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• W2022157670 abstract "Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [13C1]propionate and [13C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[2H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings. Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [13C1]propionate and [13C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[2H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings. CoA thioesters, which are members of the fatty acyl class of lipids (1Fahy E. Subramaniam S. Murphy R.C. Nishijima M. Raetz C.R. Shimizu T. Spener F. van Meer G. Wakelam M.J. Dennis E.A. Update of the LIPID MAPS comprehensive classification system for lipids.J. Lipid Res. 2009; 50: S9-S14Abstract Full Text Full Text PDF PubMed Scopus (1053) Google Scholar), are critical for adequate control of cellular bioenergetics (2Sauer S.W. Okun J.G. Schwab M.A. Crnic L.R. Hoffmann G.F. Goodman S.I. Koeller D.M. Kölker S. Bioenergetics in glutaryl-coenzyme A dehydrogenase deficiency. A role for glutaryl-coenzyme A.J. Biol. Chem. 2005; 280: 21830-21836Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), as well as the regulation of important cellular functions (3Ellis J.M. Frahm J.L. Li L.O. Coleman R.A. Acyl-coenzyme A synthetases in metabolic control.Curr. Opin. Lipidol. 2010; 21: 212-217Crossref PubMed Scopus (148) Google Scholar). Furthermore, anabolic and catabolic carbon processing is extensively dependent on a pool of acyl-CoA intermediates, which are available as activated and localized substrates (4Grevengoed T.J. Klett E.L. Coleman R.A. Acyl-CoA metabolism and partitioning.Annu. Rev. Nutr. 2014; 34: 1-30Crossref PubMed Scopus (226) Google Scholar). Specifically, synthesis of fatty acids in mammalian cells is initiated through activation of acetyl- and malonyl-CoA, with elongation of the nascent chain via addition of the acetyl moiety from acetyl-CoA (3Ellis J.M. Frahm J.L. Li L.O. Coleman R.A. Acyl-coenzyme A synthetases in metabolic control.Curr. Opin. Lipidol. 2010; 21: 212-217Crossref PubMed Scopus (148) Google Scholar). This system is in contrast to the ability of certain bacteria, which condense propionate, a three-carbon precursor, to a six-carbon product directly (5de Bok F.A. Stams A.J. Dijkema C. Boone D.R. Pathway of poropionate oxidation by a syntrophic culture of Smithella propionica and Methanospirillum hungatei.Appl. Environ. Microbiol. 2001; 67: 1800-1804Crossref PubMed Scopus (139) Google Scholar). Catabolism of diverse molecules including cholesterol, branched chain amino acids, and odd chain fatty acids is accomplished through propionyl-CoA (6Halarnkar P.P. Blomquist G.J. Comparative aspects of propionate metabolism.Comp. Biochem. Physiol. B. 1989; 92: 227-231Crossref PubMed Scopus (57) Google Scholar). Thus, a fundamental understanding of the acyl-CoA chemical space is relevant to a wide spectrum of cellular biochemical transformations. Perturbations of acyl-CoA biosynthesis are associated with diseases that have high morbidity and mortality, including a spectrum of inborn errors of metabolism. For example, dysregulated propionyl-CoA metabolism caused by deficiency or aberrant function of propionyl-CoA carboxylase results in metabolic acidosis, coma, mental developmental delays, and cardiac complications (7Sweetman L. Weyler W. Nyhan W.L. de Cespedes C. Loria A.R. Estrada Y. Abnormal metabolites of isoleucine in a patient with propionyl-CoA carboxylase deficiency.Biomed. Mass Spectrom. 1978; 5: 198-207Crossref PubMed Scopus (52) Google Scholar). Modulation of levels of other acyl-CoAs can result in cellular responses such as histone acetylation, which modulates transcriptional activation (8Lee J.V. Carrer A. Shah S. Snyder N.W. Wei S. Venneti S. Worth A.J. Yuan Z.F. Lim H.W. Liu S. et al.Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation.Cell Metab. 2014; 20: 306-319Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). In addition, exogenous exposures such as the pesticide and complex I inhibitor, rotenone, disrupt specific acyl-CoA metabolism and cause Parkinson's disease-like symptoms (9Basu S.S. Blair I.A. Rotenone-mediated changes in intracellular coenzyme A thioester levels: implications for mitochondrial dysfunction.Chem. Res. Toxicol. 2011; 24: 1630-1632Crossref PubMed Scopus (18) Google Scholar). Increasing use of LC-MS/MS, LC-selected reaction monitoring (SRM)/MS, and LC-high-resolution MS (HRMS) provides orthogonal separations and structural information that are ideal for analysis of complex analytes such as acyl-CoAs that are present in biological matrices (10King M.T. Reiss P.D. Separation and measurement of short-chain coenzyme-A compounds in rat liver by reversed-phase high-performance liquid chromatography.Anal. Biochem. 1985; 146: 173-179Crossref PubMed Scopus (79) Google Scholar). Development of new model systems, such as isolated human platelets, may provide a future platform integrating acyl-CoA analysis into quantitative metabolic studies (11Basu S.S. Deutsch E.C. Schmaier A.A. Lynch D.R. Blair I.A. Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.Bioanalysis. 2013; 5: 3009-3021Crossref PubMed Scopus (14) Google Scholar). Furthermore, coupling of stable isotopic labels, including [13C]- and [2H]labeled precursors, to metabolic analysis can provide new insight into cellular metabolism in humans, as well as in animal models (12Zhang G.F. Sadhukhan S. Tochtrop G.P. Brunengraber H. Metabolomics, pathway regulation, and pathway discovery.J. Biol. Chem. 2011; 286: 23631-23635Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). When employing LC-MS/MS to examine the utilization of isotopically labeled precursors including [13C1]propionate and [13C3]propionate for acyl-CoA biosynthesis, we discovered a high abundance unknown chromatographic spectral feature which was inconsistent with existing mammalian metabolic models. The present study was designed to test the hypothesis that the unknown feature resulted from propionate metabolism to a previously undescribed acyl-CoA. 5-Sulfosalicylic acid, ammonium formate solution, glacial acetic acid, formic acid, and DMSO were purchased from Sigma-Aldrich (St. Louis, MO). [13C3]propionate, [13C1]propionate, and 2,2-[2H2]propionate were purchased from Cambridge Isotopes (Tewksbury, MA). Optima LC-MS grade methanol, acetonitrile (ACN), 2-propanol, and water were purchased from Fisher Scientific (Pittsburgh, PA). DMEM:F12 medium, FBS, streptomycin, and penicillin were purchased from Invitrogen (Carlsbad, CA). Charcoal stripped FBS was from Gemini Bioproducts (West Sacramento, CA). RPMI1640 medium omitting pantothenate was purchased from Athena ES (Baltimore, MD). [13C315N1]calcium pantothenate was purchased from Isosciences (King of Prussia, PA). HepG2 cells were grown and maintained in DMEM:F12 with 2% FBS and 2 mM glutamine with 100,000 units/l penicillin and 100 mg/l streptomycin. For all experiments, cells were grown to 80% confluence prior to treatment. For dose response studies, solutions of sodium propionate in maintenance medium were prepared by serial dilution from the most concentrated stock after filtration through a 0.2 μm sterile filter (Corning Inc., Corning, NY). Spent medium was aspirated and new medium containing 0, 1 μM, 100 μM, 1 mM, 10 mM, or 100 mM propionate was added. After incubations lasting 1 h, the cells were extracted and analyzed. For time-course studies, cells were grown as above, and spent medium was replaced with 1 mM propionate-containing medium. At indicated time-points, cells were taken for extraction and analysis. For all mass isotopologue