*3.3. Influence of Sex and Age in the Plasma Markers of Oxidative Stress Evolution of the Runners*

Data depicted in Figure 1, demonstrate that female runners have a significantly higher CG content when compared to males at the end of the race and 48 h later. To the contrary, MDA concentration 48 h post-race is higher in male compared to female runners. The antioxidant defenses, measured as GR and GPx enzymatic activity, are not conditioned by runner's sex.

**Figure 1.** Changes in plasma markers of oxidative status throughout the study according to runners' gender. CG (carbonyl groups) content (**A**), MDA (malondialdehyde) (**B**), GR (glutathione reductase) activity (**C**) and GPx (glutathione peroxidase) activity (**D**).

Data from Figure 2 shows the effect of the runner's age. A tendency can be observed in the plasma oxidative status of the senior runners which appears to be globally worse compared to the in the younger runner's values. Briefly, less antioxidant defenses and higher levels of oxidative damage to lipids and proteins. However, only the lipid peroxidation (MDA concentration) was significantly affected by the age. Thus, the MDA concentration was significantly higher in the senior runners when compared to the middle age and young competitors at the finish line and 48 h post-race.

**Figure 2.** Changes in plasma markers of oxidative status throughout the study according to runners' gender. CG (carbonyl groups) content (**A**), MDA (malondialdehyde) (**B**), GR (glutathione reductase) activity (**C**) and GPx (glutathione peroxidase) activity (**D**).

### *3.4. Correlation between the Plasma Markers of Oxidative Stress and the Skeletal Muscle Force Production, Muscle Damage and Systemic Inflammatory Response*

Regarding the post-race skeletal muscle strength, Table 3 shows that there was significant negative correlation between basal MDA concentration and the fold increase in HG values in the finish line. We also observed a significant positive correlation between basal GR activity and the fold increase in SJ values at the finish line.

**Table 3.** Significant correlations between baseline plasma markers of oxidative stress and Delta values of muscle strength (SJ and HG) and muscle damage (CK and LDH).


In addition, basal GR activity negatively correlated with the delta values of LDH at the finish line, 24 h and 48 h post-race. Moreover, basal GR activity negatively correlated with the fold increase of CK at the finish line. It was also demonstrated a significant positive correlation between basal CG content (oxidative damage to proteins) and the rise in LDH observed 24 h and 48 h post-race (Table 3). The inflammatory response observed after the race did not correlate significantly with any of the plasma markers of oxidative stress parameters measured.

#### *3.5. Multiple Regression Analysis*

Results of the multiple regression analysis are listed in Table 4. When performing the multiple linear regression analysis using Age and GR as the predictive variables, a significant regression equation was obtained for the SJ delta Value dependent variable. This analysis would indicate that younger age as main predictor and higher basal GR concentration, a lower SJ delta value after finishing the ultramarathon was obtained. This regression model predicts the 28.3% of the variance. Another multiple linear regression model was obtained in which the dependent variable was the Δ CK finish line value and the main predictive variables were Age and GR. In this scenario, the regression analysis model predicts a 18.3% of the variance.


Abbreviations: Δ SJ (Fold increase Squat Jump); Δ CK (Fold increase Creatine Kinase); GR (Glutathione reductase).

As we have previously mentioned, according to the criterion proposed by Cohen [38], our regression models might be considered for their predictive value, explaining within a multicausal model context the influence of oxidative stress on muscle damage and fatigue after a severe effort such as running an ultramarathon.

#### **4. Discussion**

The present investigation aimed to ascertain the relationship between several athlete's plasma markers of oxidative stress and the degree of muscle strength and damage after ultraendurance exercise. Results of this study were also extended to investigate the possible runner's sex and age influence. It is important to remark that this short of studies present limitations regarding the sample size due to the difficulty to finish the competition by the runners. The demographic/anthropometric characteristics of the present study (age, body composition and resistance training) as well as muscle damage, acute inflammation and muscle strength variables have been previously analyzed and discussed by our group [3] (See Supplementary Table S1).

Briefly, male participants showed significant higher pre-race values for weight, BMR, BMI and percentage of muscular mass when compared to women runners [31,32]. The muscular membrane disruption variables, LDH and CK release, peaked after the race and returned to normal values after 24 h. In the Supplementary material section, we also have included results about acute inflammatory processes (CRP), muscle damage (CK and LDH) and muscle strength (SJ and HG), previously published by our research group [4]. No changes were observed in the performance of SJ and HG before the race when compared with the finish line. Both acute inflammation and muscle damage were observed in the finish line, as well as 24 h and 48 h post race.

