Abstract
though physical activity is a health-promoting, popular global pastime, regular engage- ment in strenuous exercises, such as long-distance endurance running races, has been associated with a variety of detrimental physiological and immunological health effects. The resulting altered physiological state has previously been associated with uctuations in various key metabolite con- centrations; however, limited literature exists pertaining to the global/holistic metabolic changes that are induced by such. This investigation subsequently aims at elucidating the metabolic changes induced by a marathon by employing an untargeted proton nuclear magnetic
variety of detrimental physiological and immunological health effects. The resulting altered physiological state has previously been associated with uctuations in various key metabolite con- centrations; however, limited literature exists pertaining to the global/holistic metabolic changes that are induced by such. This investigation subsequently aims at elucidating the metabolic changes induced by a marathon by employing an untargeted proton nuclear magnetic resonance ( 1 H-NMR) spectrometry metabolomics approach. A principal component analysis (PCA) plot revealed a nat- ural differentiation between pre- and post-marathon metabolic pro les of the 30-athlete cohort, where17 metabolite uctuations were deemed to be statistically signi cant. These included reduced concentrations of various amino acids (AA) along with elevated concentrations of ketone bodies, glycolysis, tricarboxylic acid (TCA) cycle, and AA catabolism intermediates. Moreover, elevated concentrations of creatinine and creatine in the post-marathon group supports previous ndings of marathon-induced muscle damage. Collectively, the results of this investigation characterize the strenuous metabolic load induced by a marathon and the consequential regulation of main energy- producing pathways to accommodate this, and a better description of the cause of the physiological changes seen after the completion of a marathon. Keywords: endurance races; marathon; metabolites; untargeted metabolomics; 1 H-NMR spectrome- try; serum metabolome 1. Introduction The year 2021 marks the 125th anniversary of the rst marathon run during the 1896 Summer Olympics in Greece. The popularity of this event sparked the conception of long-distance ( 5 km) endurance running races, generally categorized as half-marathons (21.1 km), marathons (42.2 km) and ultra-marathons ( 42.2 km) [1]. Not only has partici- pation in marathons become increasingly common, but it has also become af liated with the many health bene ts that are associated with aerobic exercise [2]. The most notable of which is the lower prevalence of cardiovascular disease [3], elevated cognitive health [4], Metabolites2021,11, 656.
Metabolites2021,11, 656 2 of 13 and increases in skeletal muscle mitochondrial volume as well as subsequent increases in muscle oxidative capacity [5]. There is, however, a large disparity between the energy expenditure and bodily demands associated with marathon running in comparison to most other aerobic exercises. As such, regular participation in these endurance running events have been found to induce numerous potentially deleterious immunological and physiological health effects. Some of these immunological effects include acute pro- and anti-in ammatory responses [6], damage to bronchial epithelial cells [7], a perturbed mu- cosal immune system, and higher susceptibility to symptoms of upper respiratory tract infections [8]. On a physiological level, short-term occurrences of muscle damage [9] and medial tibial stress syndrome [10] are common for the average marathon participant, while more acute effects such as an increased risk for myocardial brosis [11], deleterious cardiac structural changes [12], as well as acute liver and renal damage [13] have been reported for extreme/elite veteran marathon athletes. Although these immunological and physiological effects have been well character- ized, there is limited literature on the impact of these races on metabolite uctuations (metabolome) of marathon runners using untargeted metabolomics. Metabolomics aims to comprehensively detect, identify, and quantify uctuations in metabolite (<1500 Da organic and inorganic chemical compounds) concentrations