Abstract
IntroductionRelative oxidation of different metabolic substrates in the heart varies both physiologically and pathologically, in order to meet metabolic demands under different circumstances. 13C labelled substrates have become a key tool for studying substrate use—yet an accurate model is required to analyse the complex data produced as these substrates become incorporated into the Krebs cycle.ObjectivesWe aimed to generate a network model for the quantitative analysis of Krebs cycle intermediate isotopologue distributions measured by mass spectrometry, to determine the 13C labelled proportion of acetyl-CoA entering the Krebs cycle.MethodsA model was generated, and validated ex vivo using isotopic distributions measured from isolated hearts perfused with buffer containing 11 mM glucose in total, with varying fractions of universally labelled with 13C. The model was then employed to determine the relative oxidation of glucose and triacylglycerol by hearts perfused with 11 mM glucose and 0.4 mM equivalent Intralipid (a triacylglycerol mixture).ResultsThe contribution of glucose to Krebs cycle oxidation was measured to be 79.1 ± 0.9%, independent of the fraction of buffer glucose which was U-13C labelled, or of which Krebs cycle intermediate was assessed. In the presence of Intralipid, glucose and triglyceride were determined to contribute 58 ± 3.6% and 35.6 ± 0.8% of acetyl-CoA entering the Krebs cycle, respectively.ConclusionThese results demonstrate the accuracy of a functional model of Krebs cycle metabolism, which can allow quantitative determination of the effects of therapeutics and pathology on cardiac substrate metabolism.
Highlights
Relative oxidation of different metabolic substrates in the heart varies both physiologically and pathologically, in order to meet metabolic demands under different circumstances. 13C labelled substrates have become a key tool for studying substrate use—yet an accurate model is required to analyse the complex data produced as these substrates become incorporated into the Krebs cycle
The model was used to predict isotopologue distributions of When the proportion of labelled glucose provided in the malate, citrate, α-ketoglutarate and succinate at every value perfusion buffer was varied, and the resulting steady-state
Isotopologue distributions determined by Liquid chromatography‐coupled mass spectrometry (LC–MS), α was fitted separately for each experimental replicate, using each of malate, glutamate and succinate independently
Summary
Relative oxidation of different metabolic substrates in the heart varies both physiologically and pathologically, in order to meet metabolic demands under different circumstances. 13C labelled substrates have become a key tool for studying substrate use—yet an accurate model is required to analyse the complex data produced as these substrates become incorporated into the Krebs cycle. Objectives We aimed to generate a network model for the quantitative analysis of Krebs cycle intermediate isotopologue distributions measured by mass spectrometry, to determine the 13C labelled proportion of acetyl-CoA entering the Krebs cycle. An important use of 13C labelled metabolites has been to investigate the proportional contribution of different substrates to the Krebs cycle under varying conditions and across different tissues This has generated many insights, when used in studies of the isolated perfused heart where there can be significant changes in substrate selection under different pathological and physiological conditions (Aubert et al 2016; Crown et al 2016; Liu et al 2016; Lloyd et al 2003, 2004). We propose an easy to apply network model which accounts for both the stoichiometry between molecules in the network, and that between isotopomers
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