Molecular Profiling of Athletes Performing High-Intensity Exercises in Extreme Environments

aim of this study was to determine the in uence of high-intensity training under extreme conditions (T = 40 C) on the metabolism and immunological reactions of athletes. Male triathletes (n= 11) with a high level of sports training performed load testing to failure (17 2.7 min ) and maximum oxygen consumption (64.1 6.4 mL/min/kg). Blood plasma samples were collected before and immediately after exercise. Mass spectrometric metabolomic analysis identi ed 30 metabo- lites and 6 hormones in the plasma, of which 21 and 4 changed after exercise, respectively. Changes in the intermediate products of tricarboxylic and amino acids were observed (FC > 1.5) after exercise. The obtained data can be associated with the effect of physical activity on metabolism in athletes. Therefore, constant monitoring of the biochemical parameters of athletes can help coaches identify individual shortcomings in a timely manner and track changes, especially as the volume of training increases. In addition, it was revealed that the immunological

were observed (FC > 1.5) after exercise. The obtained data can be associated with the effect of physical activity on metabolism in athletes. Therefore, constant monitoring of the biochemical parameters of athletes can help coaches identify individual shortcomings in a timely manner and track changes, especially as the volume of training increases. In addition, it was revealed that the immunological reaction (manifestation of a hyperactive reaction to food components) is personalized in nature. Therefore, it is important for coaches and sports doctors to analyze and control the eating behavior of athletes to identify food intolerances or food allergies in a timely manner and develop an individual elimination diet. Keywords: metabolome; athletes; ELISA; mass spectrometric analysis; IgG; food allergens;amino acids 1. Introduction Studies on the molecular mechanisms that accompany adaptation to physical activity are becoming increasingly popular [1,2]. Changes in the blood metabolic pro le in professional athletes are preceded by over- training, which largely depends on the variant and duration of the training mode [3]. During training, various behavioral, biochemical, hormonal, and immune markers are used to assess the physiological condition of athletes [4]. However, some studies have demon- strated that standard tests cannot accurately detect physiological pre- and postexercise differences between endurance athletes and controls [5]. The aim of metabolomics is to quantify the pro les of endogenous non-molecular components associated with exercise in professional athletes in order to identify biomarkers of performance, fatigue responses, and possibly sport-related disorders [5,6]. Metabolomics allows for the detection of changes in response to various physiological stimuli and helps to identify metabolic traits with potential translational impacts in professional athletes [7]. These changes include the metabolic products associated with the metabolism of glucose, lipids, amino acids, and energy [3,5]. In addition, metabolomic pro ling of athletes per- forming intense exercise revealed changes in plasma lactate [8] and breakdown products of nitrogenous bases, in particular adenine [9], indicating anaerobic metabolism and ATP Sports2023,11, 36.

ling of athletes per- forming intense exercise revealed changes in plasma lactate [8] and breakdown products of nitrogenous bases, in particular adenine [9], indicating anaerobic metabolism and ATP Sports2023,11, 36.

Sports2023,11, 36 2 of 15 production, respectively. Howarth et al. examined changes in the level of the Krebs cycle or the TCA cycle (tricarboxylic acid cycle) intermediates [10]. Increases in serum sex steroid hormones have also been reported in endurance athletes in response to high-intensity exercise [11]. To determine the hyper-reactive reaction to food allergens mediated by immune processes, an enzyme-linked immunosorbent assay (ELISA) was performed before and after exercise to determine the concentration of class G immunoglobulins (IgG). ELISA is widely used in allergology and immunology for the diagnosis of antigen–antibody- type immune reactions with high selectivity, reproducibility, and speci city [12,13]. In immunological tests, the IgG or G4 subclass is a marker of a speci c humoral immune response, which is speci c to the food antigen being tested [14,15]. The choice of this class of antibodies is based on the fact that IgG makes up 70–80% of all blood immunoglobulins and plays a fundamental role in providing long-term humoral immunity. Although IgGs are not classical secretory antibodies, they are present in the intestinal lumen, with a protective function. IgGs have been shown to bind to Fc receptors on intestinal epithelial cells in acidic environments. This transcytosis-mediating receptor mediates protective IgG transport and release on the basal side of enterocytes, where neutral pH causes them to dissociate from the receptor [14,15]. IgG is the main antibody of the secondary immune response to most antigens and manifests itself in the form of type III immunopathological reactions. Moreover, IgG ELISA is characterized by a sensitivity of 92–95%, a speci city of 86–89%, and a reproducibility of 95–97%, as well as the possibility of studying the dynamics of the state of the immune system before and after an elimination diet [16]. Elite-level athletes often perform under extreme environmental conditions, such as high altitude and extremely high or low air temperatures. Performance under such condi- tions may be accompanied by changes in the molecular pro le of athletes, as well as the development of stress characterized by low performance and low ef ciency of recovery. Furthermore, the adaptation of athletes to extreme

