Metabolic flux and metabolic network analysis of Penicillium chrysogenum using 2D [13C, 1H] COSY NMR measurements and cumulative bondomer simulation

At present two alternative methods are available for analyzing the fluxes in a metabolic network: (1) combining measurements of net conversion rates with a set of metabolite balances including the cofactor balances, or (2) leaving out the cofactor balances and fitting the resulting free fluxes to measured (13)C-labeling data. In this study these two approaches are applied to the fluxes in the glycolysis and pentose phosphate pathway of Penicillium chrysogenum growing on either ammonia or nitrate as the nitrogen source, which is expected to give different pentose phosphate pathway fluxes. The presented flux analyses are based on extensive sets of 2D [(13)C, (1)H] COSY data. A new concept is applied for simulation of this type of (13)C-labeling data: cumulative bondomer modeling. The outcomes of the (13)C-labeling based flux analysis substantially differ from those of the pure metabolite balancing approach. The fluxes that are determined using (13)C-labeling data are shown to be highly dependent on the chosen metabolic network. Extending the traditional nonoxidative pentose phosphate pathway with additional transketolase and transaldolase reactions, extending the glycolysis with a fructose 6-phosphate aldolase/dihydroxyacetone kinase reaction sequence or adding a phosphoenolpyruvate carboxykinase reaction to the model considerably improves the fit of the measured and the simulated NMR data. The results obtained using the extended version of the nonoxidative pentose phosphate pathway model show that the transketolase and transaldolase reactions need not be assumed reversible to get a good fit of the (13)C-labeling data. Strict statistical testing of the outcomes of (13)C-labeling based flux analysis using realistic measurement errors is demonstrated to be of prime importance for verifying the assumed metabolic model.     

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.     

Innovations in generation and analysis of 2D [(13)C,(1)H] COSY NMR spectra for metabolic flux analysis purposes.

2D [(13)C,(1)H] COSY NMR is used by the metabolic engineering community for determining (13)C-(13)C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [(13)C,(1)H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range (13)C-(13)C scalar couplings in 2D [(13)C,(1)H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Innovations in Generation and Analysis of 2D [C,H] COSY NMR Spectra for Metabolic Flux Analysis Purposes

2D [¹³C,¹H] COSY NMR is used by the metabolic engineering community for determining ¹³C–¹³C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [¹³C,¹H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range ¹³C–¹³C scalar couplings in 2D [¹³C,¹H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Quantitative analysis of metabolite concentrations in human urine samples using 13C{1H} NMR spectroscopy

Targeted profiling is a library-based method of using mathematically modeled reference spectra for quantification of metabolite concentrations in NMR mixture analysis. Metabolomics studies of biofluids, such as urine, represent a highly complex problem in this area, and for this reason targeted profiling of ¹H NMR spectra can be hampered. A number of the issues relating to ¹H NMR spectroscopy can be overcome using ¹³C{¹H} NMR spectroscopy. In this work, a ¹³C{¹H} NMR database was created using Chenomx NMR Suite, incorporating 120 metabolites. The ¹³C{¹H} NMR database was standardized through the analysis of a series of metabolite solutions containing varying concentrations of 19 distinct metabolites, where the metabolite concentrations were varied across a range of values including biological ranges. Subsequently, the NMR spectra of urine samples were collected using ¹³C{¹H} NMR spectroscopy and profiled using the ¹³C{¹H} NMR library. In total, about 30 metabolites were conclusively identified and quantified in the urine samples using ¹³C{¹H} NMR targeted profiling. The proton decoupling and larger spectral window provided easier identification and more accurate quantification for specific classes of metabolites, such as sugars and amino acids with overlap in the aliphatic region of the ¹H NMR spectrum. We discuss potential application areas in which ¹³C{¹H} NMR targeted profiling may be superior to ¹H NMR targeted profiling.     

2D [1H,13C] NMR study of carbon fluxes during glucose utilization by Escherichia coli MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells--glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)--were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655(edd-eda), MG1655(zwf, edd-eda), and MG1655(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.     

