In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis—Technical Aspects and Model Interpretation
Ketogenesis occurs in liver mitochondria where acetyl-CoA molecules, derived from lipid oxidation, are condensed into acetoacetate (AcAc) and reduced to β-hydroxybutyrate (BHB). During carbohydrate scarcity, these two ketones are released into circulation at high rates and used as oxidative fuels in...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-04-01
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| Series: | Metabolites |
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| Online Access: | https://www.mdpi.com/2218-1989/11/5/279 |
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| author | Stanislaw Deja Blanka Kucejova Xiaorong Fu Jeffrey D. Browning Jamey D. Young Shawn Burgess |
| author_facet | Stanislaw Deja Blanka Kucejova Xiaorong Fu Jeffrey D. Browning Jamey D. Young Shawn Burgess |
| author_sort | Stanislaw Deja |
| collection | DOAJ |
| description | Ketogenesis occurs in liver mitochondria where acetyl-CoA molecules, derived from lipid oxidation, are condensed into acetoacetate (AcAc) and reduced to β-hydroxybutyrate (BHB). During carbohydrate scarcity, these two ketones are released into circulation at high rates and used as oxidative fuels in peripheral tissues. Despite their physiological relevance and emerging roles in a variety of diseases, endogenous ketone production is rarely measured in vivo using tracer approaches. Accurate determination of this flux requires a two-pool model, simultaneous BHB and AcAc tracers, and special consideration for the stability of the AcAc tracer and analyte. We describe the implementation of a two-pool model using a metabolic flux analysis (MFA) approach that simultaneously regresses liquid chromatography-tandem mass spectrometry (LC-MS/MS) ketone isotopologues and tracer infusion rates. Additionally, <sup>1</sup>H NMR real-time reaction monitoring was used to evaluate AcAc tracer and analyte stability during infusion and sample analysis, which were critical for accurate flux calculations. The approach quantifies AcAc and BHB pool sizes and their rates of appearance, disposal, and exchange. Regression analysis provides confidence intervals and detects potential errors in experimental data. Complications for the physiological interpretation of individual ketone fluxes are discussed. |
| first_indexed | 2024-03-10T11:53:02Z |
| format | Article |
| id | doaj.art-6564245739284e7ca315cb3f38beba41 |
| institution | Directory Open Access Journal |
| issn | 2218-1989 |
| language | English |
| last_indexed | 2024-03-10T11:53:02Z |
| publishDate | 2021-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Metabolites |
| spelling | doaj.art-6564245739284e7ca315cb3f38beba412023-11-21T17:33:32ZengMDPI AGMetabolites2218-19892021-04-0111527910.3390/metabo11050279In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis—Technical Aspects and Model InterpretationStanislaw Deja0Blanka Kucejova1Xiaorong Fu2Jeffrey D. Browning3Jamey D. Young4Shawn Burgess5Center for Human Nutrition, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USACenter for Human Nutrition, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USACenter for Human Nutrition, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USADepartment of Clinical Nutrition, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USADepartment of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN 37235, USACenter for Human Nutrition, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USAKetogenesis occurs in liver mitochondria where acetyl-CoA molecules, derived from lipid oxidation, are condensed into acetoacetate (AcAc) and reduced to β-hydroxybutyrate (BHB). During carbohydrate scarcity, these two ketones are released into circulation at high rates and used as oxidative fuels in peripheral tissues. Despite their physiological relevance and emerging roles in a variety of diseases, endogenous ketone production is rarely measured in vivo using tracer approaches. Accurate determination of this flux requires a two-pool model, simultaneous BHB and AcAc tracers, and special consideration for the stability of the AcAc tracer and analyte. We describe the implementation of a two-pool model using a metabolic flux analysis (MFA) approach that simultaneously regresses liquid chromatography-tandem mass spectrometry (LC-MS/MS) ketone isotopologues and tracer infusion rates. Additionally, <sup>1</sup>H NMR real-time reaction monitoring was used to evaluate AcAc tracer and analyte stability during infusion and sample analysis, which were critical for accurate flux calculations. The approach quantifies AcAc and BHB pool sizes and their rates of appearance, disposal, and exchange. Regression analysis provides confidence intervals and detects potential errors in experimental data. Complications for the physiological interpretation of individual ketone fluxes are discussed.https://www.mdpi.com/2218-1989/11/5/279ketogenesisstable isotope<sup>13</sup>C MFALC-MS/MS<sup>1</sup>H NMRin vivo |
| spellingShingle | Stanislaw Deja Blanka Kucejova Xiaorong Fu Jeffrey D. Browning Jamey D. Young Shawn Burgess In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis—Technical Aspects and Model Interpretation Metabolites ketogenesis stable isotope <sup>13</sup>C MFA LC-MS/MS <sup>1</sup>H NMR in vivo |
| title | In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis—Technical Aspects and Model Interpretation |
| title_full | In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis—Technical Aspects and Model Interpretation |
| title_fullStr | In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis—Technical Aspects and Model Interpretation |
| title_full_unstemmed | In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis—Technical Aspects and Model Interpretation |
| title_short | In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis—Technical Aspects and Model Interpretation |
| title_sort | in vivo estimation of ketogenesis using metabolic flux analysis technical aspects and model interpretation |
| topic | ketogenesis stable isotope <sup>13</sup>C MFA LC-MS/MS <sup>1</sup>H NMR in vivo |
| url | https://www.mdpi.com/2218-1989/11/5/279 |
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