in a biological system in response to a perturbation (disease, environmental factors, drug-intake, lifestyle, dietary, etc.), as a means of providing information regarding the altered physical state [1416]. Previous investigations [1720] that have employed targeted and/or semi-targeted metabolomics approaches have provided credible information on the metabolic effects of strenuous exercises. In short, energy production takes place in a hierarchical manner during physical activity [21], where the contribution of each metabolic pathway is deter- mined by factors such as the overall intensity, duration, and frequency of exercise [22]. The various metabolic processes at play include: (1) substrate-level phosphorylation via the phosphocreatine system [23], providing suf cient ATP for only a few seconds of run- ning activity [18]; (2) anaerobic glycolysis and homolactic fermentation of pyruvic acid, producing suf cient ATP for an additional few minutes of running activity [22];
the overall intensity, duration, and frequency of exercise [22]. The various metabolic processes at play include: (1) substrate-level phosphorylation via the phosphocreatine system [23], providing suf cient ATP for only a few seconds of run- ning activity [18]; (2) anaerobic glycolysis and homolactic fermentation of pyruvic acid, producing suf cient ATP for an additional few minutes of running activity [22]; and (3) aerobic catabolism of dietary substrates by means of oxidative phosphorylation, which is of high energetic value to endurance athletes, since it is able to supply suf cient ATP to support several hours of exercise, provided suf cient nutrient store availability [22]. Carbohydrates are widely recognized as the primary aerobic ATP source utilized during endurance running events [18,24,25], although the capacity of aerobic glycolysis can be limited during continuous running activity [26]. Insuf cient free glucose and glycogen stores reportedly lead to a gluconeogenic in ux, reduced insulin secretion, and an elevated glucagon/insulin ratio, subsequently activating alternative energy-producing pathways such as lipolysis and protein catabolism [25]. According to Hawley and Leckey [27], the aerobic carbohydrate utilization rate is reduced, while an upregulated fatty acid oxidation is observed in skeletal muscles during endurance exercise [28]. This is supported by various previous metabolomics studies [18,20,24,29] that observed elevated concentrations of fatty acids, glycerol, acyl- carnitines, and ketone bodies, concurrent with upregulated lipolysis and ketogenesis activity. Moreover, saturation of beta-oxidation (elevated 3-hydroxy acids) and subsequent upregulation of omega-oxidation (elevated dicarboxylic acids), which is normally consid- ered to be a minor pathway capable of compensating for incomplete beta-oxidation, has been reported [24,25] following a marathon. In addition to the utilization of lipids as an alternative fuel substrate, a general reduction in amino acids (AA) and elevation in their associated catabolism intermediates has also been observed in previous metabolomics investigations [19,20,24], further indicating the utilization of proteins/AA as yet another alternative fuel substrate. Although previous studies provide credible information pertaining to endurance exercise-induced metabolic changes, most are based on studies done using targeted and/or semi-targeted approaches (biased) that were performed in controlled environments (cy-
been observed in previous metabolomics investigations [19,20,24], further indicating the utilization of proteins/AA as yet another alternative fuel substrate. Although previous studies provide credible information pertaining to endurance exercise-induced metabolic changes, most are based on studies done using targeted and/or semi-targeted approaches (biased) that were performed in controlled environments (cy-
