**1. Introduction**

Competitive cycling is highly stressful on both aerobic and anaerobic metabolisms. Road cycling races require the riders to produce high relative power output (W/kg) for short duration (i.e., less than 1 min at the start, during steep climb and at the end of the race) while also sustain efforts that last for several minutes to several hours [1]. Overall, professional cyclists (PRO) perform high training volumes (~32,500 Km) during the competitive season, which include 90–100 race days [2]. On the other hand, amateur competitive cyclists (AMA) can be defined as cyclists that train 3–7 times per week, with daily training volumes of 60–120 min and that compete about 20 times in a year [3]. During training sessions and competitions (aerobic and anaerobic exercise), there is a rise in reactive oxygen species (ROS) and subsequent oxidative stress, which can lead to a favorable adaptation in the body's antioxidant defense system [4]. This improvement in the endogenous antioxidant system is generally associated with lower levels of oxidative stress biomarkers [5].

Within the endogenous antioxidant system, superoxide dismutase (SOD) is the first line of enzymatic defense that transforms the superoxide radical(O2 •−) into hydrogen peroxide (H2O2) [6]. Then, H2O2, which is also harmful to cells, can be metabolized in a couple of ways: (1) conversion into water by glutathione peroxidase (GPx) with the

**Citation:** Martínez-Noguera, F.J.; Alcaraz, P.E.; Ortolano-Ríos, R.; Dufour, S.P.; Marín-Pagán, C. Differences between Professional and Amateur Cyclists in Endogenous Antioxidant System Profile. *Antioxidants* **2021**, *10*, 282. https:// doi.org/10.3390/antiox10020282

Academic Editors: Gareth Davison and Conor McClean

Received: 16 January 2021 Accepted: 10 February 2021 Published: 12 February 2021

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reduced glutathione consumption (GSH) being converted into oxidized glutathione (GSSG) and (2) when the production of H2O2 exceeds the capacity of glutathione peroxidase, catalase (CAT) takes over to remove the excess H2O2 [7]. However, un-neutralized H2O2 can interact with transition metals, such as Fe2+ and Cu+, and result in the production of the hydroxyl radical (•OH; i.e., Fenton reaction), which is an extremely powerful oxidizing agent that reacts with all biological macromolecules. However, currently it is not clear what the end product of the Fenton reaction •OH or FeO2+ is [8]. Finally, •OH can elicit damage to DNA, oxidation of the thiol group of proteins and peroxidation of lipids [9].

Exercise increases oxygen uptake and almost 0.15% of the oxygen consumed can be converted into ROS, which can be harmful to muscle and mitochondrial function [10]. Recent findings show that the main source of ROS during exercise is nicotinamide adenine dinucleotide phosphate oxidases [11]. It is known that long-duration strenuous exercise and extensive sprint training can exceed our ability to detoxify the action of reactive oxygen species within the blood cells, as well as at the muscle level [12]. Conversely, prevention of oxidative stress to enhance performance in professional athletes can be done by the adaptation mechanisms (hormesis) and detoxifying function of antioxidant enzymes (SOD, CAT, GPx, glutathione reductase (GR), glutathione-s-transferase), as well as via non-enzymatic antioxidants (such as vitamins E, A, C, and GSH and GSSG [13]. Therefore, it has been suggested that higher levels of the endogenous antioxidant system may improve performance of skeletal muscle contraction [5]. Cordova et al. [14] analyzed antioxidant markers and showed an average GSH of 3.24 <sup>μ</sup>mol·g−<sup>1</sup> Hb, GSSG of 1.54 <sup>μ</sup>mol·g−<sup>1</sup> Hb, GSSG/GSH ratio of 0.56 %, catalase (CAT) of 172.0 mmol·min−1·g−<sup>1</sup> Hb and superoxide dismutase (SOD) of 1983.0 U·g−<sup>1</sup> Hb activity. In a previous study [15], average values of GSH of ~4.7 <sup>μ</sup>mol·g−<sup>1</sup> Hb, GSSG of ~0.7 <sup>μ</sup>mol·g−<sup>1</sup> Hb and GSSG/GSH ratio of 0.15% were observed in PRO cyclists in February in of the same competitive season, suggesting that training season can modify the levels of these components of the endogenous antioxidant system [5,12,15].

At rest, endogenous antioxidant enzymes (EAE) levels are generally lower in athletes than in sedentary subjects, although higher or unchanged levels have been observed [6,16]. Several factors may explain this discrepancy, the most important being differences in the methods used to estimate the state of oxidative stress, the characteristics of the sample population (high-level athlete, sedentary, etc.) and the time of measurement (period of the sport's season) [13]. However, what is clearly evident is that acute exercise can lead to an imbalance between ROS and endogenous antioxidants, causing what is known as oxidative stress [7]. A recent study has shown that acute exercise at low, moderate or high intensity has the capacity to reduce GSH concentration and increase SOD and CAT activity compared to baseline, in addition to increasing F2-isoprostanes (markers of oxidative stress) at all levels of exercise. [17]. In addition, it is generally known that chronic exercise causes an increase in enzymatic and non-enzymatic antioxidant defense, leading to adaptations to the training response and improving the protection against ROS [18]. Chronic exercise (6-week cycling training) has the ability to increase the concentration and activity of GSH, SOD and CAT, while maintaining the levels of F2-isoprostanes [17]. This same study also found that moderate and high intensity exercise promoted greater adaptations in antioxidant markers than low intensity exercise at baseline.

Despite the large amount of information on the EAE status and their relationship to the effects of acute and chronic exercise, to our knowledge, there are no studies that have compared the status of antioxidant enzymes between professional and amateur cyclists and their relationship to performance. Therefore, the main objective of this research was to determine the differences in endogenous antioxidants enzymes and hemogram levels between PRO and AMA, and whether these might be related to differences in performance (power output at VT1, VT2 and VO2MAX) in an incremental test. Finally, the secondary objective was to assess whether differences between endogenous antioxidant enzymes and hematological were associated with differences in performance between PRO and AMA.
