Stable Isotope Guided Metabolomics

A special issue of Metabolites (ISSN 2218-1989). This special issue belongs to the section "Advances in Metabolomics".

Deadline for manuscript submissions: closed (15 September 2020) | Viewed by 25891

Special Issue Editor

Diabetes and Obesity Center, Christina Lee Brown Envirome Institute, Louisville, KY 40202, USA
Interests: stable isotope resolved metabolomics; method development; fluxomics; metabolic pathway activity; high-resolution mass spectrometry
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The use of stable isotope-labeled compounds in studying metabolism and biological processes is widespread. The Special Issue of Metabolites, "Stable Isotope-Guided Metabolomics", will focus on cutting-edge and newly established metabolomics technologies and workflows that significantly benefit from the use of stable isotopically labeled compounds. This includes methodologies for in vitro and in vivo stable metabolite pathway tracing and flux analyses, as well as applications that improve quality, confidence, and validation of metabolomics analyses and aid in metabolite assignments and quantification. In this Special Issue, we aim to cover a variety of topics, including experimental design, workflows for the analysis of isotopically enriched data, statistical and computational approaches for data analysis and interpretation, and other important topics.

Dr. Pawel Lorkiewicz
Guest Editor

Manuscript Submission Information

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Keywords

  • stable isotopes
  • flux analysis
  • pathway tracing
  • isotope labeling

Published Papers (8 papers)