distribution (MID) analysis, samples were treated with an equal concentration to the tracer with unlabeled propionate (as a control) and processed in parallel. The untreated samples were then analyzed and used for isotope correction as previously described (13Worth A.J. Basu S.S. Snyder N.W. Mesaros C. Blair I.A. Inhibition of neuronal cell mitochondrial complex I with rotenone increases lipid β-oxidation supporting acetyl-coenzyme A levels.J. Biol. Chem. 2014; 289: 26895-26903Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells were cultured in similar conditions to the HepG2 cells, except the base medium was RPMI1640 for the H358 cells and F-12 K for the LoVo cells. Treatment and extraction conditions were identical to those used for HepG2 cells. Blood was donated by healthy volunteers under the Children's Hospital of Philadelphia protocol #01-002609. Platelet-rich plasma (PRP) was isolated as previously described (11Basu S.S. Deutsch E.C. Schmaier A.A. Lynch D.R. Blair I.A. Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.Bioanalysis. 2013; 5: 3009-3021Crossref PubMed Scopus (14) Google Scholar). Briefly, donor blood was collected into acid-citrate-dextrose tubes, gently mixed by inversion then transferred to 15 ml conical tubes. Whole blood was spun at 170 g for 15 min at 25°C with no breaks. The resulting upper layer of PRP was transferred, avoiding the buffy coat and red blood cells, to a 1.5 ml Eppendorf tube. The PRP was centrifuged at 400 g for 10 min to pellet the platelets. The supernatant was aspirated and the platelets were gently resuspended in Tyrode's buffer with or without propionate, [13C3]propionate, [13C1]propionate, or 2,2-[2H2]propionate. The samples were incubated for 1 h at 37°C, and then centrifuged at 10,000 g for 2 min to pellet the platelets. The resulting pellet was then taken for extraction and analysis. The procedures for cell extraction have been described in detail previously (11Basu S.S. Deutsch E.C. Schmaier A.A. Lynch D.R. Blair I.A. Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.Bioanalysis. 2013; 5: 3009-3021Crossref PubMed Scopus (14) Google Scholar, 14Basu S.S. Mesaros C. Gelhaus S.L. Blair I.A. Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and Its thioesters.Anal. Chem. 2011; 83: 1363-1369Crossref PubMed Scopus (39) Google Scholar, 15Basu S.S. Blair I.A. SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards.Nat. Protoc. 2012; 7: 1-12Crossref Scopus (41) Google Scholar, 16Snyder N.W. Basu S.S. Zhou Z. Worth A.J. Blair I.A. Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-CoA thioesters.Rapid Commun. Mass Spectrom. 2014; 28: 1840-1848Crossref PubMed Scopus (19) Google Scholar, 17Snyder N.W. Khezam M. Mesaros C.A. Worth A. Blair I.A. Untargeted metabolomics from biological sources using ultraperformance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS).J. Vis. Exp. 2013; 75: e50433Google Scholar). Briefly, cells or platelets were gently lifted and transferred to 15 ml conical tubes, centrifuged at 500 g for 5 min and resuspended in 1 ml of ice-cold 10% TCA. They were then pulse-sonicated for 30 s on ice using a sonic dismembrator (Fisher), followed by a 5 min centrifugation at 15,000 g at 4°C. The supernatant was transferred to a fresh tube, and the pellet was discarded. The supernatant was purified by solid-phase extraction as follows: Oasis HLB 1 cm3 (30 mg) solid-phase extraction columns (Waters) were conditioned with 1 ml of methanol followed by 1 ml of water. The collected supernatant was applied, washed with 1 ml of water, and finally eluted using three subsequent applications of 0.5 ml of methanol containing 25 mM ammonium acetate. Eluted compounds were dried down under nitrogen and resuspended in 50 μl of 5% 5-sulfosalicylic acid. Injections of 10 μl were made for LC-ESI/MS analysis. Acyl-CoAs