Quantification of compartmented metabolic fluxes in developing soybean embryos by employing biosynthetically directed fractional (13)C labeling, two-dimensional [(13)C, (1)H] nuclear magnetic resonance, and comprehensive isotopomer balancing

Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis tool that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-(13)C Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [(13)C, (1)H] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 (13)C isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain sds. A high flux was found through the oxidative pentose phosphate pathway (19.99 +/- 4.39 micromol d(-1) cotyledon(-1), or 104.2 carbon mol +/- 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 +/- 5.00 micromol d(-1) cotyledon(-1), or 113.2 carbon mol +/-26.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.     

Combining metabolic flux analysis tools and 13C NMR to estimate intracellular fluxes of cultured astrocytes

In this work, brain cell metabolism was investigated by (13)C NMR spectroscopy and metabolic flux analysis (MFA). Monotypic cultures of astrocytes were incubated with labeled glucose for 38 h, and the distribution of the label was analyzed by (13)C NMR spectroscopy. The analysis of the spectra reveals two distinct physiological states characterized by different ratios of pyruvate carboxylase to pyruvate dehydrogenase activities (PC/PDH). Intracellular flux distributions for both metabolic states were estimated by MFA using the isotopic information and extracellular rate measurements as constraints. The model was subsequently checked with the consistency index method. From a biological point of view, the occurrence of the two physiological states appears to be correlated with the presence or absence of extracellular glutamate. Concerning the model, it can be stated that the metabolic network and the set of constraints adopted provide a consistent and robust characterization of the astrocytic metabolism, allowing for the calculation of central intracellular fluxes such as pyruvate recycling, the anaplerotic flux mediated by pyruvate carboxylase, and the glutamine formation through glutamine synthetase.     

13C metabolic flux analysis (MFA) to find out the metabolic fluxes of biomass production and lipid accumulation in Neochloris oleoabundans UTEX 1185

Journal of Applied Phycology - Neochloris oleoabundans UTEX 1185 is a green microalga that is considered as a promising feedstock for microalgal biodiesel production as it contains high amount of...     

Quantifying the effects of long-range 13C-13C dipolar coupling on measured relaxation rates in RNA

Journal of Biomolecular NMR - Selective stable isotope labeling has transformed structural and dynamics analysis of RNA by NMR spectroscopy. These methods can remove 13C-13C dipolar couplings that...     

Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13C- and 1H-MR spectroscopy.

Information displayed by homonuclear and heteronuclear spin-coupling patterns in 13C- and 1H-MR spectra allowed us to identify the major lactate isotopomers produced either from [1-(13)C]-glucose or from [2-(13)C]-glucose by human erythrocytes. Relative concentrations of detectable isotopomers were determined by integrating the corresponding MR signals. The interpretation of these data in terms of the fractional glucose metabolised through glycolysis and pentose phosphate pathway was performed by a computer simulation of the metabolism that took into account metabolic schemes pertaining to glycolysis and to the F-type of pentose phosphate pathway. The simulation was organised in a way to anticipate the populations of the isotopomers produced from any precursor at a priori established metabolic steady state. By the simulation, isotopomer populations were determined according to different values of pentose cycle, defined as the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. The populations of the isotopomers originating from [2-(13)C]-glucose were described by polynomials, and ratios between the polynomials were used in conjunction with 13C- and 1H-MR data to determine pentose cycle values. The knowledge of glucose uptake and of pentose cycle value allowed us to perform accurate measurement of the pentose phosphate pathway flux, of the hexokinase and phosphofructokinase fluxes as well as, indirectly, of the carbon dioxide production.     

In vivo 13C NMR determines metabolic fluxes and steady state in linseed embryos

The dynamics of developing linseed embryo metabolism was investigated using (13)C-labelling experiments where the real-time kinetics of label incorporation into metabolites was monitored in situ using in vivo NMR. The approach took advantage of the occurrence in this plant tissue of large metabolite pools - such as sucrose or lipids - to provide direct and quantitative measurement of the evolution of the labelling state within central metabolism. As a pre-requisite for the use of steady state flux measurements it was shown that isotopic steady state was reached within 3 h at the level of central intermediates whereas it took a further 6h for the sucrose pool. Complete isotopic and metabolic steady state took 18 h to be reached. The data collected during the transient state where label was equilibrated but the metabolic steady state was incomplete, enabled the rates of lipid and sucrose synthesis to be measured in situ on the same sample. This approach is suitable to get a direct assessment of metabolic time-scales within living plant tissues and provides a valuable complement to steady state flux determinations.     