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Research

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15 pages, 3866 KiB  
Article
NMR Methods for Determining Lipid Turnover via Stable Isotope Resolved Metabolomics
by Penghui Lin, Li Dai, Daniel R. Crooks, Leonard M. Neckers, Richard M. Higashi, Teresa W-M. Fan and Andrew N. Lane
Metabolites 2021, 11(4), 202; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11040202 - 29 Mar 2021
Cited by 7 | Viewed by 2634
Abstract
Lipids comprise diverse classes of compounds that are important for the structure and properties of membranes, as high-energy fuel sources and as signaling molecules. Therefore, the turnover rates of these varied classes of lipids are fundamental to cellular function. However, their enormous chemical [...] Read more.
Lipids comprise diverse classes of compounds that are important for the structure and properties of membranes, as high-energy fuel sources and as signaling molecules. Therefore, the turnover rates of these varied classes of lipids are fundamental to cellular function. However, their enormous chemical diversity and dynamic range in cells makes detailed analysis very complex. Furthermore, although stable isotope tracers enable the determination of synthesis and degradation of complex lipids, the numbers of distinguishable molecules increase enormously, which exacerbates the problem. Although LC-MS-MS (Liquid Chromatography-Tandem Mass Spectrometry) is the standard for lipidomics, NMR can add value in global lipid analysis and isotopomer distributions of intact lipids. Here, we describe new developments in NMR analysis for assessing global lipid content and isotopic enrichment of mixtures of complex lipids for two cell lines (PC3 and UMUC3) using both 13C6 glucose and 13C5 glutamine tracers. Full article
(This article belongs to the Special Issue Stable Isotope Guided Metabolomics)
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9 pages, 1320 KiB  
Article
Application of Carbon-13 Isotopomer Analysis to Assess Perinatal Myocardial Glucose Metabolism in Sheep
by Mukundan Ragavan, Mengchen Li, Anthony G. Giacalone, Charles E. Wood, Maureen Keller-Wood and Matthew E. Merritt
Metabolites 2021, 11(1), 33; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11010033 - 05 Jan 2021
Cited by 2 | Viewed by 2603
Abstract
Ovine models of pregnancy have been used extensively to study maternal–fetal interactions and have provided considerable insight into nutrient transfer to the fetus. Ovine models have also been utilized to study congenital heart diseases. In this work, we demonstrate a comprehensive assessment of [...] Read more.
Ovine models of pregnancy have been used extensively to study maternal–fetal interactions and have provided considerable insight into nutrient transfer to the fetus. Ovine models have also been utilized to study congenital heart diseases. In this work, we demonstrate a comprehensive assessment of heart function and metabolism using a perinatal model of heart function with the addition of a [U-13C]glucose as tracer to study central energy metabolism. Using nuclear magnetic resonance spectroscopy, and metabolic modelling, we estimate myocardial citric acid cycle turnover (normalized for oxygen consumption), substrate selection, and anaplerotic fluxes. This methodology can be applied to studying acute and chronic effects of hormonal signaling in future studies. Full article
(This article belongs to the Special Issue Stable Isotope Guided Metabolomics)
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18 pages, 2684 KiB  
Article
Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics
by Holden C. Williams, Margaret A. Piron, Grant K. Nation, Adeline E. Walsh, Lyndsay E. A. Young, Ramon C. Sun and Lance A. Johnson
Metabolites 2020, 10(12), 501; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo10120501 - 08 Dec 2020
Cited by 13 | Viewed by 5279
Abstract
Stable isotope-resolved metabolomics (SIRM) is a powerful tool for understanding disease. Advances in SIRM techniques have improved isotopic delivery and expanded the workflow from exclusively in vitro applications to in vivo methodologies to study systemic metabolism. Here, we report a simple, minimally-invasive and [...] Read more.
Stable isotope-resolved metabolomics (SIRM) is a powerful tool for understanding disease. Advances in SIRM techniques have improved isotopic delivery and expanded the workflow from exclusively in vitro applications to in vivo methodologies to study systemic metabolism. Here, we report a simple, minimally-invasive and cost-effective method of tracer delivery to study SIRM in vivo in laboratory mice. Following a brief fasting period, we orally administered a solution of [U-13C] glucose through a blunt gavage needle without anesthesia, at a physiological dose commonly used for glucose tolerance tests (2 g/kg bodyweight). We defined isotopic enrichment in plasma and tissue at 15, 30, 120, and 240 min post-gavage. 13C-labeled glucose peaked in plasma around 15 min post-gavage, followed by period of metabolic decay and clearance until 4 h. We demonstrate robust enrichment of a variety of central carbon metabolites in the plasma, brain and liver of C57/BL6 mice, including amino acids, neurotransmitters, and glycolytic and tricarboxylic acid (TCA) cycle intermediates. We then applied this method to study in vivo metabolism in two distinct mouse models of diseases known to involve dysregulation of glucose metabolism: Alzheimer’s disease and type II diabetes. By delivering [U-13C] glucose via oral gavage to the 5XFAD Alzheimer’s disease model and the Lepob/ob type II diabetes model, we were able to resolve significant differences in multiple central carbon pathways in both model systems, thus providing evidence of the utility of this method to study diseases with metabolic components. Together, these data clearly demonstrate the efficacy and efficiency of an oral gavage delivery method, and present a clear time course for 13C enrichment in plasma, liver and brain of mice following oral gavage of [U-13C] glucose—data we hope will aid other researchers in their own 13C-glucose metabolomics study design. Full article
(This article belongs to the Special Issue Stable Isotope Guided Metabolomics)
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14 pages, 3342 KiB  
Article
Isotope Fractionation during Gas Chromatography Can Enhance Mass Spectrometry-Based Measures of 2H-Labeling of Small Molecules
by Daniel P. Downes, Takhar Kasumov, Natalie A. Daurio, Neil B. Wood, Michael J. Previs, Payal R. Sheth, David G. McLaren and Stephen F. Previs
Metabolites 2020, 10(11), 474; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo10110474 - 20 Nov 2020
Cited by 3 | Viewed by 1945
Abstract
Stable isotope tracers can be used to quantify the activity of metabolic pathways. Specifically, 2H-water is quite versatile, and its incorporation into various products can enable measurements of carbohydrate, lipid, protein and nucleic acid kinetics. However, since there are limits on how [...] Read more.
Stable isotope tracers can be used to quantify the activity of metabolic pathways. Specifically, 2H-water is quite versatile, and its incorporation into various products can enable measurements of carbohydrate, lipid, protein and nucleic acid kinetics. However, since there are limits on how much 2H-water can be administered and since some metabolic processes may be slow, it is possible that one may be challenged with measuring small changes in isotopic enrichment. We demonstrate an advantage of the isotope fractionation that occurs during gas chromatography, namely, setting tightly bounded integration regions yields a powerful approach for determining isotope ratios. We determined how the degree of isotope fractionation, chromatographic peak width and mass spectrometer dwell time can increase the apparent isotope labeling. Relatively simple changes in the logic surrounding data acquisition and processing can enhance gas chromatography-mass spectrometry measures of low levels of 2H-labeling, this is especially useful when asymmetrical peaks are recorded at low signal:background. Although we have largely focused attention on alanine (which is of interest in studies of protein synthesis), it should be possible to extend the concepts to other analytes and/or hardware configurations. Full article