Metabolism of [2-13C]succinate in renal cells determined by 13C NMR

[2-13C]Succinate has been used to examine the metabolic carbon flux from the Krebs cycle in rat renal proximal convoluted tubular (PCT) cells under physiological and pathophysiological conditions. Therefore, we developed a mathematical model that enabled us to determine the metabolic fluxes of the Krebs cycle. A mathematical model for the calculation of flux from [2-13C]succinate was used to determine fluxes in rat PCT cells during chronic acidosis in the presence and absence of 0.1 mM angiotensin II. The relative carbon efflux via glutamate dehydrogenase in rat renal PCT cells increases during chronic acidosis from 0.27 to 0.39, whereas this carbon flux is not affected by the presence of peptide hormone angiotensin II in the incubation medium. The fraction of intermediate 13C-labelled oxaloacetate transformed into the phosphoenolpyruvate and aspartate pools increases significantly from 0.41 to 0.57 in the case of chronic acidosis. The carbon efflux is not affected by angiotensin II. The 13C-NMR data also show that the carbon efflux through phosphoenolpyruvate carboxykinase increases from 0.35 to 0.56 in rat renal PCT cells derived from chronic acidotic animals, as well as in the presence of angiotensin II. The present results indicate that angiotensin II affects only the flux through phosphoenolcarboxykinase, whereas chronic acidosis increases the flux through phosphoenolpyruvate carboxykinase as well as the gluconeogenic flux.     

Gluconeogenesis in hepatocytes determined with [2-13C] acetate and quantitative 13C NMR spectroscopy

• 1.1. In the present study the major metabolic pathways of glucose metabolism were determined in isolated liver cells using [2-¹³C]acetate and ¹³C magnetic resonance spectroscopy.
• 2.2. The relative reaction rates of glucose synthesis to the TCA cycle were determined from the ¹³C distribution in glucose where the overall ¹³C enrichment of glucose was 6.41 ± 1.94% (mean ± SD; n = 6) and the mean ¹³C enrichment of C1, C2, C5, C6 to C3, C4 was 2.63 ± 0.30.
• 3.3. Since the distribution of tracer in glucose is a function of the relative entry rates of pyruvate to acetyl-CoA into the oxaloacetate pool this was calculated to be 0.32 ± 0.15 and the factor for carbon exchange (1/P) between the gluconeogenic pathway and the TCA cycle was calculated to be 1.03 ± 0.20.
• 4.4. With this carbon exchange factor and the approximated ¹³C enrichment of acetyl-CoA the intramitochondrial ¹³C enrichment of phosphoenolpyruvate was calculated and the “true” rate of hepatic gluconeogenesis from phosphoenolpyruvate estimated.
• 5.5. Since acetate was metabolized solely in liver cells the ¹³C enrichment of acetyl-CoA could be approximated from that of 3-hydroxybutyrate.
• 6.6. The carbon 13 enrichment of 3-hydroxybutyrate and phosphoenolpyruvate was 5.89 ± 0.90% and 5.96 ± 1.67%, respectively.
• 7.7. The per cent gluconeogenesis from phosphoenolpyruvate calculated as the ratio of the ¹³C enrichment of glucose to that of 3-hydroxybutyrate times 1/P was 107 ± 8%.
• 8.8. In this study the validity of assessing isotopic exchange at oxaloacetate as suggested by Katz [Katz J. (1985) Am. J. Physiol.248, R391–R399] when interpretation of the data are not obscured by pseudoketogenesis.
• 9.9. Magnetic resonance spectroscopy provides direct information about intramolecular tracer distribution by which flux rates in major metabolic pathways are derived.

     

In vitro metabolism of [2-13C]-ethanol by 1H NMR spectroscopy using 13C decoupling with the reverse dept polarization-transfer pulse sequence

The metabolism of [2-13C]-ethanol by alcohol dehydrogenase purified from Drosophila melanogaster has been observed by proton nuclear magnetic resonance spectroscopy (NMR). The reverse-DEPT pulse sequence, with composite pulse 13C decoupling to simplify and increase the signal-to-noise of spectra, has been used to eliminate the strong water signal while still observing the proton signals of metabolites of interest. Using these techniques the rates of synthesis of acetaldehyde, its diol and acetate from [2-13C] ethanol by alcohol dehydrogenase were measured simultaneously.     

13C tracer experiments and metabolite balancing for metabolic flux analysis: comparing two approaches

Conventional metabolic flux analysis uses the information gained from determination of measurable fluxes and a steady-state assumption for intracellular metabolites to calculate the metabolic fluxes in a given metabolic network. The determination of intracellular fluxes depends heavily on the correctness of the assumed stoichiometry including the presence of all reactions with a noticeable impact on the model metabolite balances. Determination of fluxes in complex metabolic networks often requires the inclusion of NADH and NADPH balances, which are subject to controversial debate. Transhydrogenation reactions that transfer reduction equivalents from NADH to NADPH or vice versa can usually not be included in the stoichiometric model, because they result in singularities in the stoichiometric matrix. However, it is the NADPH balance that, to a large extent, determines the calculated flux through the pentose phosphate pathway. Hence, wrong assumptions on the presence or activity of transhydrogenation reactions will result in wrong estimations of the intracellular flux distribution. Using 13C tracer experiments and NMR analysis, flux analysis can be performed on the basis of only well established stoichiometric equations and measurements of the labeling state of intracellular metabolites. Neither NADH/NADPH balancing nor assumptions on energy yields need to be included to determine the intracellular fluxes. Because metabolite balancing methods and the use of 13C labeling measurements are two different approaches to the determination of intracellular fluxes, both methods can be used to verify each other or to discuss the origin and significance of deviations in the results. Flux analysis based entirely on metabolite balancing and flux analysis, including labeling information, have been performed independently for a wild-type strain of Aspergillus oryzae producing alpha-amylase. Two different nitrogen sources, NH4+ and NO3-, have been used to investigate the influence of the NADPH requirements on the intracellular flux distribution. The two different approaches to the calculation of fluxes are compared and deviations in the results are discussed. Copyright 1998 John Wiley & Sons, Inc.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Metabolic profiling by 13C-NMR spectroscopy: [1,2-13C2]glucose reveals a heterogeneous metabolism in human leukemia T cells

