In vivo estimation of ketogenesis using metabolic flux analysis-Technical aspects and model interpretation

Stanislaw Deja, Blanka Kucejova, Xiaorong Fu, Jeffrey D. Browning, Jamey D. Young, Shawn Burgess

Research output: Contribution to journalArticlepeer-review

Abstract

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, 1H 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.

Original languageEnglish (US)
Article number279
JournalMetabolites
Volume11
Issue number5
DOIs
StatePublished - May 2021

Keywords

  • C MFA
  • H NMR
  • Acetoacetate
  • BHB
  • Flux
  • In vivo
  • Ketogenesis
  • LC-MS/MS
  • Liver
  • Metabolism
  • Metabolomics
  • Stable isotope

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Biology

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