(This article belongs to the Special Issue Stable Isotope Guided Metabolomics)
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16 pages, 2741 KiB  
Article
Enhanced Metabolome Coverage and Evaluation of Matrix Effects by the Use of Experimental-Condition-Matched 13C-Labeled Biological Samples in Isotope-Assisted LC-HRMS Metabolomics
by Asja Ćeranić, Christoph Bueschl, Maria Doppler, Alexandra Parich, Kangkang Xu, Marc Lemmens, Hermann Buerstmayr and Rainer Schuhmacher
Metabolites 2020, 10(11), 434; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo10110434 - 27 Oct 2020
Cited by 4 | Viewed by 1937
Abstract
Stable isotope-assisted approaches can improve untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomics studies. Here, we demonstrate at the example of chemically stressed wheat that metabolome-wide internal standardization by globally 13C-labeled metabolite extract (GLMe-IS) of experimental-condition-matched biological samples can help to improve [...] Read more.
Stable isotope-assisted approaches can improve untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomics studies. Here, we demonstrate at the example of chemically stressed wheat that metabolome-wide internal standardization by globally 13C-labeled metabolite extract (GLMe-IS) of experimental-condition-matched biological samples can help to improve the detection of treatment-relevant metabolites and can aid in the post-acquisition assessment of putative matrix effects in samples obtained upon different treatments. For this, native extracts of toxin- and mock-treated (control) wheat ears were standardized by the addition of uniformly 13C-labeled wheat ear extracts that were cultivated under similar experimental conditions (toxin-treatment and control) and measured with LC-HRMS. The results show that 996 wheat-derived metabolites were detected with the non-condition-matched 13C-labeled metabolite extract, while another 68 were only covered by the experimental-condition-matched GLMe-IS. Additional testing is performed with the assumption that GLMe-IS enables compensation for matrix effects. Although on average no severe matrix differences between both experimental conditions were found, individual metabolites may be affected as is demonstrated by wrong decisions with respect to the classification of significantly altered metabolites. When GLMe-IS was applied to compensate for matrix effects, 272 metabolites showed significantly altered levels between treated and control samples, 42 of which would not have been classified as such without GLMe-IS. Full article
(This article belongs to the Special Issue Stable Isotope Guided Metabolomics)
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15 pages, 2566 KiB  
Article
Robust Moiety Model Selection Using Mass Spectrometry Measured Isotopologues
by Huan Jin and Hunter N.B. Moseley
Metabolites 2020, 10(3), 118; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo10030118 - 21 Mar 2020
Cited by 5 | Viewed by 2333
Abstract
Stable isotope resolved metabolomics (SIRM) experiments use stable isotope tracers to provide superior metabolomics datasets for metabolic flux analysis and metabolic modeling. Since assumptions of model correctness can seriously compromise interpretation of metabolic flux results, we have developed a metabolic modeling software package [...] Read more.
Stable isotope resolved metabolomics (SIRM) experiments use stable isotope tracers to provide superior metabolomics datasets for metabolic flux analysis and metabolic modeling. Since assumptions of model correctness can seriously compromise interpretation of metabolic flux results, we have developed a metabolic modeling software package specifically designed for moiety model comparison and selection based on the metabolomics data provided. Here, we tested the effectiveness of model selection with two time-series mass spectrometry (MS) isotopologue datasets for uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc) generated from different platforms utilizing direct infusion nanoelectrospray and liquid chromatography. Analysis results demonstrate the robustness of our model selection methods by the successful selection of the optimal model from over 40 models provided. Moreover, the effects of specific optimization methods, degree of optimization, selection criteria, and specific objective functions on model selection are illustrated. Overall, these results indicate that over-optimization can lead to model selection failure, but combining multiple datasets can help control this overfitting effect. The implication is that SIRM datasets in public repositories of reasonable quality can be combined with newly acquired datasets to improve model selection. Furthermore, curation efforts of public metabolomics repositories to maintain high data quality could have a huge impact on future metabolic modeling efforts. Full article
(This article belongs to the Special Issue Stable Isotope Guided Metabolomics)
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12 pages, 1803 KiB  
Article
Addressing Glutathione Redox Status in Clinical Samples by Two-Step Alkylation with N-ethylmaleimide Isotopologues
by Tamara Tomin, Matthias Schittmayer and Ruth Birner-Gruenberger
Metabolites 2020, 10(2), 71; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo10020071 - 16 Feb 2020
Cited by 20 | Viewed by 4066
Abstract
Determination of the ratio of reduced to oxidized glutathione is of profound clinical interest in assessing the oxidative status of tissues and body fluids. However, this ratio is not yet a routine clinical parameter due to the analytically challenging interconversion of reduced (free) [...] Read more.
Determination of the ratio of reduced to oxidized glutathione is of profound clinical interest in assessing the oxidative status of tissues and body fluids. However, this ratio is not yet a routine clinical parameter due to the analytically challenging interconversion of reduced (free) glutathione to oxidized (bound) glutathione. We aimed to facilitate this ratio determination in order to aid its incorporation as a routine clinical parameter. To this end, we developed a simple derivatization route that yields different isotopologues of N-ethylmaleimide alkylated glutathione from reduced and oxidized glutathione (after its chemical reduction) for mass spectrometric analysis. A third isotopologue can be used as isotopic standard for simultaneous absolute quantification. As all isotopologues have similar chromatographic properties, matrix effects arising from different sample origins can only impact method sensitivity but not quantification accuracy. Robustness, simplified data analysis, cost effectiveness by one common standard, and highly improved mass spectrometric sensitivity by conversion of oxidized glutathione to an alkylated glutathione isotopologue are the main advantages of our approach. We present a method fully optimized for blood, plasma, serum, cell, and tissue samples. In addition, we propose production of N-ethylmaleimide customized blood collection tubes to even further facilitate the analysis in a clinical setting. Full article
(This article belongs to the Special Issue Stable Isotope Guided Metabolomics)
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Review

