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.
Highlights
When carbohydrates are scarce, mammalian metabolism resorts to the use of ketones as a compensatory energy source [1]
Ketogenesis occurs in liver mitochondria where longchain fatty acids (LCFAs) are oxidized to 2-carbon acetyl-CoA units and condensed into the 4-carbon acetoacetate (AcAc)
We found that most ketogenic fluxes were characterized by high precision scores, with values around 0.8
Summary
Mammalian metabolism resorts to the use of ketones as a compensatory energy source [1]. Ketogenesis occurs in liver mitochondria where longchain fatty acids (LCFAs) are oxidized to 2-carbon acetyl-CoA units and condensed into the 4-carbon acetoacetate (AcAc). AcAc, a β-ketoacid, is chemically unstable and is, in large part, reduced to its stable β-hydroxy form, β-hydroxybutyrate (BHB). Hepatic ketogenesis allows the rapid systemic oxidation of lipids by converting insoluble and transport-restricted LCFAs into freely soluble ketones, which are avidly oxidized by most tissues [1,2]. During fasting or carbohydrate restriction, ketone production increases by an order of magnitude and plasma ketone concentrations can rise from 100 μM to several mM. Glucose production and its plasma concentration rarely change by more than 50%. Unlike endogenous glucose production, which is routinely quantified using stable isotope tracers [3], endogenous ketone production is rarely measured using tracer approaches
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