Untargeted metabolomics reveals a mild impact of remote ischemic conditioning on the plasma metabolome and α-hydroxybutyrate as a possible cardioprotective factor and biomarker of tissue ischemia

Abstract

IntroductionRemote ischemic conditioning (RIC) is a maneuver by which short non-lethal ischemic events are applied on distant organs or limbs to reduce ischemia and reperfusion injuries caused by e.g. myocardial infarct. Although intensively investigated, the specific mechanism of this protective phenomenon remains incompletely understood and in particular, knowledge on the role of small metabolites is scarce.ObjectivesIn this study, we aimed to study perturbations in the plasma metabolome following RIC and gain insight into metabolic changes by the intervention as well as to identify potential novel cardio-protective metabolites.MethodsBlood plasma samples from ten healthy males were collected prior to and after RIC and tested for bioactivity in a HL-1 based cellular model of ischemia–reperfusion damage. Following this, the plasma was analyzed using untargeted LC-qTOF-MS and regulated metabolites were identified using univariate and multivariate statistical analysis. Results were finally verified in a second plasma study from the same group of volunteers and by testing a metabolite ester in the HL-1 cell model.ResultsThe analysis revealed a moderate impact on the plasma metabolome following RIC. One metabolite, α-hydroxybutyrate (AHB) however, stood out as highly significantly upregulated after RIC. AHB might be a novel and more sensitive plasma-biomarker of transient tissue ischemia than lactate. Importantly, it was also found that a cell permeable AHB precursor protects cardiomyocytes from ischemia–reperfusion damage.ConclusionUntargeted metabolomics analysis of plasma following RIC has led to insight into metabolism during RIC and revealed a possible novel metabolite of relevance to ischemic-reperfusion damage.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-017-1202-2) contains supplementary material, which is available to authorized users.

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“…Protective pathways of preconditioning-induced ischemic tolerance have been identified by transcriptomics, proteomics and metabolomics-based data sets (Stenzel-Poore et al, 2003 ; Scornavacca et al, 2012 ; Laursen et al, 2017 ). It is postulated that integration of RNA, protein and metabolite changes culminates in an ischemia tolerant cellular phenotype, yet few studies have attempted to identify the common pathways across omics platforms.…”

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“… 16 Cells were cultivated and subjected to 5.5-hrs simulated ischemia and 2 hrs reperfusion as previously described. 17 In short, the cells (~1x10 6 cells/well) were seeded into six-well plates (n=3-5 wells for each group) and cultivated for 48 hrs prior to induction of ischemia. Upon induction of ischemia, the growth medium was substituted by a stabilizing buffer solution (CaCl 2 : 1.25 mM, MgCl: 1mM, NaCl: 137 mM, KCl: 6 mM, Glucosemonohydrate: 10 mM, HEPES: 6 mM and NaH 2 PO 4 : 0.9 mM).…”

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“…Exogenous application of nicotinamide mononucleotide (NMN), an intermediate of NAD + synthesis, mimics the protective effect of IPC under ischemia and reperfusion injury. An untargeted metabolomics study has revealed that α-hydroxybutyrate (α-HB) stands out as highly significantly upregulated after IPC [ 78 ], while previous studies have shown that an elevation in the cytosolic NADH/NAD + ratio would promote α-HB formation. α-HB is a biomarker of the cytosolic NADH/NAD + ratio [ 79 ], indicating that IPC can regulate the NADH/NAD + ratio.…”