*Differences in Antioxidant Enzymes and Hemogram*

When intense physical exercise is performed (especially in untrained or those not familiar with the exercise), there is an increase in the production of reactive oxygen species, which are neutralized by our complex endogenous antioxidant defense system (GSH, GSSG, CAT, SOD, GPx and GR) and by exogenous antioxidants (vitamin C, vitamin E, carotenes) [30].

Regarding EAE, we observed higher levels of CAT activity, GSSG and GSSG/GSH, but lower levels of SOD activity in PRO versus AMA. Mena et al. [31] found higher resting levels of SOD, CAT and GPx in a sample of PRO cyclists compared to sedentary people. Tauler et al. [32] also showed differences in antioxidant enzyme activity in erythrocyte between PRO and AMA at rest. In the same study, a decrease in CAT (−12%), GPx (−14%) and GR activity (−16%) but an increase in SOD activity of about 25% after a submaximal test (80% VO2MAX; 1 h 30 min) was reported [32].

Long distance runners have been shown to have a three-fold higher CAT activity compared to short distance runners [18]. Similarly, it was observed that marathon runners had twice as high catalase activity compared to sprinters [12]. In this study, we also demonstrated higher levels of CAT in PRO than in AMA, and this may be largely explained by the fact that PRO perform greater volume, intensity and competitions (higher aerobic load and prolonged periods of exercise) than AMA, which induces higher levels of exposure to ROS and, consequently, adaptations of EAE [6]. When CAT levels increase, it is possible that GPx activity is not sufficient to neutralize high levels H2O2 (endurance exercise) [7].

Regarding SOD, Mena et al. [31] observed lower levels of SOD activity (−32.1%) in PRO than in elite cyclist, but in the case of CAT (80.0%) and GPx (149.0%), the levels were higher, reporting an ascending behavior of SOD during a stage race (2800 km in 17 stages) in PRO. Tauler et al. [32] has also found lower levels of SOD activity in PRO (−19.8%) than in AMA at baseline, which are in line with the results of our study. Antioxidant enzyme activity can be modified either by an initial increase (adaptation) or a decrease if the oxidative stress of long duration (utilization) [33]. Therefore, the low basal levels of SOD activity in professional cyclists could be overwhelmed and the high concentration of superoxide anions could activate CAT, allowing compensated metabolization of H2O2. This may be the reason why PRO has lower levels of SOD activity than AMA, as PRO have higher levels of exercise exigency that sometimes get close to exhaustion, which can lead a decrease in the working capacity of SOD.

On the other hand, there is evidence to suggest that GSH or GSH/GSSG decreases during exercise because of its utilization against ROS [33]. Ultra-endurance exercise depletes erythrocyte GSH levels by ∼66% for 24 h and levels remain ∼33% lower than normal 1 month later [34]. PRO frequently compete in longer distance events than AMA, which can lead to lower levels of GSH in PRO than in AMA, although no differences were observed in GSH between PRO vs. AMA in our study. In addition, the muscle can import GSH from plasma during exercise, and as a result, there is a change in the GSH/GSSG ratio after exercise with a decrease in the GSH/GSSG ratio at the time of exhaustion [35]. Furthermore, it is important to mention that tissues are not only capable of importing GSH but also exporting GSSG under oxidative stress [35]. Moreover, GSH is a molecule that is key in cellular redox status regulation, and consequences of prolonged GSH depletion may include a compromise in immunity, where lower GSH is associated with decreased lymphocyte proliferation and increased viral reactivation [34].