Metabolites2021,11, 656 3 of 13 cling, treadmill, rowing activities) [30]. As such, the current study is aimed at investi- gating the effects of a marathon (42.2 km) on the serum metabolome of 30 recreational marathon runners by using an untargeted proton nuclear magnetic resonance ( 1 H-NMR) metabolomics approach. Considering this, we aim to not only con rm the previously pro- posed marathon-induced metabolic changes, but to possibly identify additionally affected metabolic pathways, allowing for a more holistic view of the global metabolome change induced by a marathon. 2. Results The principal component analysis (PCA) plot (Figure) shows clear separation of the pre-marathon and post-marathon metabolome data. Upon employing the rst round of the multi-statistical approaches, 67 of the original 132 1 H-NMR spectral bins were deemed signi cant, while the second round identi ed 17 statistically signi cant metabolites associated with these bins. These metabolites are listed in Table, and uctuations are discussed in detail thereafter (associated PCA loading plot is illustrated in Figure S1).Metabolites 2021, 11, x FOR PEER REVIEW 3 of 14 Although previous studies provide credible information pertaining to endurance ex- ercise-induced metabolic changes, most are based on studies done using targeted and/or semi-targeted approaches (biased) that were performed in controlled environments (cy- cling, treadmill, rowing activities) [30]. As such, the current study is aimed at investigating the effects of a marathon (42.2 km) on the serum metabolome of 30 recreational marathon runners by using an untargeted proton nuclear magnetic resonance ( 1 H-NMR) metabo- lomics approach. Considering this, we aim to not only confirm the previously proposed marathon-induced metabolic changes, but to possibly identify additionally affected met- abolic pathways, allowing for a more holistic view of the global metabolome change in- duced by a marathon. 2.Results The principal component analysis (PCA) plot (Figure 1) shows clear separation of the pre-marathon and post-marathon metabolome data. Upon employing the first round of the multi-statistical approaches, 67 of the original 132 1 H-NMR spectral bins were deemed significant, while the second round identified 17 statistically significant metabolites asso- ciated with these bins. These metabolites are listed in Table
2.Results The principal component analysis (PCA) plot (Figure 1) shows clear separation of the pre-marathon and post-marathon metabolome data. Upon employing the first round of the multi-statistical approaches, 67 of the original 132 1 H-NMR spectral bins were deemed significant, while the second round identified 17 statistically significant metabolites asso- ciated with these bins. These metabolites are listed in Table 1, and fluctuations are dis- cussed in detail thereafter (associated PCA loading plot is illustrated in Figure S1). Figure 1. Principal component analysis (PCA) plot illustrating the natural differentiation of the pre- marathon (red circles) and post-marathon (green triangles) serum metabolic profiles of the 30 mar- athon participants. Table 1. Statistically significant marathon-induced metabolite changes. Metabolite (PubChem ID) Pre-Marathon Post-Marathon Pre- vs. Post-Marathon Figure 1. Principal component analysis (PCA) plot illustrating the natural differentiation of the pre-marathon (red circles) and post-marathon (green triangles) serum metabolic pro les of the 30 marathon participants.
Metabolites2021,11, 656 4 of 13 Table 1.Statistically signi cant marathon-induced metabolite changes. Metabolite (PubChem ID) Pre-Marathon Post-Marathon Pre- vs. Post-Marathon Average Concentration in M (Standard Deviation) p-Value (<0.05) d-Value ( 0.5) 3-Hydroxybutyric acid (441) c 56.7 (32.4) 424.8 (268.8) 1.0 10 12 3.7 3-Hydroxyisobutyric acid (87) a * 19.4 (5.9) 38.5 (9.4) 9.1 10 10 1.9 3-Methyl-2-oxovaleric acid (47) b 35.7 (16.3) 70.5 (18.2) 7.7 10 8 1.4 Acetoacetic acid (96) b 21.3 (6.2) 55.0 (26.2) 2.4 10 8 2.5 Acetone (180) b 6.7 (2.1) 17.2 (11.2) 7.7 10 7 2.3 Citric acid (311) c 137.5 (33.6) 221.9 (55.0) 2.9 10 10 2.0 Creatine (586) b 67.9 (21.9) 100.2 (51.8) 9.7 10 5 1.1 Creatinine (588) b 50.7 (9.5) 70.1 (18.5) 2.5 10 7 1.3 Glucose (5793) 1426.1 (382.1) 1927.3 (469.6) 1.4 10 5 1.1 Histamine (774) a * 93.9 (26.6) 68.9 (26.8) 2.5 10 3 1.5 Isoleucine (6306) 72.3 (20.4) 49.5 (10.9) 1.7 10 8 1.1 Lactic acid (612) 2472.0 (851.5) 4423.3 (1182.7) 2.0 10 8 1.9 Leucine (6106) 159.0 (36.8) 119.0 (22.0) 1.7 10 8 1.2 Lysine (5962) 161.4 (42.0) 127.6 (30.4) 1.4 10 5 0.9 Proline (145742) c 284.1 (73.3) 219.2 (59.0) 5.8 10 7 1.0 Pyruvic acid (1060) b 60.9 (28.1) 112.5 (38.5) 6.3 10 8 1.4 Valine (6287) 267.0 (53.3) 200.3 (35.1) 1.8 10 10 1.3 a No JRES or COSY con rmation; b JRES 2D con rmation only; c COSY 2D con rmation only; * level 2 identi cation. 