Ultramarathon induces metabolite changes associated with the pathophysiology of cardiovascular diseases- in silico and in vivo analysis
Authors: Wicik Z , Ciechanowicz A , Marszalek M , Keswani D , Wiecha S , Eyileten-Postula C , Malek M , Kucia M , Postula M 
Published: May 2023

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre (NCN) and Internal source of Medical University of Warsaw.

Introduction
Regular physical exercise is analogous to improved cardiovascular health and increased longevity. However, if in excess it can act as a double edged sword. Acute bouts of extreme physical activity trigger complex molecular responses reflected by changes in inflammatory markers and metabolic pathways that are believed to yield detrimental effects on the cardiovascular system (CVS)[1].

Purpose
We aimed to assess the effects of high-intensity and long-lasting exercise prototype (100km run) on the metabolomic changes in a unique population of high-level long-term ultra-marathon runners.

Methods
Plasma samples were collected from ultramarathon runners (n=22) with 2-time points: before and after the run (Completion (100 km) or exhaustion (52 - 91 km, median 74 km)).
For untargeted metabolomic analysis we used ultra-high resolution Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry with an electrospray ion source in direct injection. Mass spectra were analyzed by T-Rex2D algorithm in the MetaboScape5.0. Identified compounds were ontologically analyzed using MetaboAnalystR. MetscapeCytoscape plugin was used for metabolite-gene interaction analysis and gene lists were obtained from Biomart and DisGeNet.

Results
Mann-Whitney test (FDR&lt;0.05) identified 1601 up and 1030 downregulated metabolites with a at least 2 fold of difference after high-intensity run. Enrichment analysis showed that the top pathways affected by analyzed metabolites were the TCA cycle (FDR=0.0442), metabolism of; Sphingolipids (p=0.00774), Glyoxylate and dicarboxylate (p=0.0249) and Butanoate (p=0.0352). Top disease phenotypes associated with differentially regulated metabolites were critical illness (major trauma, severe septic shock, or cardiogenic shock) (p=0.00154) and multiple CVS-related diseases (Fig1).
Top after the run up-regulated metabolites identified in the study included heart failure associated acetylcarnitines[2] CAR(14:2) (log2FC=11.57; AUC=0.97), Dodecanoylcarnitine (2.00;0.960), 9-Decenoylcarnitine (1.78;0.95), CAR(DC8:0) (14.99;0.94), but also 2-Carboxy-5,7-dimethyl-4-octanolide (8.04;0.98), Methylmalonic acid (9.33;0.96) and cardiotoxic Cyclophosphamide (16.29;0.940)[3]. Top down-regulated metabolites after the run included potentially cardio-protective Gamma Glutamylglutamic acid (log2FC=-1.81; AUC=0.85), Fructose 1,6-bisphosphate (-2.42;0.88)[4], Oleamide (-1.82;0.86) and gamma-Glutamylglycine (-7.98;0.85).
Interaction network analysis focused on genes associated with CVS identified atherosclerosis/ obesity-related Phospholipase A2 Group IB (PLA2G1B) as potentially the most affected gene by analyzed metabolites (Fig2).

Conclusion
Results show a twofold post run increase in metabolites that are both potential markers for acute exercise and that are strongly associated with CV diseases. This calls for consideration of the detrimental effects of excessive exercise and implementation of monitoring in amateur athletes.