Elite-level athletes often perform under extreme environmental conditions, such as high altitude and extremely high or low air temperatures. Performance under such condi- tions may be accompanied by changes in the molecular pro le of athletes, as well as the development of stress characterized by low performance and low ef ciency of recovery. Furthermore, the adaptation of athletes to extreme conditions is probably individual and depends on many factors. Many studies have been conducted with the aim of monitoring the state of the bodies of athletes training under normal conditions. However, physiological pro les are mostly compared by competitive level or across disciplines [17,18]. The content of various xenobiotics or other additives in biological samples of athletes in various sports is often studied [19,20]. Studies on changes in the molecular pro le of athletes training in extreme conditions are limited. The aim of this study was to identify the metabolic characteristics associated with endurance loading under extreme conditions. It was hypothesized that evaluating these metrics can provide valuable insights into athletes' current tness and training adaptations. A deeper understanding of the quantitative changes in low-molecular-weight (endoge- nous) participants in biochemical processes (tricarboxylic acid cycle, protein metabolism, ornithine cycle, etc.) during physical activity can allow for the design of comprehen- sive training programs that prevent potential overexercise disorders and improve general performance by changing the signature of endogenous metabolites. 2. Materials and Methods 2.1. Study Participants This study involved 11 male athletes involved in a multisport endurance race consist- ing of swimming, cycling, and running over various distances (triathlon). The anthropo- metric characteristics of the participants are listed in Table.

Sports2023,11, 36 3 of 15 Table 1.Anthropometric characteristics of study participants. Sample No. # Age (Years) Weight (kg) Height (cm) BMI (kg/m 2 ) 1 28 82.5 179 25.7 2 28 67 176 21.6 3 28 76.4 180 23.6 4 29 70.8 181 21.4 5 26 72.4 176 23.4 6 28 76.3 180 23.5 7 28 69.0 177 22.0 8 29 80.0 178 22.9 9 32 63.6 171 21.8 10 27 72.9 171 24.9 11 32 82.2 179 25.6 The functional characteristics of the participants are presented in Table. Table 2.Functional indicators of study participants. Indicator Unit Mean (SD) VO 2max mL/min/kg 64.1 6.4 RQ relative units 1.14 0.1 METs relative units 18 1.9 Resting heart rate bpm 82 6.5 Aerobic threshold VO 2 mL/min 47.2 9 VE L/min 90 17.8 Heart rate bpm 154 12 Anaerobic threshold VO 2 mL/min 58 9.2 VE L/min 138 21.5 Heart rate bpm 176 7.6 Maximal oxygen consumption VE L/min 174 11.4 Heart rate bpm 186 5.5 Abbreviations: bpm—beats per minute; METs—metabolic equivalents; RQ—respiratory quotient; VO2—oxygen consumption. Inclusion criteria: (1) Quali cations of an athlete must be proven during the year before inclusion by participating in competitions during the winter season up to three times and in summer races up to four times. (2) High-intensity physical activity at aerobic and anaerobic thresholds (80% and 20%, respectively) carried out for 2 h three times a week, with speed–strength training carried out for 2.5 h twice a week. (3) At the time of the study, the doctor performed a comprehensive assessment of athletes' health status to issue a conclusion about their health. The conclusion was obtained based on an in-depth medical examination, which included X-ray examination of the chest, ultrasound diagnostics of the abdominal cavity and pelvic organs, diagnostics of the car- diovascular system, echocardiography, electrocardiography, biochemical tests of urine and blood, and examinations by specialized physicians (ophthalmologists, otolaryngologists, surgeons, cardiologists, neurologists, dentists, endocrinologists, and therapists). (4) Training was absent for 3 days prior to the start of the study. All participants were informed of the risks and discomforts associated with the inves- tigation and

and pelvic organs, diagnostics of the car- diovascular system, echocardiography, electrocardiography, biochemical tests of urine and blood, and examinations by specialized physicians (ophthalmologists, otolaryngologists, surgeons, cardiologists, neurologists, dentists, endocrinologists, and therapists). (4) Training was absent for 3 days prior to the start of the study. All participants were informed of the risks and discomforts associated with the inves- tigation and signed a written consent to participate. The study was approved by the Board