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21 pages, 2832 KiB  
Review
Metabolic Flux Analysis—Linking Isotope Labeling and Metabolic Fluxes
by Yujue Wang, Fredric E. Wondisford, Chi Song, Teng Zhang and Xiaoyang Su
Metabolites 2020, 10(11), 447; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo10110447 - 06 Nov 2020
Cited by 24 | Viewed by 4156
Abstract
Metabolic flux analysis (MFA) is an increasingly important tool to study metabolism quantitatively. Unlike the concentrations of metabolites, the fluxes, which are the rates at which intracellular metabolites interconvert, are not directly measurable. MFA uses stable isotope labeled tracers to reveal information related [...] Read more.
Metabolic flux analysis (MFA) is an increasingly important tool to study metabolism quantitatively. Unlike the concentrations of metabolites, the fluxes, which are the rates at which intracellular metabolites interconvert, are not directly measurable. MFA uses stable isotope labeled tracers to reveal information related to the fluxes. The conceptual idea of MFA is that in tracer experiments the isotope labeling patterns of intracellular metabolites are determined by the fluxes, therefore by measuring the labeling patterns we can infer the fluxes in the network. In this review, we will discuss the basic concept of MFA using a simplified upper glycolysis network as an example. We will show how the fluxes are reflected in the isotope labeling patterns. The central idea we wish to deliver is that under metabolic and isotopic steady-state the labeling pattern of a metabolite is the flux-weighted average of the substrates’ labeling patterns. As a result, MFA can tell the relative contributions of converging metabolic pathways only when these pathways make substrates in different labeling patterns for the shared product. This is the fundamental principle guiding the design of isotope labeling experiment for MFA including tracer selection. In addition, we will also discuss the basic biochemical assumptions of MFA, and we will show the flux-solving procedure and result evaluation. Finally, we will highlight the link between isotopically stationary and nonstationary flux analysis. Full article
(This article belongs to the Special Issue Stable Isotope Guided Metabolomics)
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