3. Discussion The majority of the metabolites listed in Table energy-producing pathways, including the phosphagen system, anaerobic and aerobic glycolysis, the tricarboxylic acid (TCA cycle), ketogenesis, and amino acid oxidation (illustrated in Figure). Anaerobic glycolysis typically involves the conversion of accumulating pyruvic acid to lactic acid, via lactic acid dehydrogenase, accepting NADH as a coenzyme, and producing NAD + [31]. This is concurrent with the elevated post-marathon lactic acid and pyruvic acid observed in the current investigation (Figure) and is further supported by previous studies [18,25]. Although this mechanism provides a more rapid method of energy produc- tion than aerobic glycolysis and aids in the
acid, via lactic acid dehydrogenase, accepting NADH as a coenzyme, and producing NAD + [31]. This is concurrent with the elevated post-marathon lactic acid and pyruvic acid observed in the current investigation (Figure) and is further supported by previous studies [18,25]. Although this mechanism provides a more rapid method of energy produc- tion than aerobic glycolysis and aids in the maintenance of the NAD + /NADH ratios [31], its performance is restricted due to the resulting lactic acidosis [32], hence coercing the transition to aerobic glycolysis and the catabolism of alternative fuel substrates [22]. It is well known that carbohydrates are preferentially oxidized by the body during endurance-type exercises [23], reportedly leading to glucose and glycogen store depletion within approximately 90 min after the start of endurance running (at >75% of maximum oxygen uptake) [26]. However, elevated serum glucose was observed immediately post- marathon in this investigation (Figure). This is supported by the studies conducted by Stander et al. [25] and Lewis et al. [18] who reported elevated post-marathon serum glucose, as well as an elevation in the gluconeogenesis-associated metabolites. A plausible explanation for this includes the initial depletion of free glucose as well as intramuscular and liver glycogen stores, resulting in downregulated insulin secretion, upregulated gluco- neogenesis, and an elevated glucagon/insulin ratio [25]. This phenomenon is thought to be regulated by a variety of factors, including altered hormone secretion (glucocorticoids) in response to the stress signals caused by the hypoxic state and the strenuous energy de- mands induced during the endurance race [33]. Cortisol is one of the major glucocorticoids associated with the latter, and results in the translocation of glucose transporters to the cell membrane, subsequently inhibiting glucose uptake during fasting and/or exercising and eventuating elevated blood glucose levels [34].
Metabolites2021,11, 656 5 of 13Metabolites 2021, 11, x FOR PEER REVIEW 5 of 14 Figure 2. A schematic description of the marathon-induced metabolic changes, showing increased (↑) and decreased (↓) concentrations of significantly altered metabolites in the post- marathon samples (in bold text) are indicated relative to the pre-marathon values. ATP adenosine triphosphate, ADP adenosine diphosphate, AMP adenosine monophosphate, NAD + nicotinamide adenine dinucleotide, NADH reduced nicotinamide adenine dinucleotide, NADP + nicotinamide adenine dinucleotide phosphate, NADPH reduced nicotinamide adenine dinucleotide phosphate, Pi inorganic phosphate, CoA coenzyme A, FAD flavin adenine dinucleotide, FADH2 reduced flavin adenine dinucleotide, GTP guanosine triphosphate, GDP guanosine diphosphate. Figure 2. A schematic description of the marathon-induced metabolic changes, showing increased (") and decreased (#) concentrations of signi cantly altered metabolites in the post-marathon samples (in bold text) are indicated relative to the pre-marathon values. ATP adenosine triphosphate, ADP adenosine diphosphate, AMP adenosine monophosphate, NAD + nicotinamide adenine dinucleotide, NADH reduced nicotinamide adenine dinucleotide, NADP + nicotinamide adenine dinucleotide phosphate, NADPH reduced nicotinamide adenine dinucleotide phosphate, P iinorganic phosphate, CoA coenzyme A, FAD avin adenine dinucleotide, FADH 2reduced avin adenine dinucleotide, GTP guanosine triphosphate, GDP guanosine diphosphate.
