The Effect of a 7-Week Training Period on Changes in Skin NADH Fluorescence in Highly Trained Athletes

udy aimed to evaluate the changes of nicotinamide adenine dinucleotide (NADH) uorescence in the reduced form in the super cial skin layer, resulting from a 7-week training period in highly trained competitive athletes (n=41). The newly, non-invasive ow mediated skin uorescence (FMSF) method was implemented to indirectly evaluate the mitochondrial activity by NADH uorescence. The FMSF measurements were taken before and after an exercise treadmill test until exhaustion. We found that athletes showed higher post-training values in basal NADH uorescence (pre-exercise: 41% increase; post-exercise: 49% increase). Maximum NADH uorescence was also higher after training both pre- (42% increase) and post-exercise (47% increase). Similar changes have been revealed before and after exercise for minimal NADH uorescence (before exercise: 39% increase; after exercise: 47% increase). In conclusion, physical training results in an increase in the skin NADH uorescence levels at rest and after exercise in athletes. Keywords:nicotinamide adenine dinucleotide; training; athletes; mitochondrion 1. Introduction Skin microcirculatory function and e ciency of blood supply to the skin can impact mitochondrial activity and the changes of nicotinamide adenine dinucleotide (NADH) uorescence in the reduced form [1]. Mitochondrial function can be indirectly evaluated by NADH uorescence [1] that has been measured in animals

rest and after exercise in athletes. Keywords:nicotinamide adenine dinucleotide; training; athletes; mitochondrion 1. Introduction Skin microcirculatory function and e ciency of blood supply to the skin can impact mitochondrial activity and the changes of nicotinamide adenine dinucleotide (NADH) uorescence in the reduced form [1]. Mitochondrial function can be indirectly evaluated by NADH uorescence [1] that has been measured in animals [2,3] and humans [1,4] at rest and under various conditions (e.g., ischemia and temperature changes). Bugaj et al. [5] were the rst to describe the time course of NADH changes in the skin in athletes at rest and after exercise. In their study, a new method of evaluating NADH uorescence— ow mediated skin uorescence (FMSF)—was utilized. The FMSF method is based on the ability of NADH to auto uorescence. The uorescence measured using the FMSF method re ects the dynamics ofin vivochanges in NADH levels in most super cial layers of the skin [5–7]. Bugaj et al. [5] have shown that exercise to exhaustion induces changes in skin NADH uorescence, in other words, the values recorded after exercise were higher than those before exercise (increase in: basal NADHfluorescence 13%, maximal NADH fluorescence 7% and minimal NADH fluorescence 12%). Nicotinamide adenine dinucleotide (NAD) is synthesized in the cytosol, mitochondria, and nucleus. This molecule is active in the cytoplasm during glycolysis and in the mitochondria during oxidative Appl. Sci.2020,10, 5133; doi:10.3390 /app10155133 /journal/applsci

Appl. Sci.2020,10, 5133 2 of 11 phosphorylation when adenosine-5 0 -triphosphate (ATP) is produced [8]. NAD occurs in two forms: oxidized NAD + and reduced NADH. NAD takes part in many biological reactions including electron transport. The reduction of NAD + to NADH occurs almost exclusively in the mitochondria at the nal stage of cellular respiration [9,10]. In the human body, there is a pool of NAD that takes reduced (NAD + ) and oxidized (NADH) forms, transforming into each other [8]. Importantly, the NAD pool is only constant for relatively short periods [8,11]. In the long term, the NAD amount changes depending on several factors such as age, diet, physical activity, medicaments, boosters, time of the day, etc. [11]. NAD + metabolism is complex and includes many NAD + -consuming pathways as well as de novo and salvage pathways [8]. Mayevsky and Barbiro-Michaely [1] have claimed that the monitoring of the NADH level in tissue provides important information about the mitochondrial metabolic state (energy production, amount of intracellular oxygen). In addition, changes in the NAD + /NADH ratio re ect cellular respiration processes in mitochondria, thus indirectly represent their function [1,5]. Studies on changes in NADH in response to physical exercise were performed on animal and human skeletal muscle samples, but not in the skin [8,9,12]. Early reports including animals did not provide a clear answer as to how NADH levels were modi ed by exercise [13,14]. Subsequent human research had shown that intensive exercise, unlike light exercise, shifted the NAD + /NADH balance toward NADH [8,15]. Only Koltai et al. [16] have examined the in uence of endurance training on changes in NAD + level in rat muscles and showed that training resulted in an increase in NAD + biosynthesis. Studies on skeletal muscle mitochondria are valuable, but usually invasive due to the use of the biopsy technique [17,18] and expensive if transmission electron microscopy is used [19]. However, it has been suggested that physical exercise brings bene cial changes not only in skeletal muscle mitochondria, but also in skin mitochondria [20]. It has been demonstrated that physical

+ biosynthesis. Studies on skeletal muscle mitochondria are valuable, but usually invasive due to the use of the biopsy technique [17,18] and expensive if transmission electron microscopy is used [19]. However, it has been suggested that physical exercise brings bene cial changes not only in skeletal muscle mitochondria, but also in skin mitochondria [20]. It has been demonstrated that physical exercise results in several bene cial mitochondrial adaptations [19,21–25]. Various changes were extensively studied in skeletal muscle mitochondria [19,21,25–27], while only one study dealt with the changes in the skin [20]. However, we do not know whether training only a ects muscle mitochondria, or the adaptations also take place in skin mitochondria that are easily accessible to study because they lie super cially. To the best of our knowledge, there is a lack of studies describing the e ect of physical training on changes in NADH uorescence in the skin. The novel, noninvasive, and cheap ow mediated skin uorescence method can be a source of valuable information about the mitochondrial activity. Therefore, the study aimed to evaluate the changes in NADH uorescence in the super cial skin layer resulting from a 7-week training period in highly trained competitive athletes. We hypothesize that physical training results in an increase in the NADH uorescence levels in athletes. 2. Materials and Methods 2.1. Subjects Forty-one highly trained athletes (28 men, 13 women), ages ranging from 18 to 35 years, participated in the study. They were members of the Polish national team or athletes taking part in national and international competitions. They represented the following sport disciplines: triathlon (Olympic distance: 1.5 km swim, 40 km bike ride, 10 km run) (seven men, four women); long-distance running (5 km, 10 km, and marathon) (six men, two women); Olympic taekwondo (six men, one woman); sprint (100 m, 200 m, and 4 100 m relay) (six men, one woman); canoeing (three men); and fencing ( ve women). Before starting the study, each participant was informed about the aim and procedures, potential risks, and the possibility to withdraw at any time without giving any reason. All athletes gave