Sports2023,11, 36 4 of 15 for Ethical Questions at the A. I. Burnazyan State Research Center of the FMBA of Russia (Protocol No. 40, 18 November 2020). 2.2. Stress Testing Introductory testing of these groups was performed on separate days. Basic load testing “to failure” was carried out at a temperature of 22 C and a relative humidity of 60%. During testing, the participants were dressed in sports shorts, socks, and shoes. Testing was continued until volitional exhaustion, de ned as the point at which participants were unable to maintain treadmill speed and continue with the load. Load testing was performed on a V-ergo PRO treadmill (Italy) in a climate-controlled room. Throughout the examination of the participants, ECG parameters were continuously recorded using a Cosmed “Quark C 12” wired recorder to measure the electrical activity of the heart. Indicators of pulmonary ventilation and metabolic response were continuously measured using Cosmed “Quark CPET” (Italy) systems for analysis of the gas composition of inhaled and exhaled air. Physical activity on the V-ergo PRO treadmill was carried out in accordance with the protocol and continued until the moment of “failure” (Table). Subsequently, the recovery phase was activated in the gas analysis program, accompanied by a slowdown in the speed of the moving web to a complete stop after 3 min. Table 3.Treadmill load protocol *.Stage Speed (km/h) Time (min) 1 5.00 0.00–1.40 2 6.00 1.40–3.20 3 7.00 3.20–5.00 4 8.00 5.00–6.40 5 8.00 6.40–8.20 6 10.00 8.20–9.00 7 11.00 9.00–10.40 8 12.00 10.40–12.20 9 13.00 12.20–14.00 10 14.00 14.00–15.40 11 15.00 15.40–17.20 12 16.00 17.20–19.00 13 17.00 19.00–20.40 14 18.00 20.40–22.00 15 19.00 22.00–23.40 16 20.00 23.40–25.00 Recovery 4.00 2.00 2.7 3.00 * Treadmill load is a modi cation of an electrocardiographic examination in which an electrocardiogram is recorded during intense physical activity. To determine the physical performance and functional reserves of the participants, the following indicators were taken into account: total time of work, time of the onset of the aerobic threshold, time of anaerobic threshold, resting heart rate (RHR) before the start of the test, RHR at aerobic threshold, RHR at

in which an electrocardiogram is recorded during intense physical activity. To determine the physical performance and functional reserves of the participants, the following indicators were taken into account: total time of work, time of the onset of the aerobic threshold, time of anaerobic threshold, resting heart rate (RHR) before the start of the test, RHR at aerobic threshold, RHR at anaerobic threshold, RHR maximum, RHR recovery after 5 min, oxygen consumption, removal of carbon dioxide (CO2.), respiratory coefficient, metabolic units, frequency of respiratory movements, minute volume of breath, tidal volume, oxygen pulse, respiratory equivalent for O2, and respiratory equivalent for CO2. 2.3. Plasma Samples Blood samples were collected on an empty stomach from the cubital vein following overnight fasting and stored in vacutainers containing 3.8% sodium citrate anticoagu- lant (IMPROVACUTER, Guangzhou Improve Medical Instruments Co., Ltd., Guangzhou, China). The samples were centrifuged at 3000 rpm for 6 min at room temperature. Each

Sports2023,11, 36 5 of 15 plasma sample (500 L) was nally collected into two dry Eppendorf-type polypropylene test tubes, frozen, and stored at 80 C prior to analysis. 2.4. Mass Spectrometric Analysis Data acquisition was performed using a high-resolution quadrupole time-of- ight (Q-TOF) Xevo G2-XS mass spectrometer (Waters, Inc., Ireland, Milford) equipped with a Z-spray ionization source coupled with a UPLC Acquity H Class (Waters, Inc., Ireland, Milford) system. Details of instrumental analysis are available in [21]. 2.5. ELISA Immune response to food ingredients was assessed by a solid-phase, non-competitive, indirect enzyme-linked immunosorbent assay (ELISA) using a commercial reagent kit for a semiquantitative enzyme immunoassay of allergen-speci c IgG antibodies (LLC NPO Immunoteks, Stavropol, Russia). This kit uses monoclonal anti-IgG antibodies included in the peroxidase conjugate that are capable of detecting antibodies of the immunoglobulin G class in human serum/plasma, which have an af nity for allergens sorbed on the surface of a polystyrene tablet. The analysis was performed according to the recommendations of manufacturer. The kit contained 2 polystyrene 96-well plates with food antigens immobi- lized on the surface. One kit was designed to determine the content of speci c IgG in eight test samples for 22 allergens (Table). Table 4. Food allergens in the reagent kit for a semiquantitative enzyme immunoassay for allergen- speci c IgG antibodies. No. Allergen No. Allergen 1. Milk protein concentrate (micellar casein)12. Beef protein hydrolysate (beef protein) 2. Dry egg white (egg protein) 13. Whey protein concentrate (whey protein) 3. Orange 14. Corn grits 4. Potato 15. Baker's yeast 5. Peanut 16. Hazelnut 6. Honey 17. Strawberry 7. Almond 18. Pomegranate 8. Oat groats 19. Coconut 9. A mixture of brown and wild rice 20. Flax seed 10. Soy protein isolate (soy protein) 21. Wheat groats 11. Barley groats 22. Brewer's yeast ELISA was performed on a Multiscan FC microplate photometer (Thermo Scienti c, Waltham, MA, USA), and the results interpreted according to the manufacturer's instruc- tions (Table). Table 5.Interpretation of ELISA results. IgG Concentration ( g/mL) Result 0 Negative 0–0.88 Mild allergic reaction 0.88–8.8 Moderate allergic reaction 8.8–88 Highly allergic reaction 88–880

21. Wheat groats 11. Barley groats 22. Brewer's yeast ELISA was performed on a Multiscan FC microplate photometer (Thermo Scienti c, Waltham, MA, USA), and the results interpreted according to the manufacturer's instruc- tions (Table). Table 5.Interpretation of ELISA results. IgG Concentration ( g/mL) Result 0 Negative 0–0.88 Mild allergic reaction 0.88–8.8 Moderate allergic reaction 8.8–88 Highly allergic reaction 88–880 Very highly allergic reaction 2.6. Statistical Analysis The metabolite concentrations after recovery and exercise were determined for each study participant. If the value was in the range of (0.67, 1.5), the data were excluded from further analysis, that is, the calculation of the mean and standard deviation. Statistical analyses were performed using R Project for Statistical Computing [22]. A semiquantitative

Sports2023,11, 36 6 of 15 assessment of hormone content was performed taking into account the chromatographic peak area. 3. Results 3.1. Analysis of the Metabolomic Pro le of Participants before and after Exercise Mass spectrometric analysis of blood plasma revealed that 21 and 4 subjects signif- icantly differed in the content of amino acids and hormones, respectively, and the levels after exercise differed by 1.5 times relative to the level “before exercise” (Table). Table 6. Amino acids and hormones detected in the blood plasma of athletes before and after exercise (FC > 1.5). No. Metabolite Mean Concentration ( M/L), before Loading Mean Concentration ( M/L), after Recovery SD before Loading SD after Loading Change 1. 2-ketoglutaric acid 1.26 2.68 1.60 4.14 "2.11 2. 3-Methylhistidine 0.77 0.13 1.01 0.17 #0.17 3. Ascorbic acid 1.86 3.19 1.88 3.87 "1.711 4. Aspartic acid 2.01 3.16 1.71 2.97 "1.56 5. Carnosine 1.26 0.40 0.96 0.18 #0.31 6. cis-Aconitic acid 1.29 3.37 1.38 4.38 "2.59 7. Citric acid 1.25 4.58 0.09 0.66 "3.66 8. Citrulline 2.87 1.48 0.76 0.50 #0.51 9. Creatinine 15.14 8.20 - - #0.54 10. Histidine 34.63 16.82 - - #0.48 11. Hydroxyproline 2.36 1.10 - - #0.46 12. Lactic acid 30.04 10.57 3.27 4.14 #0.35 13. Lysine 14.97 5.32 - - #0.35 14. Ornithine 5.07 1.91 - - #0.37 15. Oxalic acid 5.33 3.13 3.22 1.38 #0.58 16. Proline 61.74 33.69 - - #0.54 17. Serine 25.67 46.73 9.84 14.37 "1.82 18. Succinic acid 0.39 0.18 0.30 0.25 #0.47 19. Taurine 14.51 28.83 - - "1.98 20. Threonine 9.64 18.68 - - "1.93 21. Tyrosine 17.03 6.90 1.60 4.14 #0.40 Hormones 22. 11-Deoxycortisol – – – – "1.22 23. Cortisol – – – – #0.47 24. Estriol (E3) – – – – "1.43 25. Dehydroepiandrosterone – – – – #0.43 Table 2 hormones and a decrease in the concentration of 13 metabolites and 2 hormones were detected in the blood plasma of the participants. 3.2. Hyper-Reactive Reaction to Food Allergens Mediated by Immune Processes Based on the detection of IgG-mediated immunological reactions to 22 food antigens in plasma samples, it was

This study analyzes metabolic and immunological responses in male triathletes during high-intensity training.