Metabolic profiling is defined as the simultaneous assessment of substrate fluxes within and among the different pathways of metabolite synthesis and energy production under various physiological conditions. The use of stable-isotope tracers and the analysis of the distribution of labeled carbons in various intermediates, by both mass spectrometry and NMR spectroscopy, allow the role of several metabolic processes in cell growth and death to be defined. In the present paper we describe the metabolic profiling of Jurkat cells by isotopomer analysis using (13)C-NMR spectroscopy and [1,2-(13)C(2)]glucose as the stable-isotope tracer. The isotopomer analysis of the lactate, alanine, glutamate, proline, serine, glycine, malate and ribose-5-phosphate moiety of nucleotides has allowed original integrated information regarding the pentose phosphate pathway, TCA cycle, and amino acid metabolism in proliferating human leukemia T cells to be obtained. In particular, the contribution of the glucose-6-phosphate dehydrogenase and transketolase activities to phosphoribosyl-pyrophosphate synthesis was evaluated directly by the determination of isotopomers of the [1'-(13)C], [4',5'-(13)C(2)]ribosyl moiety of nucleotides. Furthermore, the relative contribution of the glycolysis and pentose cycle to lactate production was estimated via analysis of lactate isotopomers. Interestingly, pyruvate carboxylase and pyruvate dehydrogenase flux ratios measured by glutamate isotopomers and the production of isotopomers of several metabolites showed that the metabolic processes described could not take place simultaneously in the same macrocompartments (cells). Results revealed a heterogeneous metabolism in an asynchronous cell population that may be interpreted on the basis of different metabolic phenotypes of subpopulations in relation to different cell cycle phases.     

Development and application of aromatic [13C, 1H] SOFAST-HMQC NMR experiment for nucleic acids

Higher sensitivity of NMR spectrometers and novel isotopic labeling schemes have ushered the development of rapid data acquisition methodologies, improving the time resolution with which NMR data can be acquired. For nucleic acids, longitudinal relaxation optimization in conjunction with Ernst angle excitation (SOFAST-HMQC) for imino protons, in addition to rendering rapid pulsing, has been demonstrated to yield significant improvements in sensitivity per unit time. Extending such methodology to other spins offers a viable prospect to measure additional chemical shifts, thereby broadening their utilization for various applications. Here, we introduce the 2D [¹³C, ¹H] aromatic SOFAST-HMQC that results in overall sensitivity gain of 1.4- to 1.7-fold relative to the conventional HMQC and can also be extended to yield long-range heteronuclear chemical shifts such as the adenine imino nitrogens N1, N3, N7 and N9. The applications of these experiments range from monitoring real-time biochemical processes, drug/ligand screening, and to collecting data at very low sample concentration and/or in cases where isotopic enrichment cannot be achieved.     

The trans labilization of cis-[PtCl2(13CH3NH2)2] by glutathione can be monitored at physiological pH by [1H,13C] HSQC NMR

In order to monitor the trans labilization of cisplatin at physiological pH we have prepared the complex cis-[PtCl₂(¹³CH₃NH₂)₂] and studied its interactions with excess glutathione in aqueous solution at neutral pH by two-dimensional [1H,13C] heteronuclear single-quantum correlation (HSQC) NMR spectroscopy. [1H,13C] HSQC spectroscopy is a good method for following the release of ¹³CH₃NH₂ but is not so good for characterizing the Pt species in solution. In the reaction of cisplatin with glutathione, Pt–S bonds are formed and Pt–NH₃ bonds are broken. The best technique for following the formation of Pt–S bonds of cisplatin is by UV spectroscopy. [1H,13C] HSQC spectroscopy is the best method for following the breaking of the Pt–N bonds. [1H,15N] HSQC spectroscopy is the best method for characterizing the different species in solution. However, the intensity of the peaks in the ¹⁵NH₃–Pt–S region, in [1H,15N] HSQC, reflects a balance between the formation of Pt–S bonds, which increases the signal intensity, and the trans labilization, which decreases the signal intensity. [1H,15N] HSQC spectroscopy and [1H,13C] HSQC spectroscopy are complementary techniques that should be used in conjunction in order to obtain the most accurate information on the interaction of platinum complexes with sulfur-containing ligands.     

