Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets

Snyder, Nathaniel W.; Basu, Sankha S.; Worth, Andrew J.; Mesaros, Clementina; Blair, Ian A.

doi:10.1194/jlr.m055384

Cited by 16 publications

(31 citation statements)

References 33 publications

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“…Several studies have also used platelets to assay the activity of the electron transport chain in multiple neurological diseases [32]. In addition to the benefit of a relatively noninvasive sample collection and isolation, platelets are mitochondria-rich and maintain metabolic activity if properly isolated [24,33]. Since inherited metabolic diseases often affect metabolism globally, we reasoned that platelets could serve as a surrogate diagnostic tissue for inherited metabolic diseases such as FRDA if these pathways are utilized in platelets.…”

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confidence: 99%

Stable Isotopes and LC–MS for Monitoring Metabolic Disturbances in Friedreich’s Ataxia Platelets

et al. 2015

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Background Friedreich’s ataxia (FRDA) is an autosomal recessive disease with metabolic abnormalities that have been proposed to play an important role in the resulting neurodegeneration and cardiomyopathy. The inability to access the highly affected neuronal and cardiac tissues has hampered metabolic evaluation and biomarker development. Methods Employment of a LC–MS-based method to determine whether platelets isolated from patients with FRDA exhibit differentiable metabolism compared with healthy controls. Results Isotopologue analysis showed a marked decrease in glucose incorporation with a concomitant increase in palmitate-derived acyl-CoA thioesters in FRDA platelets compared with controls. Conclusion Our findings demonstrate that platelets can be used as a surrogate tissue for in vivo biomarker studies to monitor new therapeutic approaches for the treatment of FRDA.

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confidence: 99%

Stable Isotopes and LC–MS for Monitoring Metabolic Disturbances in Friedreich’s Ataxia Platelets

et al. 2015

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“…We make use of data from a previously published experiment of isotopologue analysis of an unknown product of propionate metabolism. This data was generated in human hepatocellular carcinoma HepG2 cells incubated in [ 2 H 2 ]-propionate or unlabeled propionate and was analyzed by MS/MS using an API-4000 triple quadrupole mass spectrometer, as described elsewhere [7]. Since, at the time of the experiment, the chemical formula of the putative metabolite was unknown, no generation of simulated spectra was possible.…”

Section: Resultsmentioning

confidence: 99%

FluxFix: automatic isotopologue normalization for metabolic tracer analysis

2016

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BackgroundIsotopic tracer analysis by mass spectrometry is a core technique for the study of metabolism. Isotopically labeled atoms from substrates, such as [13C]-labeled glucose, can be traced by their incorporation over time into specific metabolic products. Mass spectrometry is often used for the detection and differentiation of the isotopologues of each metabolite of interest. For meaningful interpretation, mass spectrometry data from metabolic tracer experiments must be corrected to account for the naturally occurring isotopologue distribution. The calculations required for this correction are time consuming and error prone and existing programs are often platform specific, non-intuitive, commercially licensed and/or limited in accuracy by using theoretical isotopologue distributions, which are prone to artifacts from noise or unresolved interfering signals.ResultsHere we present FluxFix (http://fluxfix.science), an application freely available on the internet that quickly and reliably transforms signal intensity values into percent mole enrichment for each isotopologue measured. ‘Unlabeled’ data, representing the measured natural isotopologue distribution for a chosen analyte, is entered by the user. This data is used to generate a correction matrix according to a well-established algorithm. The correction matrix is applied to labeled data, also entered by the user, thus generating the corrected output data. FluxFix is compatible with direct copy and paste from spreadsheet applications including Excel (Microsoft) and Google sheets and automatically adjusts to account for input data dimensions. The program is simple, easy to use, agnostic to the mass spectrometry platform, generalizable to known or unknown metabolites, and can take input data from either a theoretical natural isotopologue distribution or an experimentally measured one.ConclusionsOur freely available web-based calculator, FluxFix (http://fluxfix.science), quickly and reliably corrects metabolic tracer data for natural isotopologue abundance enabling faster, more robust and easily accessible data analysis.

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“…Samples were kept in a temperature controlled autosampler at 6 °C and LC separation was performed as previously described on a Waters XBridge 3.5 μm particle size C18 2.1 × 150 mm column. LC conditions were as follows modified from previous studies[22, 20]; column oven temperature 25 °C, solvent A water with 5 mM ammonium acetate, solvent B 95:5 acetonitrile: water with 5 mM ammonium acetate, solvent C (wash solvent) 80:20 acetonitrile: water with 0.1% formic acid. The gradient was as follows: 0.2 mL/min flow at 98% A and 2% B for 1.5 min, 80% A 20% B at 5 min, 100% B at 12 min, 0.3 mL/min 100% B at 16 min, 0.2 mL/min 100% C at 17 min, held to 21 min, then re-equilibrated at 0.2 mL/min flow at 98% A and 2% B from 22 to 28 min.…”

Section: Methodsmentioning

confidence: 99%

LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and 13C-isotopic labeling of acyl-coenzyme A thioesters

et al. 2016

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Acyl-coenyzme A thioesters (acyl-CoAs) are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoAs in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoAs. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoAs can be labeled with either a fixed [13C315N1] label derived from pantothenate into the CoA moiety, or via variable [13C] labeling into the acyl-chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoAs by stable isotope dilution using the [13C315N1] label and measurement of the incorporation of labeled carbon atoms derived from [13C6]-glucose, [13C515N2]-glutamine and [13C3]-propionate. As a proof-of-principle we applied this method to human B-cell lymphoma (WSU-DLCL2) cells in culture, to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoAs.

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Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets

Cited by 16 publications

References 33 publications

Stable Isotopes and LC–MS for Monitoring Metabolic Disturbances in Friedreich’s Ataxia Platelets

Stable Isotopes and LC–MS for Monitoring Metabolic Disturbances in Friedreich’s Ataxia Platelets

FluxFix: automatic isotopologue normalization for metabolic tracer analysis

LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and 13C-isotopic labeling of acyl-coenzyme A thioesters

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