Isotopic tracing with carbon-13 in primary hepatocytes

2019 ◽  
Author(s):  
Katherine A Overmyer
Keyword(s):  
Culture Medium ◽  
Metabolic Flux ◽  
Isotopic Labeling ◽  
Flux Analysis ◽  
Isotopic Enrichment ◽  
Primary Hepatocytes ◽  
Expected Time ◽  
Isotopic Tracing ◽  
Intracellular Metabolism ◽  
Genetic Mutants

Abstract Isotopic labeling is commonly applied for investigating intracellular metabolism. The general workflow is to first introduce isotopically-labeled metabolites into the culture medium, then at defined time points wash and harvest cells, process samples for metabolomics analysis, and analyze the samples for isotopic enrichment in specified metabolite pools. Here we apply this technique to primary hepatocytes from mice. We introduce either 13C5 glutamine or 13C6 glucose at the typical media concentrations, 1:1 replacing the 12C version with 13C version. Cells are harvested at 0 and 30 min after isotope introduction, metabolites are extracted and then analyzed by GC-MS and LC-MS. The resulting data are used to compare relative 13C isotopic labeling in metabolites between different genetic mutants. This strategy is not suitable for directly quantifying metabolic flux (i.e., Metabolic flux analysis), but is useful for describing relative metabolic flux between two models. The expected time to complete is ~3-5 days.

scholarly journals The metabolic origins of non-photorespiratory CO2 release during photosynthesis: A metabolic flux analysis

2021 ◽  
Author(s):  
Yuan Xu ◽  
Xinyu Fu ◽  
Thomas D Sharkey ◽  
Yair Shachar-Hill ◽  
Berkley J Walker
Keyword(s):  
Gas Exchange ◽  
Goodness Of Fit ◽  
Time Course ◽  
Metabolic Flux ◽  
Carbon Fixation ◽  
Tca Cycle ◽  
Isotopic Labeling ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Oilseed Crop

Abstract Respiration in the light (RL) releases CO2 in photosynthesizing leaves and is a phenomenon that occurs independently from photorespiration. Since RL lowers net carbon fixation, understanding RL could help improve plant carbon-use efficiency and models of crop photosynthesis. Although RL was identified more than 75 years ago, its biochemical mechanisms remain unclear. To identify reactions contributing to RL, we mapped metabolic fluxes in photosynthesizing source leaves of the oilseed crop and model plant camelina (Camelina sativa). We performed a flux analysis using isotopic labeling patterns of central metabolites during 13CO2 labeling time course, gas exchange and carbohydrate production rate experiments. To quantify the contributions of multiple potential CO2 sources with statistical and biological confidence, we increased the number of metabolites measured and reduced biological and technical heterogeneity by using single mature source leaves and quickly quenching metabolism by directly injecting liquid N2; we then compared the goodness-of-fit between these data and data from models with alternative metabolic network structures and constraints. Our analysis predicted that RL releases 5.2 μmol CO2 g−1 FW hr−1 of CO2, which is relatively consistent with a value of 9.3 μmol CO2 g−1 FW hr−1 measured by CO2 gas exchange. The results indicated that ≤10% of RL results from TCA cycle reactions, which are widely considered to dominate RL. Further analysis of the results indicated that oxidation of glucose-6-phosphate to pentose phosphate via 6-phosphogluconate (the G6P/OPP shunt) can account for >93% of CO2 released by RL.

scholarly journals Accumulation of Homolanthionine and Activation of a Novel Pathway for Isoleucine Biosynthesis in Corynebacterium glutamicum McbR Deletion Strains

2006 ◽  
Vol 188 (2) ◽  
pp. 609-618 ◽  
Author(s):  
Jens Olaf Krömer ◽  
Elmar Heinzle ◽  
Hartwig Schröder ◽  
Christoph Wittmann
Keyword(s):  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Side Reaction ◽  
Isotopic Labeling ◽  
Gas Chromatography Mass Spectrometry ◽  
Substrate Affinity ◽  
Flux Analysis ◽  
Methionine Biosynthesis ◽  
Metabolome Analysis ◽  
Isoleucine Biosynthesis

ABSTRACT In the present work, the metabolic consequences of the deletion of the methionine and cysteine biosynthesis repressor protein (McbR) in Corynebacterium glutamicum, which releases almost all enzymes of methionine biosynthesis and sulfate assimilation from transcriptional regulation (D. A. Rey, A. Pühler, and J. Kalinowski, J. Biotechnol. 103:51-65, 2003), were studied. C. glutamicum ATCC 13032 ΔmcbR showed no overproduction of methionine. Metabolome analysis revealed drastic accumulation of a single metabolite, which was not present in the wild type. It was identified by isotopic labeling studies and gas chromatography/mass spectrometry as l-homolanthionine {S-[(3S)-3-amino-3-carboxypropyl]-l-homocysteine}. The accumulation of homolanthionine to an intracellular concentration of 130 mM in the ΔmcbR strain was accompanied by an elevated intracellular homocysteine level. It was shown that cystathionine-γ-synthase (MetB) produced homolanthionine as a side reaction. MetB showed higher substrate affinity for cysteine (Km = 260 μM) than for homocysteine (Km = 540 μM). The cell is able to cleave homolanthionine at low rates via cystathionine-β-lyase (MetC). This cleavage opens a novel threonine-independent pathway for isoleucine biosynthesis via 2-oxobutanoate formed by MetC. In fact, the deletion mutant exhibited an increased intracellular isoleucine level. Metabolic flux analysis of C. glutamicum ΔmcbR revealed that only 24% of the O-acetylhomoserine at the entry of the methionine pathway is utilized for methionine biosynthesis; the dominating fraction is either stored as homolanthionine or redirected towards the formation of isoleucine. Deletion of metB completely prevents homolanthionine accumulation, which is regarded as an important step in the development of C. glutamicum strains for biotechnological methionine production.

scholarly journals OpenMebius: An Open Source Software for Isotopically Nonstationary13C-Based Metabolic Flux Analysis

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Shuichi Kajihata ◽  
Chikara Furusawa ◽  
Fumio Matsuda ◽  
Hiroshi Shimizu
Keyword(s):  
Open Source ◽  
Open Source Software ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Simulated Data ◽  
Isotopic Labeling ◽  
Metabolic Model ◽  
Cell Physiology ◽  
Flux Analysis

Thein vivomeasurement of metabolic flux by13C-based metabolic flux analysis (13C-MFA) provides valuable information regarding cell physiology. Bioinformatics tools have been developed to estimate metabolic flux distributions from the results of tracer isotopic labeling experiments using a13C-labeled carbon source. Metabolic flux is determined by nonlinear fitting of a metabolic model to the isotopic labeling enrichment of intracellular metabolites measured by mass spectrometry. Whereas13C-MFA is conventionally performed under isotopically constant conditions, isotopically nonstationary13C metabolic flux analysis (INST-13C-MFA) has recently been developed for flux analysis of cells with photosynthetic activity and cells at a quasi-steady metabolic state (e.g., primary cells or microorganisms under stationary phase). Here, the development of a novel open source software for INST-13C-MFA on the Windows platform is reported. OpenMebius (Open source software for Metabolic flux analysis) provides the function of autogenerating metabolic models for simulating isotopic labeling enrichment from a user-defined configuration worksheet. Analysis using simulated data demonstrated the applicability of OpenMebius for INST-13C-MFA. Confidence intervals determined by INST-13C-MFA were less than those determined by conventional methods, indicating the potential of INST-13C-MFA for precise metabolic flux analysis. OpenMebius is the open source software for the general application of INST-13C-MFA.

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