Metabolic profiling of Rhodiola rosea rhizomes by 1H NMR spectroscopy

Introduction – Rhodiola rosea is a broadly used medicinal plant with largely unexplored natural variability in secondary metabolite levels. Objective – The aim of this work was to develop a non‐target procedure for ¹H NMR spectroscopic fingerprinting of rhizome extracts for pattern recognition analysis and identification of secondary metabolites responsible for differences in sample composition. To achieve this, plants from three different geographic areas (Swiss Alps, Finland, and Altai region in Siberia) were investigated. Results – A sample preparation procedure was developed in order to remove polymeric polyphenols as the ¹H NMR analysis of low‐molecular‐weight metabolites was hampered by the presence of tannins. Principal component analysis disclosed tight clustering of samples according to population. PCA models based on the aromatic region of the spectra showed that the first two components reflected changes in the content of salidroside and rosavin, respectively, the rosavin content being negatively correlated to that of rhodiocyanoside A and minor aromatics. Score plots and non‐parametric variance tests demonstrated population‐dependent changes according to harvest time. Data consistency was assessed using score plots and box‐and‐whisker graphs. In addition, a procedure for presenting loadings of PCA models based on bucketed data as high‐resolution plots, which are reminiscent of real ¹H NMR spectra and help to identify latent biomarkers, is presented. Conclusion – This study demonstrated the usefulness of the established procedure for multivariate non‐target ¹H NMR metabolic profiling of Rhodiola rosea. Copyright © 2010 John Wiley & Sons, Ltd.     

Assignment of overlapping (1)H NMR signals in carp seminal plasma by proton-detected 2D C,H correlation spectroscopy

The (1)H NMR spectrum of the perchloric acid extract of carp seminal plasma was heavily congested. It is demonstrated that proton-detected C,H chemical shift correlation spectroscopy (HSQC, HSQC-TOCSY) allows an unequivocal identification of proline, glutamate, taurine, and methionine sulfoxide, although several key proton signals were strongly overlapped.     

Bioreaction network topology and metabolic flux ratio analysis by biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy

Biosynthetically directed fractional 13C labeling of the proteinogenic amino acids is achieved by feeding a mixture of uniformly 13C-labeled and unlabeled carbon source compounds into a bioreaction network. Analysis of the resulting labeling pattern enables both a comprehensive characterization of the network topology and the determination of metabolic flux ratios. Attractive features with regard to routine applications are (i) an inherently small demand for 13C-labeled source compounds and (ii) the high sensitivity of two-dimensional [13C,1H]-correlation nuclear magnetic resonance spectroscopy for analysis of 13C-labeling patterns. A user-friendly program, FCAL, is available to allow rapid data analysis. This novel approach, which recently also has been employed in conjunction with metabolic flux balancing to obtain reliable estimates of in vivo fluxes, enables efficient support of metabolic engineering and biotechnology process design.     

Measurements of the anaplerotic rate in the human cerebral cortex using 13C magnetic resonance spectroscopy and [1-13C] and [2-13C] glucose

Recent studies in rodent and human cerebral cortex have shown that glutamate-glutamine neurotransmitter cycling is rapid and the major pathway of neuronal glutamate repletion. The rate of the cycle remains controversial in humans, because glutamine may come either from cycling or from anaplerosis via glial pyruvate carboxylase. Most studies have determined cycling from isotopic labeling of glutamine and glutamate using a [1-(13)C]glucose tracer, which provides label through neuronal and glial pyruvate dehydrogenase or via glial pyruvate carboxylase. To measure the anaplerotic contribution, we measured (13)C incorporation into glutamate and glutamine in the occipital-parietal region of awake humans while infusing [2-(13)C]glucose, which labels the C2 and C3 positions of glutamine and glutamate exclusively via pyruvate carboxylase. Relative to [1-(13)C]glucose, [2-(13)C]glucose provided little label to C2 and C3 glutamine and glutamate. Metabolic modeling of the labeling data indicated that pyruvate carboxylase accounts for 6 +/- 4% of the rate of glutamine synthesis, or 0.02 micromol/g/min. Comparison with estimates of human brain glutamine efflux suggests that the majority of the pyruvate carboxylase flux is used for replacing glutamate lost due to glial oxidation and therefore can be considered to support neurotransmitter trafficking. These results are consistent with observations made with arterial-venous differences and radiotracer methods.