13C‐metabolic flux analysis for batch culture of Escherichia coli and its pyk and pgi gene knockout mutants based on mass isotopomer distribution of intracellular metabolites

Since most bio‐production processes are conducted in a batch or fed‐batch manner, the evaluation of metabolism with respect to time is highly desirable. Toward this aim, we applied ¹³C‐metabolic flux analysis to nonstationary conditions by measuring the mass isotopomer distribution of intracellular metabolites. We performed our analysis on batch cultures of wild‐type Escherichia coli, as well as on Pyk and Pgi mutants, obtained the fluxes and metabolite concentrations as a function of time. Our results for the wild‐type indicated that the TCA cycle flux tended to increase during growth on glucose. Following glucose exhaustion, cells controlled the branch ratio between the glyoxylate pathway and the TCA cycle, depending on the availability of acetate. In the Pyk mutant, the concentrations of glycolytic intermediates changed drastically over time due to the dumping and feedback inhibition caused by PEP accumulation. Nevertheless, the flux distribution and free amino acid concentrations changed little. The growth rate and the fluxes remained constant in the Pgi mutant and the glucose‐6‐phosphate dehydrogenase reaction was the rate‐limiting step. The measured fluxes were compared with those predicted by flux balance analysis using maximization of biomass yield or ATP production. Our findings indicate that the objective function of biosynthesis became less important as time proceeds on glucose in the wild‐type, while it remained highly important in the Pyk mutant. Furthermore, ATP production was the primary objective function in the Pgi mutant. This study demonstrates how cells adjust their metabolism in response to environmental changes and/or genetic perturbations in the batch cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Quantitative analysis of metabolic fluxes in Escherichia coli, using two-dimensional NMR spectroscopy and complete isotopomer models.

Complete isotopomer models that simulate distribution of label in 13C tracer experiments are applied to the quantification of metabolic fluxes in the primary carbon metabolism of E. coli under aerobic and anaerobic conditions. The concept of isotopomer mapping matrices (IMMs) is used to simplify the formulation of isotopomer mass balances by expressing all isotopomer mass balances of a metabolite pool in a single matrix equation. A numerically stable method to calculate the steady-state isotopomer distribution in metabolic networks in introduced. Net values of intracellular fluxes and the degree of reversibility of enzymatic steps are estimated by minimization of the deviations between experimental and simulated measurements. The metabolic model applied includes the Embden-Meyerhof-Parnas and the pentose phosphate pathway, the tricarboxylic acid cycle, anaplerotic reaction sequences and pathways involved in amino acid synthesis. The study clearly demonstrates the value of complete isotopomer models for maximizing the information obtainable from 13C tracer experiments. The approach applied here offers a completely general and comprehensive analysis of carbon tracer experiments where any set of experimental data on the labeling state and extracellular fluxes can be used for the quantification of metabolic fluxes in complex metabolic networks.     

The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK-PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.     

Quantification of the concentration and 13C tracer enrichment of long-chain fatty acyl-coenzyme A in muscle by liquid chromatography/mass spectrometry

Recent diabetes and obesity research has been focused on the role of intracellular lipids in insulin resistance. Fatty acyl-coenzyme A (CoA) esters play a central role in the trafficking of intracellular lipids, but there has not previously been a method with which to quantify their kinetics using tracer methodology. We have therefore developed a high-performance liquid chromatography (HPLC)-mass spectrometry method to simultaneously measure the (13)C stable isotopic enrichment of palmitoyl-acyl-CoA ester and the concentrations of five individual long-chain fatty acyl-CoA esters extracted from muscle tissue samples. The long-chain fatty acyl-CoA can be effectively extracted from frozen muscle tissue samples and baseline separated by a reverse-phase HPLC with the presence of a volatile reagent-triethylamine. Negative ion electrospray mass spectrometry with selected ion monitoring was used to analyze the fatty acyl-CoAs to achieve reliable quantification of their concentrations and (13)C isotopic enrichment. Applying this protocol to rabbit muscle samples demonstrates that it is a sensitive, accurate, and precise method for the quantification of long-chain fatty acyl-CoA concentrations and enrichment.     

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using ¹³C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that ¹³C-[2] acetate or ¹³C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using ¹³C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/¹³C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.     

Metabolic studies of pyruvate- and lactate-perfused guinea pig hearts by 13C NMR. Determination of substrate preference by glutamate isotopomer distribution

Guinea pig hearts perfused in vitro with 99% enriched [3-13C]pyruvate and [3-13C]lactate have been examined by 13C NMR spectroscopy at 75.45 MHz. Resonances from the intracellular metabolites, glutamate, aspartate, alanine, citrate, malate, lactate, and acetylcarnitine are detected in the [3-13C]pyruvate-perfused hearts while glutamate is the only metabolite observed in the [3-13C]lactate-perfused hearts. Extracts obtained from individual freeze-clamped hearts run under high resolution conditions show similar distributions of metabolites indicating that the intracellular concentrations of these metabolites are indeed quite dependent upon the substrate being utilized. The excellent signal to noise in the spectra of lactate-perfused hearts allows observation of spin-spin coupling between 13C-enriched nuclei in the various glutamate isotopomers within the intact heart. It is shown that the steady-state distribution of glutamate isotopomers is determined by the amount of 12C- versus 13C-enriched acetyl-CoA entering the citric acid cycle and this provides the basis for a direct determination of substrate utilization by the heart in the presence of competing substrates. Similar information may be derived from the 13C spectrum of an extract prepared from a single [3-13C]pyruvate-perfused heart. Our results indicate that lactate is preferred over glucose by the guinea pig heart even in the presence of insulin.     

