR and GCMS 13C-isotopomer analysis in cardiac metabolism

NMR spectroscopy and gas chromatography-mass spectrometry (GCMS) have both been used to study cardiac metabolism using substrates labeled with the stable isotope carbon-13. ¹³C-NMR studies of substrate oxidation are based on the assumption that the ¹³C-enrichment of glutamate reflects that of 2-ketoglutarate (2-KG). This assumption appears reasonable; however, it has not been thoroughly validated. The higher sensitivity of GCMS enables the direct determination of ¹³C-enrichment of 2-KG and other tricarboxylic acid (TCA) cycle intermediates. Therefore, using extracts from normal and diabetic hearts perfused with physiological concentrations of unlabeled glucose and ¹³C-labeled substrates, [3-¹³C](lactate + pyruvate) and [U-¹³C]palmitate, we compared the mass isotopomer distribution (MID) of citrate, 2-KG, succinate and malate measured directly by GCMS with that extrapolated from ¹³C-NMR glutamate isotopomer analysis. A significant correlation between the absolute molar percent enrichments (MPE) of the various mass isotopomers of glutamate determined by ¹³C-NMR and 2-KG determined by GCMS was observed for all sixteen-heart samples. This correlation was improved if the contribution from unlabeled 2-KG was removed (i.e. relative MPE) indicating that ¹³C-NMR under estimated the unlabeled fraction. We attribute this discrepancy in the measurement of unlabeled 2-KG to the fact that GCMS measures M0 directly, while the NMR analysis calculates it by difference, since unlabeled glutamate is not detected by ¹³C-NMR spectroscopy. Despite the differences between the two methods, ¹³C-MID of glutamate determined by NMR provides a simple and reliable indicator of fluxes of ¹³C-enriched substrates through the TCA cycle. It is also clear that MID analysis of TCA cycle intermediates by GCMS is a sensitive and direct approach to assess substrate selection for citrate synthesis as well as a potential indicator of sites and extent of anaplerosis and/or compartmentation. This study demonstrates that the alliance of NMR and GCMS represents a powerful approach for investigating the control and regulation of cardiac carbon metabolism.     

Reverse flux through cardiac NADP(+)-isocitrate dehydrogenase under normoxia and ischemia

Little is known about the role of mitochondrial NADP(+)-isocitrate dehydrogenase (NADP(+)-ICDH) in the heart, where this enzyme shows its highest expression and activity. We tested the hypothesis that in the heart, NADP(+)-ICDH operates in the reverse direction of the citric acid cycle (CAC) and thereby may contribute to the fine regulation of CAC activity (Sazanov and Jackson, FEBS Lett 344: 109-116, 1994). We documented a reverse flux through this enzyme in rat hearts perfused with the medium-chain fatty acid octanoate using [U-(13)C(5)]glutamate and mass isotopomer analysis of tissue citrate (Comte et al., J Biol Chem 272: 26117-26124, 1997). In this study, we assessed the significance of our previous finding by perfusing hearts with long-chain fatty acids and tested the effects of changes in O(2) supply. We showed that under all of these conditions citrate was enriched in an isotopomer containing five (13)C atoms. This isotopomer can only be explained by substrate flux through reversal of the NADP(+)-ICDH reaction, which is evaluated at 3-7% of flux through citrate synthase. Small variations in reversal fluxes induced by low-flow ischemia that mimicked hibernation occurred despite major changes in contractile function and O(2) consumption of the heart as well as citrate and succinate release rates and tissue levels. Our data show a reverse flux through NADP(+)-ICDH and support its hypothesized role in the fine regulation of CAC activity in the normoxic and O(2)-deprived heart.     

Phenylalanine-independent biosynthesis of 1,3,5,8-tetrahydroxyxanthone. A retrobiosynthetic NMR study with root cultures of Swertia chirata.

Root cultures of Swertia chirata (Gentianaceae) were grown with supplements of [1-13C]glucose, [U-13C6]glucose or [carboxy-13C]shikimic acid. 1,3,5,8-Tetrahydroxyxanthone was isolated and analysed by quantitative NMR analysis. The observed isotopomer distribution shows that 1,3,5,8-tetrahydroxyxanthone is biosynthesized via a polyketide-type pathway. The starter unit, 3-hydroxybenzoyl-CoA, is obtained from an early shikimate pathway intermediate. Phenylalanine, cinnamic acid and benzoic acid were ruled out as intermediates.     

Bidirectional reaction steps in metabolic networks: IV. Optimal design of isotopomer labeling experiments

This article generalizes the statistical tools for the evaluation of carbon-labeling experiments that have been developed for the case of positional enrichment systems in part II of this series to the general case of isotopomer systems. For this purpose, a new generalized measurement equation is introduced that can describe all kinds of measured data, like positional enrichments, relative (13)C nuclear magnetic resonance ((13)C NMR) multiplet intensities, or mass isotopomer fractions produced with mass spectroscopy (MS) instruments. Then, to facilitate the specification of the various measurement procedures available, a new flexible textual notation is introduced from which the complicated generalized measurement equations are generated automatically. Based on these measurement equations, a statistically optimal flux estimator is established and parameter covariance matrices for the flux estimation are computed. Having implemented these tools, different kinds of labeling experiments can be compared by using statistical quality measures. A general framework for the optimal design of carbon-labeling experiments is established on the basis of this method. As an example it is applied to the Corynebacterium network from part II extended by various NMR and MS measurements. In particular, the positional enrichment, multiplet, or mass isotopomer measurements with the greatest information content for flux estimation are computed (measurement design) and various differently labeled input substrates are compared with respect to flux estimation (input design). It is examined in detail how the measurement procedure influences the estimation quality of specific fluxes like the pentose phosphate pathway influx.     

