In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis—Technical Aspects and Model Interpretation

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.

Detecting de novo Hepatic Ketogenesis Using Hyperpolarized [2-13C] Pyruvate

“…The serum or perfusate ratio of BHB/AcAc has been typically measured between ~0.6-3 in fasting and non-fasting animals with fasting increasing the ratio of BHB/AcAc even up to 7 (Krebs et al, 1969;Deja et al, 2021). It is canonically appreciated that the increase in the BHB/AcAc ratio during fasting relates to the change in redox state from excessive oxidative metabolism and has important connotation in diseases, such as NAFLD (Fletcher et al, 2019).…”

NMR Metabolomics Methods for Investigating Disease

“…Using 1 H NMR, real-time metabolism in blood was monitored to evaluate the stability of the acetoacetate tracer during infusion and sample analysis. 52 The research implemented a two-pool model using metabolic flux analysis to describe the results. The study provides insight into the rates of appearance, disposal, and exchange of the tracers.…”

Gender-Specific Metabolomics Approach to Kidney Cancer

“…Ketones, BHB, and AcAc as well as acetone were all detected in greater concentration among RCC patients than in controls. Ketogenesis is generally thought to occur solely in the liver [52,53] and this would indicate that changes in ketones among RCC patients may be associated with secondary effects such as dietary habits, and fasting duration. However, the kidney also expresses 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), the key enzyme of ketogenesis [54].…”

“…A decrease in local pH caused by a tumor releasing lactate could contribute to elevated levels of acetone. AcAc is unstable and can spontaneously decarboxylate to acetone but this process is more rapid in lower pH [53]. Therefore, regardless of the source of AcAc, formation of acetone could be associated with the tumor itself (Figure 10).…”