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Related Experiment Video

Updated: Nov 7, 2025

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes
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In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis-Technical Aspects and Model Interpretation.

Stanislaw Deja1,2, Blanka Kucejova1, Xiaorong Fu1,3

  • 1Center for Human Nutrition, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Metabolites
|April 30, 2021
PubMed
Summary

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This study introduces a novel metabolic flux analysis method to accurately measure endogenous ketone production in vivo. The approach enhances understanding of ketone bodies (AcAc and BHB) metabolism and their roles in health and disease.

Area of Science:

  • Biochemistry
  • Metabolic Research
  • Physiology

Background:

  • Ketogenesis, the production of ketone bodies like acetoacetate (AcAc) and β-hydroxybutyrate (BHB), is crucial during carbohydrate scarcity.
  • Ketones serve as vital oxidative fuels for peripheral tissues but their endogenous production in vivo is challenging to quantify.
  • Accurate measurement of ketone flux is essential for understanding their physiological roles and implications in various diseases.

Purpose of the Study:

  • To develop and implement a robust two-pool metabolic flux analysis (MFA) model for quantifying in vivo ketogenesis.
  • To simultaneously measure acetoacetate and β-hydroxybutyrate tracers for accurate flux determination.
  • To address challenges related to tracer stability and analytical accuracy in ketone body flux measurements.

Main Methods:

Keywords:
13C MFA1H NMRBHBLC-MS/MSacetoacetatefluxin vivoketogenesislivermetabolismmetabolomicsstable isotope

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  • Utilized a two-pool metabolic flux analysis (MFA) model integrating liquid chromatography-tandem mass spectrometry (LC-MS/MS) data of ketone isotopologues.
  • Employed simultaneous infusion of acetoacetate and β-hydroxybutyrate tracers.
  • Incorporated 1H NMR for real-time monitoring of tracer and analyte stability during infusion and sample analysis.

Main Results:

  • The developed MFA approach accurately quantifies acetoacetate and β-hydroxybutyrate pool sizes and their interconversion rates.
  • Simultaneous tracer analysis and stability monitoring enabled reliable calculation of ketone appearance and disposal rates.
  • Regression analysis provided confidence intervals, enhancing the reliability and error detection of flux data.

Conclusions:

  • This study presents a validated method for precise in vivo measurement of endogenous ketone production.
  • The approach offers critical insights into ketone body metabolism, essential for research in metabolic disorders and related diseases.
  • Accurate flux determination advances the physiological interpretation of ketone dynamics in various metabolic states.