Cumulative bondomers: a new concept in flux analysis from 2D [13C,1H] COSY NMR data

A well-established way of determining metabolic fluxes is to measure 2D [(13)C,(1)H] COSY NMR spectra of components of biomass grown on uniformly (13)C-labeled carbon sources. When using the entire set of measured data to simultaneously determine all fluxes in a proposed metabolic network model, the (13)C-labeling distribution in all measured compounds has to be simulated. This requires very large sets of isotopomer or cumomer balances. This article introduces the new concept of bondomers; entities that only vary in the numbers and positions of C-C bonds that have remained intact since the medium substrate molecule entered the metabolism. Bondomers are shown to have many analogies to isotopomers. One of these is that bondomers can be transformed to cumulative bondomers, just like isotopomers can be transformed to cumomers. Similarly to cumomers, cumulative bondomers allow an analytical solution of the entire set of balances describing a metabolic network. The main difference is that cumulative bondomer models are considerably smaller than corresponding cumomer models. This saves computational time, allows easier identifiability analysis, and yields new insights in the information content of 2D [(13)C,(1)H] COSY NMR data. We illustrate the theoretical concepts by means of a realistic example of the glycolytic and pentose phosphate pathways. The combinations of 2D [(13)C,(1)H] COSY NMR data that allow identification of all metabolic fluxes in these pathways are analyzed, and it is found that the NMR data contain less information than was previously expected.     

NMR-based stable isotope resolved metabolomics in systems biochemistry

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.     

Identification of hexose hydrolysis products in metabolic flux analytes: a case study of levulinic acid in plant protein hydrolysate

Biosynthetically directed fractional ¹³C labeling, a popular methodology of metabolic flux analysis, involves culture on a mixture of ¹³C and ¹²C substrates and preparation a ‘metabolic flux analyte’ (typically protein hydrolysate) from the biomass. Metabolic flux analytes prepared from complex eukaryotes may contain additional compounds than those prepared from microorganisms. We report the presence of such compounds (hexose hydrolysis products) in a plant metabolic flux analyte (acid hydrolyzed protein from soybean embryos). We designed NMR experiments to systematically identify these compounds, and found that they were levulinic acid (LVA; major) and hydroxyacetone (HyA; minor). These acid hydrolysis products of hexoses (glucose and mannose) were generated during acid hydrolysis of glycosylating sugars (glucosamine and mannose) associated with soybean embryo protein. Analysis of LVA by two-dimensional [¹³C, ¹H] NMR and measurement of its J-coupling constants revealed long-range coupling between atoms C3 and C5, which enables LVA to provide more isotopomer information than its precursor hexose. Furthermore, we found that LVA and HyA preserve the isotopomeric composition of the metabolic hexose from which they are derived. An important consequence of these results is that comparison of LVA and HyA isotopomers from two separate metabolic flux analytes (protein hydrolysate and starch hydrolysate) from the same plant tissue can distinguish between parallel glycolysis and pentose phosphate pathways in different subcellular compartments.     

An efficient method for the production of isotopically enriched cholesterol for NMR

(13)C-Cholesterol was produced with high efficiency by a genetically engineered yeast strain. The method produces ～ 1 mg of cholesterol per gram of glucose using 100 ml of culture medium. Uniform 94% enrichment where the most abundant product is the fully enriched isotopomer (u-(13)C(27)) is obtained using (u-(13)C(6), 99%) glucose medium. High enrichment is very important for relaxation experiments, but for NMR applications where carbon-carbon couplings are measured, this is problematic. A good compromise between sensitivity and cost consists in diluting (u-(13)C(6), 25%) with natural-abundance glucose. With a 2:3 ratio, the maximal amount of singlets can be obtained in 1 dimensional (D) carbon and 2D heteronuclear single-quantum correlation (HSQC) spectra with 6× intensity increase relative to natural-abundance samples. The use of (1-(13)C(1)-glucose, 99%) or (2-(13)C(1)-glucose, 99%) as isotope sources allows the labeling of the cholesterol in multiple mostly nonvicinal positions and reach 45× intensity increase. As an alternative, the dilution of (u-(13)C(6), 99%) glucose can be used to simultaneously enrich eleven pairs of (13)C up to ～ 1,000× natural-abundance probability, which should be very beneficial to double-quantum NMR experiments including the INADEQUATE and related pulse sequences. The flexibility of the method and the potential to adapt the culture protocol to specific needs should find many applications in chemistry and biology and in different fields of NMR and MS.     

Simultaneous Steady-state and Dynamic 13C NMR Can Differentiate Alternative Routes of Pyruvate Metabolism in Living Cancer Cells

Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of ¹³C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized ¹³C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined ¹³C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-¹³C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-¹³C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[¹³C]O₃⁻ and [1-¹³C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-¹³C]pyruvate. Quantitation of hyperpolarized H[¹³C]O₃⁻ provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[¹³C]O₃⁻ appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1-¹³C]pyruvate metabolism, enhancing exchanges with [1-¹³C]lactate and suppressing H[¹³C]O₃⁻ formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining ¹³C isotopomer analyses and dynamic hyperpolarized ¹³C spectroscopy may enable quantitative flux measurements in living tumors.     

4D Non-uniformly sampled C,C-NOESY experiment for sequential assignment of 13C,15N-labeled RNAs

¹³C spin diluted protein samples can be produced using [1-¹³C] and [2-¹³C]-glucose (Glc) carbon sources in the bacterial growth medium. The ¹³C spin dilution results in favorable ¹³C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-¹³C]- and [2-¹³C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-¹³C]-glycerol (¹³C labeled Cα sites on a ¹²C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17–26, 2011 ). Inspired by this approach and our own recent results using [2-¹³C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of ¹³C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle.