Seasonal Oxy-Inflammation and Hydration Status in Non-Elite Freeskiing Racer: A Pilot Study by Non-Invasive Analytic Method

Freeskiing is performed in an extreme environment, with significant physical effort that can induce reactive oxygen species (ROS) generation and dehydration. This study aimed to investigate the evolution of the oxy-inflammation and hydration status during a freeskiing training season with non-invasive methods. Eight trained freeskiers were investigated during a season training: T0 (beginning), T1-T3 (training sessions), and T4 (after the end). Urine and saliva were collected at T0, before (A) and after (B) T1-T3, and at T4. ROS, total antioxidant capacity (TAC), interleukin-6 (IL-6), nitric oxide (NO) derivatives, neopterin, and electrolyte balance changes were investigated. We found significant increases in ROS generation (T1A-B +71%; T2A-B +65%; T3A-B +49%; p < 0.05–0.01) and IL-6 (T2A-B +112%; T3A-B +133%; p < 0.01). We did not observe significant variation of TAC and NOx after training sessions. Furthermore, ROS and IL-6 showed statistically significant differences between T0 and T4 (ROS +48%, IL-6 +86%; p < 0.05). Freeskiing induced an increase in ROS production, which can be contained by antioxidant defense activation, and in IL-6, as a consequence of physical activity and skeletal muscular contraction. We did not find deep changes in electrolytes balance, likely because all freeskiers were well-trained and very experienced.


Introduction
Freeskiing is a specific type of alpine skiing, where athletes twist and throw the bodyweight in addition to the weight of the equipment, leading to a rapid transition of the leg tension that propels the athletes and allows for tricks and rotations in the vertical and horizontal planes of rotations [1]. Physical exercise is a well-known source of reactive oxygen species (ROS) in response to raised O 2 utilization to sustain the effort, especially in skeletal muscles and, to a lesser extent, in several tissues, including heart, lungs, or blood [2]. ROS play a key role in the modulation of cell redox status and signaling processes, and they are involved in the regulation of several patho-physiological mechanisms [3,4]. Nevertheless, a prolonged duration as well as a high intensity of endurance training can lead to increased ROS production, possible macromolecule damage, and, thereby, to oxidative stress [5][6][7][8], compromising the efficacy of immune system and inflammatory response [9]. Furthermore, oxidative stress might induce programmed cell death, typically through intrinsic apoptotic pathways [10]. These effects can damage the skeletal muscle, reducing physical performance [11].
All the athletes received an explanation of the study's purposes, risks, and benefits, were familiarized with the experimental protocol, and read and signed a specific informed consent form before the experiment. The study was conducted in accordance with the Helsinki Declaration and was approved by the Ethical Committee of Università degli Studi di Milano, Italy (Aut. n 37/17).

Experimental Design
This observational study was carried on during the ski season, from 8 December 2021, to 17 April 2022, at the Prato Nevoso Ski resort, at 1480 m (Frabosa Sottana, CN, Italy), see Figure 1. Freeskiers performed their training sessions in the Prato Nevoso snow park, an area of the ski resort track or ski trail, created for snowboarders and freeskiers to perform tricks.
were familiarized with the experimental protocol, and read and signed a specific informed consent form before the experiment. The study was conducted in accordance with the Helsinki Declaration and was approved by the Ethical Committee of Università degli Studi di Milano, Italy (Aut. n 37/17).

Experimental Design
This observational study was carried on during the ski season, from 8 December 2021, to 17 April 2022, at the Prato Nevoso Ski resort, at 1480 m (Frabosa Sottana, CN, Italy), see Figure 1. Freeskiers performed their training sessions in the Prato Nevoso snow park, an area of the ski resort track or ski trail, created for snowboarders and freeskiers to perform tricks.
Biological samples were collected, and measurements were carried out as follows:  In December (T0), before the season beginning. These were basal measures;  In February (T1), at the start of training session;  In March (T2), during the middle period of the season;  In April (T3), at the end of the season;  In June (T4), two months after the end of the season.
All the sessions were performed on Saturdays. Figure 1. Study protocol design, seasonal circle, and session training. Freeskiing athletes were tested five times throughout a competitive season: at the beginning (T0); every month (T1, T2, and T3); and two months after the end of the season (T4). Furthermore, as shown in the scheme on the right of the figure, from T1 to T3, the athletes were tested pre-(A) and post-(B) training session.

Training Exercise Session
As shown in Figure 1, from T1 to T3, the freeskiing athletes were tested pre-and posttraining session. Daily training consisted of 4 h (9.00 a.m.-1.00 p.m.), distributed as follows: . Study protocol design, seasonal circle, and session training. Freeskiing athletes were tested five times throughout a competitive season: at the beginning (T0); every month (T1, T2, and T3); and two months after the end of the season (T4). Furthermore, as shown in the scheme on the right of the figure, from T1 to T3, the athletes were tested pre-(A) and post-(B) training session.
Biological samples were collected, and measurements were carried out as follows: • In December (T0), before the season beginning. These were basal measures; • In February (T1), at the start of training session; • In March (T2), during the middle period of the season; • In April (T3), at the end of the season; • In June (T4), two months after the end of the season.
All the sessions were performed on Saturdays.

Training Exercise Session
As shown in Figure 1, from T1 to T3, the freeskiing athletes were tested pre-and post-training session. Daily training consisted of 4 h (9.00 a.m.-1.00 p.m.), distributed as follows: After warm-up, the rides in snow park were performed following a check of the snow consistency. In every training session, all the athletes respected the training plan and the timing. Freeskiers rode the snow park line of the Prato Nevoso Ski resort six times, with a break between the third and the fourth ride.
The snow park line consists of an area of a ski trail with dedicated structures (jumps, rails, jibbing, and slang), created for freeskiers to perform tricks; the snow park line includes 2 jumps, 2 rails, 2 slang, and 1 jibbing area, for a total length of 500 m: Chairlift takes 9 min to reach the top of the snow park. During the recorded training sessions, the air temperature ranged from −9 to −2 • C, wind from 11 to 13 km/h, and humidity from 57 to 85%.

Sample Collection
Approximately 1 mL of saliva was collected before (A) and after (B) of the training session using a Salivette device (Sarstedt, Nümbrecht, Germany). Before storage, saliva samples were centrifuged at 3000 rpm for 20 min. [31]. In addition, urine samples were collected by voluntary voiding in a sterile container before and after the training session. The sample collection at the pre-session (baseline) was always conducted at approximately 8.30 am.
All biological samples were initially stored at 4 • C in a portable cooler during the transport back to the laboratory, aliquoted, then stored at −20 • C until assayed, and thawed only once before analysis.
All samples were stabilized at 37 • C using a Temperature Controller unit (Noxigen Science Transfer & Diagnostics GmbH, Elzach, Germany), interfaced with the spectrometer.

Interleukin-6 (IL-6)
IL-6 urinary levels were determined by ELISA kit (Cayman Chemical, Ann Arbor, MI, USA, Item No. 501030), according to the manufacturer's instructions. The determinations were assessed in duplicate, and the inter-assay coefficient of variation was in the range indicated by the manufacturer.

NO Metabolites
NO derivatives, nitrate and nitrite (NO 2 + NO 3 = NOx), were measured in urine samples by a colorimetric method based on the Griess reaction, using a commercial kit (Cayman Chemical, Ann Arbor, MI, USA) previously described [35]. Samples were read at 545 nm, and the concentration was assessed by a standard curve.

Creatinine, Uric Acid, and Electrolytes
Creatinine, urea, uric acid, sodium (Na + ), potassium (K + ), chlorine (Cl − ), magnesium (Mg 2+ ), phosphorus (P), Calcium (Ca 2+ ), and total proteins were investigated in urine samples. Urine samples were defrizzed, at room temperature, shacked with the Vortex for 5 s for homogenization before the analysis. Briefly, 500µL of each sample was placed in a plastic test tube. The test tubes were allocated in the auto sampler of a Roche Cobas ®® 6000 analyzer (Roche Diagnostics, Basel, Switzerland). The reported total imprecision was <2.8%, while the intra assay CV% was <1.8%.

Neopterin
Neopterin urinary concentrations were measured by high-performance liquid chromatography (HPLC) method, as previously described [33,36]. The calibration curve was linear over the range of 0.125-1 µmol/L. Inter-assay and intra-assay coefficients of variation were <5%.

Urine Test Strip
The Urine Test Strip (Siemens Healthcare S.r.l. 10sys Multistix, Italy) was used for semiquantitative determinations of pH, urobilinogen, bilirubin, ketones, specific gravity/density, and leukocytes in urine. Test was immediately performed after sample collection in duplicate for each subject.

Scale for Assessment of Physical Fatigue and Recovery
The subject-perceived exertion was assessed immediately after every training session on the basis of the physical sensations, and muscle fatigue was assessed by the Borg Rate of Perceived Exertion scale (RPE) [37].
The quality of training recovery was assessed at pre-session training and compared with the last training by Total Quality of Recovery scale (TQR) proposed by Kenttä and Hassmén [38].
To study the subjective mood, general wellness (happy/unhappy, rested/tired), general sensation (hot/cold, calm/agitation), and head no pain/pain were evaluated using a 0-100 mm visual analog scale (VAS) to test the subjective perception [39]. If pain was present, its location was requested.
The Profile of Mood States (POMS) is a popular tool among sport psychologists who have used it to compare the prevailing moods of elite athletes and non-athletes. In short-form, this tool was administered to measure certain psychological traits, focusing on tension-anxiety, depression-dejection, anger-hostility, vigor-activity, fatigue-inertia, and confusion-bewilderment on a five-point scale from 0 to 4 [40][41][42]. High vigor scores reflect a good mood or emotion, and low scores in the other subscales reflect a good mood or emotion.

Statistical Analysis
Analysis was performed using the GraphPad Prism package (GraphPad Prism 9.5.0, GraphPad Software Inc., San Diego, CA, USA) and SPSS statistics software (IBM corporation). Data are presented as mean ± SD. Statistical analyses were performed using non-parametric tests; Wilcoxon matched-pairs signed-rank test for independent samples due to the small sample size for comparing pre-vs. post-training session; and ANOVA repeated measures, with Dunn's multiple comparison tests to further check the amonggroups significance. A p < 0.05 was considered statistically significant. dCohen with 95% CI was used for calculating the size effect. Moreover, we used the Hopkins scale for classification of the effect size. Change ∆% estimation (((pre value − post value)/pre value) × 100) is also reported in the text.
Finally, no significant differences were found in total mood score of POMS scale and in VAS item score during a single training session (A vs. B) or during the entire training season. Particularly, we observed percentage changes in the general wellness measure with respect to T0: T1A +79%, T2A +100%, T3A −47%, and T4A −2% ( Figure 4C); the pain measure with respect to T0: T1A −10%, T2A −25%, T3A −65%, and T4A −65% ( Figure 4D); and the anxiety measure with respect to T0: T1A −12%, T2A −40%, T3A −96%, and T4A −33%. In order to understand the results obtained and then evaluate the effects of training vs. seasonality in freeskiers, we applied the Hopkin scale, as shown in Figure 5. Significant group differences and large effect sizes were found for uric acid, TAC, and IL-6 during the season, while moderate effect sizes were found for ROS, SBP, and urea at the end of the freeski season. Significant group difference and a very large effect size was found for IL-6 after the T3 training session, and large effect sizes were found for neopterin, IL-6, Finally, no significant differences were found in total mood score of POMS scale and in VAS item score during a single training session (A vs. B) or during the entire training season. Particularly, we observed percentage changes in the general wellness measure with respect to T0: T1A +79%, T2A +100%, T3A −47%, and T4A −2% ( Figure 4C); the pain measure with respect to T0: T1A −10%, T2A −25%, T3A −65%, and T4A −65% ( Figure 4D); and the anxiety measure with respect to T0: T1A −12%, T2A −40%, T3A −96%, and T4A −33%.
In order to understand the results obtained and then evaluate the effects of training vs. seasonality in freeskiers, we applied the Hopkin scale, as shown in Figure 5. Significant group differences and large effect sizes were found for uric acid, TAC, and IL-6 during the season, while moderate effect sizes were found for ROS, SBP, and urea at the end of the freeski season. Significant group difference and a very large effect size was found for IL-6 after the T3 training session, and large effect sizes were found for neopterin, IL-6, TAC, ROS, and TQR during the training session. Finally, moderate effect sizes were found for ROS at T1, K at T2, and P at T3 training sessions.
Int. J. Environ. Res. Public Health 2023, 20, 3157 10 of 16 TAC, ROS, and TQR during the training session. Finally, moderate effect sizes were found for ROS at T1, K at T2, and P at T3 training sessions.

Discussion
This was the first study that attempted to investigate the evolution of oxy-inflammation and hydration parameters among freeskiers during a training season, and importantly, to do so by noninvasive measurements.
Exercise-induced ROS generation is typical of prolonged and/or high training loads, such as those of endurance activities [36,[43][44][45]. In muscle fiber during physical effort, the rate of ROS generation, such as O2•, is increased, influencing the fiber contraction [46]. An excess of ROS in skeletal muscle results in a decrease in the ability to generate force, leading to a fatigue condition [47].
We observed a significant increase in ROS production after every training session as a result of the physical effort. Importantly, ROS production plays a key role in cell signaling pathways involved in muscle adaptation to effort [48] with multi-protein pathways and signaling [49]. Furthermore, we found an increase of ROS basal value at T3A and at the end of the training season (T4), likely due to a decrease in the efficiency of the endogenous antioxidant defenses and a rising soreness. Athletes can activate the endogenous antioxidant system to control exercise-induced oxidative stress, avoiding the macromolecules damage [50][51][52][53][54]. A significant decrease in TAC was measured only after the first training session (T 3B), while other sessions did not exhibit significant change, despite a reduction of TAC level. During the first training session, the TAC value was higher than during the other sessions, resulting in a higher starting antioxidant defense level. This may explain the significant training-induced decrease in TAC value, attributable to a higher baseline, as observed by some authors [55]. Training may also induce a converging of TAC values towards an optimal level, adapting the body to the exercise-related fatigue and leading to a smaller variation after the successive session.
IL-6 is produced mainly within the working skeletal muscles [56], with its released