were separated using a reversed-phase HPLC Phenomenex Luna C18 column (2.0 × 150 mm, particle size 5 μm) with 5 mM ammonium acetate in water as solvent A, 5 mM ammonium acetate in 95/5 ACN/water (v/v) as solvent B, and 80/20/0.1 ACN/water/formic acid (v/v/v) as solvent C. Gradient conditions were as follows: 2% B for 1.5 min, increased to 25% over 3.5 min, increased to 100% B in 0.5 min and held for 8.5 min, washed with 100% C for 5 min before equilibration to 2% B for 5 min. The flow rate was 200 μl/min. Samples were analyzed using an API 4000 triple-quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) in the positive ESI mode. Samples (10 μl) were injected using a Leap CTC autosampler (CTC Analytics, Switzerland) where they were maintained at 4°C, and data were analyzed with Analyst 1.4.1 software. The column effluent was diverted to the mass spectrometer from 8 to 23 min and to waste for the remainder of the run. The mass spectrometer operating conditions were as follows: ion spray voltage (5.0 kV), nitrogen as curtain gas (15 units), ion source gas 1 (8 units), gas 2 (15 units), and collision-induced dissociation gas (5 units). The ESI probe temperature was 450°C, the declustering potential was 105 V, the entrance potential was 10 V, the collision energy was 45 eV, and the collision exit potential was 15 V. A constant neutral loss of 507 Da was monitored for each acyl-CoA. LC-SRM/MS analysis was conducted using similar instrument parameters and specific selected ions as noted in the text. LC-HRMS was conducted on a LTQ-Orbitrap XL operated in positive ion mode at a resolution of 100,000 as previously described (17Snyder N.W. Khezam M. Mesaros C.A. Worth A. Blair I.A. Untargeted metabolomics from biological sources using ultraperformance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS).J. Vis. Exp. 2013; 75: e50433Google Scholar), except that an ESI source was used and the mass spectrometer was coupled to the LC system described above. Treatment of HepG2 cells with 10 mM propionate revealed an upregulation of certain acyl-CoA species after analysis by the constant neutral loss 507 Da scans to survey short chain acyl-CoA content (Fig. 1). The expected increase of propionyl-CoA with a protonated molecule (MH+) at m/z 824 was coupled to an unexpected increase in an intense ion at m/z 864 corresponding to MH+ of a new C6 mono-unsaturated acyl-CoA (Fig. 1B). Treatment under the same conditions with [13C1]propionate revealed a similar acyl-CoA profile except that MH+ for the new acyl-CoA appeared at m/z 866 (Fig. 1C) corresponding to a C6 acyl-CoA isotopologue containing both [13C]labeled carbon atoms. Furthermore, [13C3]propionate treatment revealed a highly abundant ion at m/z 870, which would correspond to MH+ of a C6 mono-unsaturated acyl-CoA containing all six [13C]atoms (Fig. 1D). Finally, treatment of the cells with trans-2-methyl-2-pentenoic acid resulted in the formation of a C6 acyl-CoA with an MH+ at m/z 864 and similar constant neutral loss mass spectrum to that observed with propionate (Fig. 1E). Treatment of HepG2 cells with other mono-unsaturated six-carbon fatty acids for 1 h resulted in the substrate-specific acyl-CoA profiles as demonstrated by constant neutral loss scans of 507 Da (supplementary Fig. 1). However, the LC-MS chromatograms observed after treatment of cells with propionate, [13C1]propionate, or [13C3]propionate could only be reproduced by the addition of trans-2-methyl-2-pentenoic acid. Importantly, the ion at m/z 838 corresponding to MH+ of butyryl-CoA was only observed after treatment with unbranched hexanoyl fatty acids. This confirmed the differential metabolism of a 2-methyl-substituted pentanoate when compared with an unbranched hexanoate (18Mannaerts G.P. Van Veldhoven P.P. Casteels M. Peroxisomal lipid degradation via α- and β-oxidation in mammals.Cell Biochem. Biophys. 