GSSG levels are a biomarker of cellular oxidative stress, since GSH is an important antioxidant in many tissues and oxidizes in the catalyzed reduction of H2O2 to H2O to become GSSG [36]. The increase in GSH (mainly) and GSSG in plasma after exercise could be explained by an efflux from the liver to other tissues, including skeletal muscle [37]. GSSG levels in skeletal muscle have previously been shown to increase by ~50% in rats after running on a treadmill at moderate intensity [38] and by ~20% after cycling in humans (workload corresponding to 90% of VO2peak; 10 × 4 min) [39]. Leonardo et al. [40] observed an increase in both GSSG and GSSG/GSH after a period of intense PRO training, which returned to their baseline levels after a period of tapering. We found similar baseline values of GSSG in our study. In addition, we found higher levels of GSSG and GSSG/GSH in PRO than in AMA.

The efforts made during cycling competitions produce oxidative stress in lymphocytes, leading to a reduction in GSH levels and an increase in GSSG levels. The decrease in GSH and increase in GSSG during exercise may be explained by an increase in H2O2 formation, as reported by Wang et al. who found that high-intensity exercise (80% VO2MAX) decreased GSH levels while lipid peroxidation increased immediately and after 24h of exercise [41]. Furthermore, in this study, lymphocytes were incubated with H2O2 for 2 and 4 h, promoting an increase in DNA fragmentation immediately and 24 h after high intensity exercise. Thus, H2O2 would cause a failure of the endogenous antioxidant system leading to DNA damage in lymphocytes. Ferrer et al. [42] found that high intensity exercise (swimming) increased GPx activity (converts GSH to GSSG) in lymphocytes, in the same way as other authors found after a cycling stage [43,44]. This supports the decrease in GSH and increase in GSSG after high intensity exercise. Therefore, the higher levels of GSSG and GSSG/GSH in PRO vs. AMA in our study may be due to a higher production of ROS, which leads to a higher production of GSSG and, consequently, of GSSG/GSH together with a decrease in GSH.

In addition, our study is the first to show correlations between GSSG/GSH with WVT1 (r = −0.657) and WVT2 (r = −0.635) in PRO. This is also supported by a trend towards a significant correlation between GSSG and WVT2 (r = −0.575; *p* = 0.06) in PRO. These relationships suggest that cyclists who generate more power at VT1 and VT2 have lower GSSG/GSH levels, and therefore, less oxidative stress, as GSSG/GSH ratio is known to be a marker of antioxidant status [20].

In response to strenuous physical working conditions, the body's antioxidant capacity may be temporarily diminished, as its components are used to scavenge the harmful radicals that are produced [45]. It is well known that exercise-induced ROS are detrimental to physiological function, including decreased performance and immune function and increased fatigue [45]. Moreover, it has been shown that the response of antioxidant capacity to exercise responds in a similar way to the activity of EAE [45]. Therefore, the antioxidant defense system may be temporarily reduced in response to increased ROS production but may increase during the recovery period as a result of the initial prooxidant insult [46]. However, contradictory findings have been reported where increases in GPx, SOD, and CAT, as well as decreases in GPx, GR, SOD have been observed [45]. Evidently, this controversy may depend on the moment of sampling (i.e., period of the season), as well as on the duration and intensity of the exercise, which varies considerably between studies.

It could be that there is an undefined optimal level of ROS production and oxidative damage required for adaptations in antioxidant defenses and other physiological parameters, leading to health and performance improvements [45]. However, overproduction of ROS and oxidative damage due to chronic long-term exercise and/or overtraining may exceed the above-mentioned optimal level, resulting in irreparable oxidative damage, which can lead to the development or progression of poor health and/or disease [47]. Therefore, the measurement of the antioxidant capacity (CAT, SOD, GSH, GSSG and GSGG/GSH) of the body is used as a marker of oxidative stress and can provide us insight on how it affects performance. Given the results of our research and the evidence shown in the scientific literature, there is no endogenous antioxidant profile defined in PRO compared to AMA.