Discussion
This was the first study that attempted to investigate the evolution of oxy-inflammation and hydration parameters among freeskiers during a training season, and importantly, to do so by noninvasive measurements.
Exercise-induced ROS generation is typical of prolonged and/or high training loads, such as those of endurance activities [36,[43][44][45]. In muscle fiber during physical effort, the rate of ROS generation, such as O 2 •, is increased, influencing the fiber contraction [46]. An excess of ROS in skeletal muscle results in a decrease in the ability to generate force, leading to a fatigue condition [47].
We observed a significant increase in ROS production after every training session as a result of the physical effort. Importantly, ROS production plays a key role in cell signaling pathways involved in muscle adaptation to effort [48] with multi-protein pathways and signaling [49]. Furthermore, we found an increase of ROS basal value at T3A and at the end of the training season (T4), likely due to a decrease in the efficiency of the endogenous antioxidant defenses and a rising soreness. Athletes can activate the endogenous antioxidant system to control exercise-induced oxidative stress, avoiding the macromolecules damage [50][51][52][53][54]. A significant decrease in TAC was measured only after the first training session (T3B), while other sessions did not exhibit significant change, despite a reduction of TAC level. During the first training session, the TAC value was higher than during the other sessions, resulting in a higher starting antioxidant defense level. This may explain the significant training-induced decrease in TAC value, attributable to a higher baseline, as observed by some authors [55]. Training may also induce a converging of TAC values towards an optimal level, adapting the body to the exercise-related fatigue and leading to a smaller variation after the successive session.
IL-6 is produced mainly within the working skeletal muscles [56], with its released amount strongly related to the exercise duration and intensity as well as the mass of muscle recruited and endurance capacity [57,58]. We found raised IL-6 values after physical exercise in every training session, in accordance with other authors [59]. IL-6 increases significantly after eccentric exercise [60] in response to long-duration exercise, independently of muscle damage. IL-6 release in relation to muscle fiber damage occurs later and is of a smaller magnitude than IL-6 production related to muscle contractions [57]. Skiing requires a lot of eccentric contractions that occur when the muscle lengthens at the same time it contracts. In our case, freeskiing implies muscle eccentric contractions associated with concentric contraction during jump execution.
We did not find any difference in NOx level after any of the training sessions. In our case, we did not introduce any NO 2 or NO 3 supplementation to the athletes. NO derivatives may improve exercise tolerance, reducing O 2 consumption and optimizing muscle contraction efficiency [61,62] through their role in the regulation of blood flow, contractility, glucose and Ca 2+ homeostasis, mitochondrial respiration, and biogenesis [63].
Despite the increase, uric acid did not change significantly after any of the sessions, similar to the data of some authors [64]. Exercise-related changes of uric acid are contrasting. During intense exercise, uric acid excretion is reduced [65,66] as a consequence of an antidiuretic hormone increase [67] and an elevated lactate production, which leads to a decrease in the urinary excretion of oxypurines [68,69]. On the other hand, other research groups found an increase of urinary uric acid [70,71], which may be the consequence of purine release from the muscle after prolonged exercise rather than a decrease in plasma purine removal rates [72]. In muscle, ATP consumption can trigger a cascade of nucleotide degradation to purine catabolic intermediates that increase uric acid level after physical activity [65].
We evaluated the hydration status of the freeskiers without any drink supplementation. We did not find deep changes in electrolytes balance, likely because all volunteers were well-trained and very experienced. Urea value did not change significantly after any of the training sessions, likely to reduce exercise-related water loss. Urea is reabsorbed during passage of the filtrate through the tubule of the nephron, activating a mechanism of water-conserving action [73].
Despite their elevated values, Na and K levels did not show significant variation during the season, with the exception of K variation after the second training session (T2B). K is involved in the regulation of osmotic pressure and plays an important role in nerve stimulation and muscle contraction during exercise [74].
Urinary P decreased after every training session, significantly after the third session (T 3B). This phosphorus excretion reduction may reflect a slight respiratory alkalosis caused by hyperventilation during exercise [75].
Cl, Mg, and Ca did not change significantly during the season. Our findings seem to indicate freeskiing might be not intense enough to induce significant differences in electrolyte concentrations between urine samples taken before and after the physical activity. Furthermore, the absence of fluid intake may also be a possible reason for a major stability in concentration of circulating electrolytes, reducing their loss through urine [76].
Neopterin can rise during systemic oxidative stress, as reported by some authors [7,32,77]. In our freeskiers, neopterin increase can be associated with ROS production, similar to other studies [29,78].
Prolonged physical activity can impair kidney function as consequence of several physiological mechanisms to manage stress [7,33]. We did not find any difference in urine standards parameters, likely due to the good health and high level of experience of the subjects to adapt to the freeskiing-related physical effort.
We observed a progressive decrease in TQR score throughout the training season. This decrease may be due to the body adapting to exercise-induced fatigue, which has a beneficial effect on the athletes' psychological condition [79]. Freeskiers showed an increase in general wellness sore and a decrease in pain score at the end of the training season, evidence of the fact that physical activity improves the psychophysical status of athletes.
Indeed, it is well known that physical activity can improve mental health, reducing symptoms of psychological distress, such as depression, anxiety [80,81], failure after competition [82], and increasing self-esteem and cognitive functions [83].

Limitation
The main limitation of our study is the small sample size. We included the entire team of Prato Nevoso freeskiers. It should be emphasized that the freeski is not a very widespread sport, unlike alpine skiing. Therefore, results must be interpreted carefully. In fact, we studied both young and non-professional subjects. As this is a pilot study, we plan to pursue professional and non/professional and national/international athlete's collaborations in the future to increase the number of subjects studied. Nevertheless, this is the first study to assess ROS production, antioxidant capacity, nitric oxide metabolites levels, inflammatory status, and electrolytes levels, in a freeskiers athletes in a real field environment.

Conclusions
Freeskiing induced an increase in ROS production that can be contained by antioxidant defense activation. Neopterin increased as a consequence of ROS generation, while IL-6 increased as a consequence of physical activity and muscular contraction type.
We did not find deep changes in electrolytes balance, likely because all subjects examined were well-trained and very experienced. Moreover, freeskiing-related physical activity can improve body adaptation to exercise-induced fatigue and mental health, reducing symptoms of psychological distress, including anxiety. Finally, despite a small sample size, the current findings show a large effect size so that this pilot study presumably may have a practical significance.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Università degli Studi di Milano, Italy (Aut. n 37/17).

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

Data Availability Statement:
Data are available at request from the authors.

Acknowledgments:
The authors are grateful to volunteer participants and for the kind logistic support given by the Prato Nevoso Ski Resort.

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