2000; 32: 73-87Crossref PubMed Scopus (100) Google Scholar). Correspondingly, propionate did not increase the formation of butyryl-CoA, confirming that neither propionyl-CoA nor its metabolites were catabolized via unbranched fatty acid β-oxidation. Treatment of other cell lines with propionate, including mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells, also resulted in generation of the new acyl-CoA with MH+ at m/z 864 (supplementary Fig. 2). Formation of the new acyl-CoA increased in a dose-dependent manner with increasing propionate treatment over the range of 0–1 mM. Its formation reduced with 10 mM and 100 mM propionate suggesting that this new metabolic pathway could be inhibited at high propionate concentrations or that the new acyl-CoA underwent further propionate-dependent metabolism (Fig. 2A). An increase of almost an order of magnitude in concentration of the new acyl-CoA was observed between 1 and 100 μM propionate with almost another order of magnitude increase between 100 μM and 1 mM. Following treatment with 1 mM propionate, the MH+ at m/z 864 increased rapidly to a maximum at 1 h, with a stable plateau of levels from 1 h to 12 h, followed by a decrease until 24 h (Fig. 2B). [13C315N1]pantothenic acid was used to label all CoA species in mouse hepatocellular carcinoma Hepa1c1c7 cells, as previously described (14Basu S.S. Mesaros C. Gelhaus S.L. Blair I.A. Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and Its thioesters.Anal. Chem. 2011; 83: 1363-1369Crossref PubMed Scopus (39) Google Scholar). Coelution experiments conducted after the addition of unlabeled propionate provided further support for the identification of the new metabolite as trans-2-methyl-2-pentenoyl-CoA. LC-SRM/MS monitoring of the transition m/z 864 (MH+) to m/z 357 (MH+-507), as well as the corresponding [13C315N1]labeled acyl-CoA transition m/z 868 (MH+) to m/z 361 (MH+-507), demonstrated coelution of the propionate-derived acyl-CoA with MH+ at m/z 864 with the [13C315N1]labeled acyl-CoA obtained from the stable isotope labeling by essential nutrients in cell culture Hepa1c1c7 cells (Fig. 3A). Likewise, the trans-2-methyl-2-pentenoic acid derived analyte with MH+ at m/z 864 coeluted with the corresponding [13C315N1]labeled acyl-CoA (Fig. 3B). Finally, LC-SRM/MS analysis of the trans-2-methyl-2-pentenoic acid-derived acyl-CoA (SRM transition m/z 864 to m/z 357), the [13C315N1]labeled acyl-CoA (SRM transition m/z 868 to m/z 361), and the [13C3]propionate-derived [13C6]acyl-CoA (SRM transition m/z 870 to m/z 363) all coeluted (Fig. 3C). LC-HRMS analysis was employed to confirm that the MH+ at m/z 864 arose from a C6 mono-unsaturated acyl-CoA (Table 1). An intense MH+ was observed at m/z 864.1776 which corresponded to a molecular formula of C27H45N7O17P3S+ (M0, Δm −2.77 ppm). The acyl-CoA from [13C1]propionate-treated cells gave an intense MH+ at m/z 866.1846 corresponding to a molecular formula of 13C212C25H45N7O17P3S+ (M2, Δm −2.42 ppm). Importantly, LC-HRMS was able to distinguish MH+ of the 13C-istopolog arising from the natural abundance of 13C, 13C212C25H45N7O17P3S+ (m/z 866.1846) from the MH+ of hexanoyl-CoA, which had the same nominal mass but different accurate mass C27H47N7O17P3S+ (m/z 866.1956, Δm 12.70 ppm). Interestingly, the acyl-CoA derived from 2,2-[2H2]propionate revealed intense MH+ at m/z 865.1840 (M1, Δm −2.65 ppm) and m/z 866.1908 (M2, Δm −2.07 ppm), indicating the incorporation of either one or two deuterium atoms into the new acyl-CoA.TABLE 1Accurate mass and isotope analysis of the putative 2-methyl-2-pentenoyl-CoA derived from treatment of HepG2 cells with propionate, [13C1]propionate, and 2,2-[2H2]propionatePredicted Formula (MH+)Found MassPredicted MassDelta (ppm)PropionateM0C27H45N7O17P3S+864.1776864.1800−2.77[13C1]propionateM213C212C25H45N7O17P3S+866.1846866.1867−2.422,2-[2H2]propionateM0C27H45N7O17P3S+864.1784864.1800−1.85M1C272H11H44N7O17P3S+865.184865.1863−2.65M2C272H21H43N7O17P3S+866.1908866.1926−2.07 Open table in a new tab HepG2 cells treated with 1 mM [13C3]propionate revealed the anticipated M3 enrichment into MH+ of propionyl-CoA (Fig. 4A). Furthermore, propionyl-CoA extracted from HepG2 cells treated with 1 mM 2,2-[2H2]propionate revealed nearly equal enrichment in M1 and M2 of the MH+, indicating a portion of deuterium was lost through protium exchange. Similar to the observations in the constant neutral loss experiments, [13C3]propionate treatment resulted in approximately 80% of the new acyl-CoA to be enriched in the M6 isotopologue (Fig. 4B); indicating direct incorporation of six carbon atoms from two molecules of propionyl-CoA without any carbon loss. Deuterium labeling in the new acyl-CoA was almost identical to the labeling in propionyl-CoA, with nearly equal enrichment of the M1 and M2 of MH+ and virtually no labeling in M3 or M4 (Fig. 4C). M3 labeling, which was present in succinyl-CoA only for the [13C3]propionate treatment, arose from anaplerosis into the Krebs cycle (Fig. 4D). Platelets from healthy volunteers were isolated for isotopic tracer studies using a previously described procedure (11Basu S.S. Deutsch E.C. Schmaier A.A. Lynch D.R. Blair I.A. Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.Bioanalysis. 2013; 5: 3009-3021Crossref PubMed Scopus (14) Google Scholar). Studies were conducted using platelets because although they are anuclear, they are rich in mitochondria, and produce much higher levels of mitochondrial metabolites than lymphocytes (11Basu S.S. Deutsch E.C. Schmaier A.A. Lynch D.R. Blair I.A. Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.Bioanalysis. 2013; 5: 3009-3021Crossref PubMed Scopus (14) Google Scholar) or neutrophils (data not shown). Thus, platelets have a much higher basal oxygen consumption rate when compared with lymphocytes or neutrophils (19Kramer P.A. Ravi S. Chacko B. Johnson M.S. Darley-Usmar V.M. A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: Implications for their use as bioenergetic biomarkers.Redox Biol. 2014; 2: 206-210Crossref PubMed Scopus (255) Google Scholar). In contrast to platelets, lymphocytes primarily only utilize oxidative phosphorylation under basal conditions and they have a limited capacity to increase glycolytic flux (19Kramer P.A. Ravi S. Chacko B. Johnson M.S. Darley-Usmar V.M. A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: Implications for their use as bioenergetic biomarkers.Redox Biol. 2014; 2: 206-210Crossref PubMed Scopus (255) Google Scholar). Neutrophils have little or no dependence on oxidative phosphorylation and glycolysis is not increased when mitochondrial ATP synthase is inhibited (19Kramer P.A. Ravi S. Chacko B. Johnson M.S. Darley-Usmar V.M. A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: Implications for their use as bioenergetic biomarkers.Redox Biol. 2014; 2: 206-210Crossref PubMed Scopus (255) Google Scholar). Furthermore, platelets are amenable to isolation and purification, and do not require culturing, which could otherwise introduce changes in cellular metabolism. Finally, alterations in platelet mitochondrial function have been demonstrated in a variety of diseases so that they have been proposed to serve as potential markers of systemic mitochondrial dysfunction (11Basu S.S. Deutsch E.C. Schmaier A.A. Lynch D.R. Blair I.A. Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.Bioanalysis. 2013; 5: 3009-3021Crossref PubMed Scopus (14) Google Scholar, 20Sjövall F. Ehinger J.K. Marelsson S.E. Morota S. Frostner E.A. Uchino H. Lundgren J. Arnbjörnsson E. Hansson M.J. Fellman V. et al.Mitochondrial respiration in human viable platelets–methodology and influence of gender, age and storage.Mitochondrion. 