Transport and intracellular distribution of components of the cortisol-asialotranscortin complex in the liver

The effect of desialation of human transcortin on the transcortin-cortisol complex transport into hepatocytes and on subsequent intracellular distribution of the complex components was studied in model experiments with perfused rat liver. It was demonstrated that in the presence of desialated transcortin the time of [3H]cortisol incubation in the perfusion medium decreased more than 200 times as compared with the native protein. [3H]cortisol and [131I]asialotranscortin trapping by hepatocytes occurs simultaneously. The content of the [3H]cortisol--[131I]asialotranscortin complex in rat liver plasma membranes reaches a maximum 3 min after beginning of perfusion. Then the intact complex is transported into lysosomes, where it dissociates with a subsequent release of asialotranscortin and cortisol metabolites into the cytoplasm.     

Detection of metabolic fluxes of o and h atoms into intracellular water in Mammalian cells

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.     

13C-1H inter-residue,coupling in disaccharides,and the orientations of glycosidic bonds

In examining orientations of glycosidic linkages, measurements of three-bond coupling between ¹³C-1 and ¹H-4′, or ¹³C-4′ and ¹H-1, have been made from natural abundance, ¹H-coupled, ¹³C-n.m r. spectra of maltose, cyclohexaamylose, and related compounds. Maltose and cyclohexaamylose in water exhibit inter-residue ¹³COC¹H couplings of close to 3 Hz. In terms of torsional angles, φ and ψ, these findings suggest that, in aqueous solution, the molecules favor conformations that are appreciably more staggered than those known to exist in the solid state. Analogous measurements on O-acetyl derivatives suggest that φ is smaller, and ψ larger, than in maltose. Data are also presented for sucrose, maltosan, and α,α-trehalose.     

Assessment of the production of 13C labeled compounds from phototrophic microalgae at laboratory scale

An integrated process for the indoor production of 13C labeled polyunsaturated fatty acids (PUFAs) from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor operating with recirculation of the exhaust gas using a low-pressure compressor. Oxygen accumulation in the system is avoided by bubbling the exhaust gas from the reactor in a sodium sulfite solution before returning to it. To achieve a high 13C enrichment in the biomass obtained, the culture medium is initially stripped of carbon, and labeled 13CO(2) is automatically injected on-demand during operation for pH control and carbon supply. The reactor was operated in both batch and semicontinuous modes. In semicontinuous mode, the reactor was operated at a dilution rate of 0.01 h(-1), resulting in a biomass productivity of 0.1 g l(-1) per day. The elemental analysis of the inlet and outlet flows of the reactor showed that 64.9% of carbon was turned into microalgal biomass, 34.9% remained in the supernatant mainly as inorganic compounds. Only 3.8% of injected carbon was effectively fixed as the target labeled product (EPA). Regarding the isotopic composition of fatty acids, results showed that fatty acids were not labeled in the same proportion, the higher the number of carbons the lower the percentage of 13C. Isotopic composition of EPA ranged from 36.5 to 53.5%, as a function of the methodology used (GC-MS, EA-IRMS or gas chromatography-combustion-isotope ratio mass spectrometry (GC-IRMS)). The low carbon uptake efficiency combined with the high cost of 13CO(2) make necessary to redefine the designed culture system to increase the efficiency of the conversion of 13CO(2) into the target product. Therefore, the possibility of removing 12C from the fresh medium, and recovering and recirculating the inorganic carbon in the supernatant and the organic carbon from the EPA depleted biomass was studied. The inorganic carbon of the fresh medium was removed by acidification and stripping with N(2). The inorganic carbon of the supernatant was recovered also by acidification and subsequent stripping with N(2). The operating conditions of this step were optimized for gas flow rate and type of contactor. A carbon recovery step for the depleted biomass was designed based on the catalytic oxidation to CO(2) using CuO (10 wt.%) as catalyst with an oxygen enriched atmosphere (80% O(2) partial pressure). In this way, the carbon losses reduced an 80.2% and the efficiency of the conversion of carbon in EPA was increased to 19.5%, which is close to the theoretical maximum. Further increase in 13CO(2) use efficiency is only possible by additionally recovering other labeled by-products present in the biomass: proteins, carbohydrates, lipids, and pigments.     

Utilization of 13C-13C Coupling in Structural and Biosynthetic Studies Double Labeling Method

A new method which utilizes ¹³C-¹³C coupling for structural and biosynthetic studies on acetate-derived metabolites is described. The ¹³C-NMR spectra of dihydrolatumcidins separately labeled with ¹³CH₃¹³C0₂Na and with a 1: 1 mixture of ¹³CH₃CO₂-Na and CH313C02-Na gave enough information to establish its structure.     

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the ¹³C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-¹³C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the ¹³C enrichment. The approach to correct for these effects in metabolic flux studies using δ¹³C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and ¹³C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of ¹³C in proteinogenic amino acids over the whole range of applied concentrations of [1-¹³C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in ¹³C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.