Prolonged strenuous running, e.g., a marathon run, induces a rise in the concentrations and/or activity of biomarkers that reflect physiological stress of the skeletal muscle, liver, heart, and some other tissues (Banfi et al., 2012). Oxidative stress is one of the processes associated with tissue damage. It can be induced by exercise, especially by prolonged forms of exercise, such as the marathon and its more demanding variations (Gomez-Cabrera et al., 2006; Kawamura and Muraoka, 2018). Consequently, high-intensity or long-duration exercise can potentially lead to major changes in the markers of tissue damage that are commonly associated with pathological states (Smith et al., 2004). For instance, the concentration of serum cardiac troponin T (cTnT), one of the markers of heart muscle damage, increase after endurance events in up to 68%, and reach levels typically diagnostic of acute myocardial infarction (Fortescue et al., 2007; Eijsvogels et al., 2016). Similarly, after a long-distance run, liver injury biomarkers, such as aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), and lactate dehydrogenase (LDH) activities, and conjugated bilirubin, are elevated (Lippi et al., 2011; Shin et al., 2016). Changes in the concentration and/or activity of markers of heart and liver damage after a marathon run are transient and most return to the baseline after several days. While there is insufficient evidence to indicate any adverse effects of exercise on the heart (Kaleta-Duss et al., 2020) and liver (Lippi et al., 2011) of amateur marathon runners, further studies investigating the impact of strenuous exercise on the markers of heart and liver damage can give insight into the physiology of adaptation of these organs to a prolonged strenuous exercise.

Multiple studies aim to identify the optimal approach of reducing the impact of strenuous exercise on markers of tissue damage (for review see Kawamura and Muraoka, 2018). Currently, increasing attention is being focused on remote ischemic preconditioning (RIPC), which has been shown to be protective against ischemia-reperfusion injury (Tapuria et al., 2008) and other stressors, and can potentially increase sports performance (Caru et al., 2019). RIPC is a procedure, whereby brief cycles of limb ischemia and reperfusion are induced by inflating and deflating a blood pressure cuff. In this manner, skeletal muscle undergoes preconditioning and distal tissues are protected. RIPC reduces the increase of cardiac troponin levels (cTn) by 42% after coronary artery bypass (Venugopal et al., 2009), and by approximately 30% after valve replacement surgery (Cao et al., 2017). Similarly, it was shown that RIPC, when applied before liver resection, decreases the AST activity and bilirubin levels in comparison to control, not pre-conditioned, patients (Rakic et al., 2018; Liu et al., 2019; Wu et al., 2020). A similar trend was observed for RIPC and liver transplants (Robertson et al., 2017). Further, RIPC effectively reduces oxidative stress in some clinical scenarios, e.g., cardiopulmonary bypass and others (Chen et al., 2015; Arvola et al., 2016; Pinheiro et al., 2016) and improve peripheral endothelial function (Maxwell et al., 2019).

The reported outcomes indicate that RIPC might impact exercise-induce changes in the markers of heart and liver damage. However, to date, the data on this topic are limited and conflicting (El Messaoudi et al., 2013; Cocking et al., 2017). In the current study, we aimed to determine the effects of RIPC on the markers of heart and liver damage, and oxidative stress induced by a marathon run in amateur runners.

The main goal of the current study was to evaluate the effects of 10-day RIPC training on serum biomarkers of liver and heart damage induced by a marathon run. The observed significant increase in the levels of TnT and CK-MB, the markers of heart damage, was attenuated in runners who underwent RIPC training prior to the run. A similar effect of RIPC was observed on the levels of ALT, AST, and GGT activity, and BIL-T, the markers of liver damage.

The effects of RIPC on the induction of cTn levels by an endurance exercise have been studied before (El Messaoudi et al., 2013; Cocking et al., 2017). El Messaoudi et al. (2013) showed that RIPC before 70min of cycling (80% maximal heart rate) and then until exhaustion (95% maximal heart rate) did not affect the cTn levels. On the other hand, Cocking et al. (2017) reported that RIPC before a 1-h time cycling trial led to an attenuation of cTn level increase after the exercise, but without any effect on the left ventricle function. In the current study, while the exercise (running vs. cycling) and load (1 vs. 3.5h) were different from those of the study of Cocking et al. (2017), the outcomes were similar in both studies. A major factor that could explain the different outcome of the study of El Messaoudi et al. (2013) is the amount of muscle tissue involved in the RIPC procedure. In work of El Messaoudi et al. (2013), the RIPC was applied 3×5min bilaterally to the upper arm, while in Cocking et al. (2017), RIPC was applied 4×5min to the ipsilateral upper and lower limb, in an alternating manner. Similar to Cocking et al. (2017), in the current study, the RIPC protocol involved 4×5min intervals of ischemia applied to both lower limbs, which have more muscle mass than the upper limbs. Moreover, lower limbs are mainly involved in tested activities (running, cycling), while upper arms are less involved in cycling. The observed attenuation of the increase of the exercise-induced markers of heart damage after RIPC might indicate its protective effect. However, the questions of whether an exercise-induced increase in cTn levels originates solely in the cardiac muscle and its clinical relevance remain unresolved (Stavroulakis and George, 2020).