Metabolites2021,11, 656 6 of 13 Endurance-induced adaptations (generally only reported for highly trained aerobic athletes) of the skeletal muscles includes a slower utilization of carbohydrates and an upregulated lipid metabolism [27]. Although the current study cohort includes both am- ateur and well-trained marathon participants, no signi cant differences were observed when comparing the respective metabolic pro les based on previous endurance running experience. Lipids are also catabolized (especially 6090 min into such endurance events) via -oxidation, contributing to the production of acetyl-CoA [28], and initially resulting in an upregulated channeling of the latter into the TCA-cycle. This acetyl-CoA in ux may account for the elevated serum concentration of citric acid observed during the cur- rent (Figure) and previous studies [ 18,25]. Additionally, the high energy demands and associated imbalanced redox state induced by participation in such activities [35] may cause an upregulation in citric acid synthase and pyruvic acid dehydrogenase activity, as previously observed by McKenzie et al. [36], in an attempt to produce the much-needed NADH/FADH 2and, ultimately, ATP via the electron transport chain. However, the con- tinuous in ux of acetyl-CoA from the various energy-producing pathways, accompanied by the aforementioned imbalanced redox state, may exceed the mitochondrial oxidative capacity, eventuating in the activation of ketogenesis [31,37]. The latter is demonstrated in this investigation by the elevated concentrations of 3-hydroxybutyric acid, acetone, and acetoacetic acid observed in the post-marathon serum samples (Figure). In accordance with previous literature [38], AA catabolism was activated as an alter- native means of producing energy during the marathon (Figure). This is supported by a reduction in concentrations of AAs (leucine, isoleucine, valine, lysine, and proline) and the elevation of the various observed serum AA catabolism intermediates (3-methyl-2- oxovaleric acid and 3-hydroxyisobutyric acid). Additionally, the reduced concentrations of serum histamine (decarboxylated form of histidine) observed in the post-marathon samples (Figure) may be ascribed to the preferred catabolism of histidine for ATP synthesis via the TCA cycle, rather than to be decarboxylated to histamine [39]. Lastly, considering the role of histamine during acute in ammatory responses, reduced post-marathon histamine may additionally be ascribed to an immune
reduced concentrations of serum histamine (decarboxylated form of histidine) observed in the post-marathon samples (Figure) may be ascribed to the preferred catabolism of histidine for ATP synthesis via the TCA cycle, rather than to be decarboxylated to histamine [39]. Lastly, considering the role of histamine during acute in ammatory responses, reduced post-marathon histamine may additionally be ascribed to an immune suppression experienced during the open window effect directly after the marathon [39,40]. Although protein catabolism normally only contributes to supplying a small amount of the total energy requirements during a marathon, branched-chain amino acids (BCAA) are preferentially oxidized [25], a situation triggered by, amongst others, a reduced ATP:ADP ratio, acidosis, and the depletion of muscle glycogen stores [36]. Furthermore, the reduced post-marathon serum concentrations of leucine are known to inhibit glutamine transport into the cells, subsequently inhibiting mTORC1 and resulting in autophagy [25] as the body's last resort to nd the necessary energy-producing substrates to comply with the massive energy demands required to complete such an event [41]. Lastly, the elevated serum levels of creatine and creatinine, are most likely indicative of muscle damage [42], or perhaps also to a lesser extent, a declining kidney function [43], or myocardial cell injury [44], all of which have been previously proposed to potentially occur during strenuous endurance exercise. In conclusion, the current study was aimed at investigating marathon-induced (42.2 km) metabolite shift using an untargeted 1 H-NMR metabolomics approach. The aforemen- tioned metabolic changes to aerobic and anaerobic glycolysis, ketogenesis, AA catabolism (in particular, BCAAs) and the TCA cycle, indicated the extent to which the body needs to adapt in order to comply with the energy demands required for the completion of a marathon. Increases in all three endogenous ketone bodies and decreases in all three BCAAs re ect a high reliance on their associated metabolic pathways for energy produc- tion, suggesting a possible target for the development of athletic performance-enhancing strategies. The decreased post-marathon histamine concentration has not been reported before and may suggest an alternative source of energy production during a marathon run. Furthermore, the presence of creatinine and creatine
Description
The study characterizes metabolic changes in marathon runners using NMR metabolomics.