Regarding the plasma markers of oxidative stress, a time-course analysis of GPx and GR activity, and the oxidative damage to lipids and proteins (MDA and CG, respectively) was performed at the finish line, 24 h and 48 h post-race. It has been showed that ultra endurance exercise is associated with a notably enhanced rate of oxygen utilization and the generation and accumulation of ROS [6]. Moreover, the glutathione system increases its activity to restore the cell redox balance when the formation of ROS is enhanced. Our data showed an increase of the oxidative damage to macromolecules (lipids and proteins) indicating an increase of ROS cellular levels. Thus, lipid peroxidation damage appears to be increased in the finish line and 48 h post-race as confirmed by the MDA levels, proving the presence of oxidative damage to lipids two days after the extreme exercise. A partially recovering effect was observed 24 h after the race as can be seen in Table 2. At this respect, previous studies have shown controversial results regarding the blood MDA levels after ultraendurance exercise. Several studies showed an increase on the cellular MDA levels [14,16,39] compared to data supporting no lipid peroxidation effect after extreme endurance exercise [12,13]. In addition, Skenderi et al., [9] demonstrated an MDA levels decrease 48 h post-race when compared to control and post-race values. However, in this study, the sport type (running, swimming), the distance, the accumulated altitude or the anthropometric characteristics of the sample might be influencing these results. It is also noteworthy to mention that these differences can be attributed to methodological aspects. Authors used the TBARS (thiobarbituric acid-reacting substances) technique which, appear to be a less robust measure of lipid peroxidation [6].

The analysis of the CG content remained significantly elevated for all the time points of the study (finish line, 24 h post-race and 48 h post-race). Interestingly, Spanidis et al., [12] did not find significative differences for this parameter after an ultramarathon mountain race. This discrepancy is explained due to a sample size effect. In contrast, Turner et al., [16] demonstrated an increase in plasma CG content after an ultramarathon race, thus confirming oxidative damage to proteins after ultraendurance exercise.

Regarding the glutathione system enzymes, no significant differences were observed for the GPx activity. Conversely, the GR activity showed a significant increase in the finish line that was even greater 24 h post race, interestingly concurring with the partial MDA concentration recovery. Although both enzymes are mainly located in the intracellular compartment, their plasmatic activity have been broadly used as a measurement of the antioxidant status [40–43]. In the case of ultraendurance exercise, previous studies show contradictory results to what concern to the antioxidant enzymes [7,12,14,39], but our results support the hypothesis of a compensatory mechanism based on a temporary increase of the antioxidant defense to compensate an oxidative insult [44,45]. Although the activity of these two enzymes has been used to evaluate the presence of oxidative stress, GR is considered the limiting factor of these antioxidative system [46]. It is plausible that the increase of GR activity is not accompanied by an enhanced GPx activity because of other antioxidant enzymes such as catalase or paraoxonase also able to degrade hydrogen peroxide that could be affected by the intense exercise [10,47].

Moreover, it is remarkable that we report a significant negative correlation between the basal GR activity of the runners and the degree of muscle membrane disruption after the race. Thus, the basal activity of this enzyme correlated with [LDH] in the finish line, after 24 h and 48 h and with the [CK] in the finish line and after 24 h. In addition, the resting levels of oxidative damage to proteins (CG content) also showed a significant positive correlation with the magnitude of post-race muscle injury ([LDH] after 24 h and 48 h). It is important to notice that we assume that serum CK and LDH assess for muscle membrane disruption and do not necessarily correlate with muscle structural damage. Furthermore, we have also reported a significant positive correlation between basal GR activity and the improvement in the SJ performance as well as, a significant negative correlation between the basal levels of lipid peroxidation (MDA concentration) and the enhancement in the HG execution. Although there is a limitation on the correlation's coefficient power to assume an evident causality between oxidative stress and muscle fatigue, these novel findings, suggest that a stronger basal plasma oxidative status might improve muscle strength during ultraendurance sports practice. However, further studies are necessary to increase the number of research volunteers and validate the present results.

Finally, we have reported sex differences in oxidative damage to macromolecules. Surprisingly, female athletes showed higher CG content and less MDA levels than male athletes, although female athletes have less muscular mass and higher body fat mass percentage. There is almost no literature considering sex differences in type of events, probably

due to the difficulty in getting sufficient sample sizes. Although several studies include female runners in their research, they do not describe sex differences in the parameters measured [48,49]. Recently Devrim-Lanpir et al., reported significant interaction between time at exhaustion and dietary antioxidant intake in males, but not in females, who underwent an acute exhaustive exercise test (a cycle ergometer) followed by a treadmill test in a laboratory [50]. Moreover, the results of the multivariate analysis show us the predictive value of basal GR concentration and sex in relation to muscle fatigue and cell damage, after ultramarathon.