To the best of our knowledge, no other study investigating the effects of RIPC on exercise-induced markers of liver damage has been published. However, the data presented in the current study are in agreement with previous reports that participation in the marathon and ultra-marathon distance events results in elevated biomarkers associated with liver injury, including GGT, AST, ALT, and LDH activities (Shin et al., 2016). As most of these enzymes are also present in skeletal muscle, some authors suggest that they reflect skeletal muscle injury rather than liver injury (Lippi et al., 2011). That might be true for other enzymes but not for GGT. First of all, GGT is produced mainly in the liver, with little or no synthesis in skeletal muscle (White et al., 1985). Further, the serum GGT levels in individuals with Duchenne muscular dystrophy are in the normal range despite the substantial skeletal muscle damage indicated by high serum CK levels (Rosales et al., 2008). In the current study, we observed a significant increase in serum CK levels after the marathon run in all participants, indicating skeletal muscle damage. The RIPC training did not significantly affect the serum CK levels. On the other hand, the GGT activity was significantly increased after the run only in the SHAM group, indicating that liver activity during a prolonged strenuous exercise can be successfully impacted by RIPC.

Another marker of liver function that responds to strenuous exercise is bilirubin (De Paz et al., 1995; Shin et al., 2016). In the current study, bilirubin levels increased after the marathon run and returned to baseline over the next 7days. This is in agreement with other studies on strenuous running exercise, including half-marathon (Lippi et al., 2011), marathon (Kratz et al., 2002), and ultramarathon (Fallon et al., 1999; Wu et al., 2004; Arakawa et al., 2016), in which an increase in bilirubin levels was also observed. The increase can be a consequence of hemolysis, which is augmented by endurance running, but also can be a result of impaired liver function in response to exercise (De Paz et al., 1995). Hence, bilirubin, as a catabolite of heme, can be a marker of hepatobiliary insufficiency. While RIPC itself did not affect bilirubin levels at rest, we observed attenuation of BIL-D increase in the RIPC group after the marathon in the current study. Hence, our assumption that RIPC would attenuate exercise-induced increase in bilirubin levels was confirmed. Importantly, low resting bilirubin levels have been associated with increased cardiovascular risk, and exercise training increases resting bilirubin levels (Swift et al., 2012; Kang et al., 2013). However, it is important to make the distinction between training and acute exercise. Collectively, the above data suggest that RIPC protects against exercise-induced liver damage, confirming clinical observations of individuals with liver disease (Rakic et al., 2018).

In eighties of twentieth century, it was demonstrated that contracting skeletal muscles generate free radicals, and that prolonged and intense exercise leads to increased free radical formation and cellular oxidative damage (Davies et al., 1982; Alessio et al., 1988; Reid et al., 1994). Prolonged exercise induces free radical damage of proteins and lipids (Turner et al., 2011; Withee et al., 2017). For example, in one study, levels of MDA, a marker of lipid peroxidation, significantly increased in men after a marathon run (Gomez-Cabrera et al., 2006). We confirmed this observation in the current study, as both CD and MDA levels significantly increased after the run. To the best of our knowledge, this is the first study demonstrating that RIPC training attenuates exercise-induced oxidative stress. The lower level of oxidative stress in RIPC trained runners than that in the SHAM control could also be associated with a lower activity of GGT. The physiological role of GGT is to counteract oxidative stress by breaking down extracellular glutathione and making cysteine available for glutathione synthesis within the cell. Conversely, cysteinylglycine, a product of a GGT-catalyzed reaction, stimulates reactive oxygen species formation and oxidative damage in cell culture in the presence of transferrin iron (Drozdz et al., 1998). This may indicate some contribution of GGT to reactive oxygen species formation during a marathon.

Arterial ketone body ratio is used as an indication of the mitochondrial redox status in hepatic cells (White and Venkatesh, 2011). Decreased AKBR levels are associated with liver dysfunction and are linked to failure of other organs (Shimada et al., 1997). A marked decrease of AKBR levels often leads to a hepatic energy crisis, followed by an impairment of metabolic homeostasis. It is important to note that deleterious changes related to the low AKBR value occur several days after of heart and liver damage (Takahashi et al., 1997). In the current study, the drop in AKBR levels was temporary and the levels rapidly recovered after the marathon run, with a complete return to the baseline value after 1week. Furthermore, the reduction in AKBR levels after the run was much smaller in the RIPC group than that in the SHAM group.

Based on the obtained results, we conclude that repeated RIPC intervention in the form of training exerts a protective effect on the heart and liver by attenuating the induction of exercise-induced markers of organ damage and reducing oxidative stress after a marathon run.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Bioethics Committee for Clinical Research at the Regional Medical Chamber in Gdańsk (decision number KB-24/16; Gdańsk, Poland). The patients/participants provided their written informed consent to participate in this study.

JM and JA contributed to the conceptualization. JA, JM, AK, BS, BN, AB, LD-S, PB, and KS contributed to the methodology, the writing of the original draft preparation, and writing – review and editing. JM, AB, AK, BS, and BN contributed to the investigation. JM contributed to the project administration. JA contributed to the funding acquisition. All authors contributed to the article and approved the submitted version.

The sponsor had no role in the design of the study; in the collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the article for publication. The study was registered as a clinical trial NCT03417700.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.