There are also other antioxidant proteins, such as peroxiredoxin (PRX) and thioredoxin (TRX) containing thiol groups, with a high capacity to neutralize reactive oxygen and nitrogen species and decrease oxidative stress [48]. One study showed how moderate and high-intensity exercise and a low volume high intensity interval training trial increased TRX (85%, 64% and 206%, respectively); however, PRX only increased during high intensity exercise (moderate: −6229%; high: 203% and low volume high intensity interval: −23%, respectively) in peripheral blood mononuclear cells [48]. In addition, an increase in nuclear transcription factor kappa B was found during all exercises, suggesting an activation of the inflammatory system, probably due to increased oxidative stress. Future studies should examine whether there are differences in these antioxidant proteins between PRO and AMA and their relationship with performance.

Regarding hematological parameters, no significant differences were found except for MCH and MCHC between PRO vs. AMA. Schumacher et al. found hematological values in elite cyclists from the German national team (blood samples collected between November and January) and the values were similar to ours in Hb (~15.5 g/dL), Hct (~45.0%) and RBC (~5.0 × 106/mm3) in PRO [49]. In addition, other studies have found hematological values of approximately 15.0 g/dL of Hb and 45% of Hct in professional cyclists [50–52]. Well-trained cyclists have found values of 14.3 g/dL in Hb and 43.1% in Hct, values lower than PRO [53]. However, Bejder et al. [54] observed amateur competitive cyclist values of 14.8 g/dL Hb, 42.8% Hct, 4.92 × 106·μL−1RBC, 87.1 fl MCV, 30.1 pg MCH and 34.6 g/dL MCHC, lower than those reported in PRO.

MCH indicates the amount of hemoglobin contained in an erythrocyte and MCHC is the average hemoglobin concentration [55]. Therefore, the red blood cells of PROs will have a higher oxygen transport capacity due to the higher levels of MCH and MCHC. Currently, no study on cyclists has examined the differences in MCH and MCHC, so we cannot draw many conclusions in this regard. These hematological parameters have mainly been used as markers of anemia both in athletes and in the general population [56], but so far, they are not associated with an athlete's performance level in this study.

#### **5. Limitations**

Our study had limitations with regards to the sample number, since it was more difficult to recruit PRO athletes than lower-level athletes (AMA).

Differences in endogenous antioxidant marker between this study and previous works may be influenced by the instrumentation and methodology used, the timing of the season at which the measurements were made, and the training status of the cyclists.

#### **6. Conclusions**

Regarding the endogenous antioxidants profile, PRO had higher values of CAT, GSSG and GSSG/GSH compared to AMA. An inverse correlation was found for the first time between WVT1 and WVT2 with GSSG/GSH at rest only in PRO. This indicates better antioxidant status that allow for higher performance with regard to power output. Future studies should examine how training adaptations affect the studied variables and how antioxidant enzymes evolve during a race stage (e.g. Tour de France), in order to

see their association with performance, recovery and fatigue, thereby helping to develop monitoring tools for medical doctors, nutritionists and coaches.

**Author Contributions:** Conceptualization, F.J.M.-N., C.M.-P., R.O.-R. and P.E.A.; methodology, F.J.M.- N., C.M.-P., R.O.-R. and P.E.A.; formal analysis, F.J.M.-N. and C.M.-P.; investigation, F.J.M.-N., C.M.-P. and R.O.-R.; resources, F.J.M.-N. and C.M.-P.; data curation, F.J.M.-N., C.M.-P. and R.O.-R.; writing original draft preparation, F.J.M.-N.; writing—review and editing, F.J.M.-N., C.M.-P. and S.P.D.; visualization, F.J.M.-N. and C.M.-P.; supervision, C.M.-P., S.P.D. and P.E.A.; project administration, F.J.M.-N. and C.M.-P. All authors have read and agreed to the published version of the manuscript.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Catholic University of Murcia (CE091802).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** All data is contained within the article.

**Acknowledgments:** This study was supported by the Research Center in High-Performance Sport of the Catholic University of Murcia (Murcia, Spain). We would like to acknowledge Linda H. Chung for her help in this project.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