2013; 13: 7-14Crossref PubMed Scopus (86) Google Scholar). The M0 through M3 isotopologue of MH+ from propionyl-CoA, and the M0 through M6 isotopologue of MH+ from the new acyl-CoA were monitored by LC-SRM/MS. After adjustment for natural isotopic abundance, treatment with unlabeled propionate gave essentially 100% M0 isotopologue at MH+ for both propionyl-CoA (Fig. 5A) and the new acyl-CoA (Fig. 5B). [13C1]propionate treatment resulted in a predominant M1 isotopologue at MH+ for propionyl-CoA (Fig. 5A). However there was a predominant M2 isotopologue at MH+ for the new acyl-CoA (Fig. 5B). Similarly, [13C3]propionate resulted in a predominant M3 isotopologue at MH+ for propionyl-CoA (Fig. 5A), whereas there was a predominant M6 isotopologue at MH+ for the new acyl-CoA (Fig. 5B). In contrast, 2,2-[2H2]propionate treatment resulted in a similar mixture of M0, M1 and M2 isotopes in MH+ for both propionyl-CoA (Fig. 5A) and the new acyl-CoA (Fig. 5B). The LC-MS/MS properties of the new six carbon mono-unsaturated acyl-CoA were identical with the acyl-CoA that was formed by the addition of trans-2-methyl-2-pentenoic acid to human cell lines and platelets. This finding together with the isotope labeling data provided compelling evidence for the structural assignment of the new acyl-CoA as trans-2-methyl-2-pentenoyl-CoA (Fig. 6). Its mechanism of formation must account for the results of three isotope-labeling experiments: 1) conservation of the [13C1]atom from each precursor propionate; 2) conservation of all [13C3]atoms from precursor propionate; and 3) exchangeable deuterium labeling with a maximum of two total conserved [2H]atoms from precursor propionate (Fig. 6). The proposed mechanism involves initial formation of propionyl-CoA through the action of a short chain CoA synthetase (Fig. 6). The limited protium/deuterium exchange observed in the formation of propionyl-CoA from 2,2-[2H2]propionate (Fig. 5A) was consistent with the labiality of the deuterium atoms on the α-carbon atom (C2) during CoA thioester formation. The propionyl-CoA was then converted to propionyl-acyl carrier protein (ACP) and 2-methyl-malonyl-CoA through the action of propionyl-CoA:ACP transacylase or propionyl-CoA carboxylase, respectively (Fig. 6). Subsequently, 3-keto-ACP synthase added an additional propionate moiety from propionyl-ACP with a concomitant loss of CO2 (containing the carbon added during the formation of 2-methyl-malonyl-CoA) and free ACP. This conserved the [13C]atom from 1-[13C]propionate and partially conserved one of the deuterium atoms at C2 derived from the first molecule of 2,2-[2H2]propionate. Sequential actions of a reductase and dehydratase resulted in the generation of an olefin, which was converted to the final trans-2-methyl-2-pentenoyl-CoA molecule by a transacylase (Fig. 6). All deuterium atoms that were originally on C2 from the first propionate molecule were lost during the dehydration step (Fig. 6). The remaining deuterium atoms at C2 from the second propionate molecule were partially exchanged for protium (Fig. 5B) in a similar manner to that observed in propion" @default.
• W2022157670 created "2016-06-24" @default.
• W2022157670 creator A5007007052 @default.
• W2022157670 creator A5037348461 @default.
• W2022157670 creator A5050679549 @default.
• W2022157670 creator A5068779075 @default.
• W2022157670 creator A5079384190 @default.
• W2022157670 date "2015-01-01" @default.
• W2022157670 modified "2023-10-17" @default.
• W2022157670 title "Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets" @default.
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• W2022157670 cites W1642029460 @default.
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