two women); Olympic taekwondo (six men, one woman); sprint (100 m, 200 m, and 4 100 m relay) (six men, one woman); canoeing (three men); and fencing ( ve women). Before starting the study, each participant was informed about the aim and procedures, potential risks, and the possibility to withdraw at any time without giving any reason. All athletes gave their written consent to participate in the examinations and ful lled a questionnaire on their health status and potential contraindications. All athletes had valid health certi cates issued by a physician who specialized in sports medicine, thus were eligible for training and competition. Exclusion criteria were illness symptoms, injuries, and taking drugs (temporarily or chronically). Only the data of those athletes who were present at both examinations was analyzed. The study was conducted in accordance

Appl. Sci.2020,10, 5133 3 of 11 with the Declaration of Helsinki. The Ethics Committee of the Poznan University of Medical Sciences in Poland approved the study protocol (approval no. 1017/16 issued on 5 October 2016). 2.2. Training Characteristics All participants attended training sessions at least six times a week. During the whole 7-week period under study (general preparation phase of the one-year cycle), the athletes had on average 57 training sessions of a total duration of 71.2 h. The average duration of a single session was 84 min. 2.3. Study Design The study was conducted in the Human Movement Laboratory of the Department of Athletics, Strength and Conditioning at the Poznan University of Physical Education (Pozna´n, Poland). Athletes arrived at the laboratory in the morning. During all measurements, the constant temperature was maintained (20-21 C) by an air conditioning system. On the day of the examination, the participants could only eat a light breakfast. It was also recommended for them to avoid co ee or tea for 12 h, alcohol for 24 h, and hard exercise for 48 h before each examination. After arriving, athletes changed into their lightweight sports clothing (without watches and wristbands potentially a ecting blood ow) and acclimatized to the laboratory conditions for at least 30 min. During this time, they completed the required questionnaires, and height and weight measurements were performed. Athletes underwent the examinations twice: at the beginning of the general preparation phase and after seven weeks, at the end of this phase. Each time, the same procedure was applied: (1) initial resting blood pressure measurement; (2) resting NADH uorescence measurement; (3) blood draw, (4) incremental exercise test; (5) second blood draw; (6) post-exercise blood pressure measurement; and (7) post-exercise NADH uorescence measurement (3 min after the end of the test). 2.4. Incremental Exercise Test The exercise test was conducted on the H/P Cosmos treadmill (h/p/cosmos sports & medical GmbH, Nussdorf – Traunstein, Germany). All participants were familiar with the treadmill test because they regularly (2-3 times a year) participated in similar tests. The purpose of this examination was to assess maximal oxygen uptake

min after the end of the test). 2.4. Incremental Exercise Test The exercise test was conducted on the H/P Cosmos treadmill (h/p/cosmos sports & medical GmbH, Nussdorf – Traunstein, Germany). All participants were familiar with the treadmill test because they regularly (2-3 times a year) participated in similar tests. The purpose of this examination was to assess maximal oxygen uptake (VO2max) and peak heart rate (HR). Respiratory gases were collected and analyzed using the MetaMax 3B ergospirometer (Cortex Biophysik BmbH, Leipzig, Germany) and the MetaSoft Studio 5.1.0 software (Cortex Biophysik BmbH, Leipzig, Germany). The exercise protocol started with a 4-min warm-up at the treadmill speed of 6 km/h. Then, the treadmill accelerated by 2 km/h every 3 min. The treadmill inclination was 1% throughout the whole test. The test terminated if the athlete signaled his/her voluntary exhaustion by raising one hand. Maximal oxygen uptake was considered to be reached if the oxygen uptake (VO2) was stabilized despite the further increase in treadmill speed. All participants were highly trained, so during the test, all of them reached a plateau in VO2uptake. We also checked three additional conditions to con rm reached maximal oxygen uptake: (i) HR reached at least 95% of the age-adjusted HR; (ii) cuto blood lactate concentration 9 mmol/L for man and 7 mmol/L for women; and (iii) respiratory exchange ratio was 1.1 [28]. Heart rate was measured using the Polar H6 Bluetooth Smart monitor (Polar Electro Oy, Kempele, Finland) attached to a chest strap. 2.5. Lactic Acid Measurements Capillary blood samples were obtained from the ngertip at rest and 2 min after the exercise test. A total of 20 L of whole blood was drawn to a micro test tube using a capillary. Biosen C-line (EKF Diagnostics, Cardi , UK) was used to measure the level of lactate.

Appl. Sci.2020,10, 5133 4 of 11 2.6. Anthropometric Measure Anthropometric measurements were performed according to standardized procedures. Body mass (kg) and height (cm) were measured with a digital measuring station Seca 285 (SECA, Hamburg, Germany). Body mass index (BMI) was calculated as body weight divided by height squared (kg/m 2 ). 2.7. Nicotinamide Adenine Dinucleotide Fluorescence NADH fluorescence was measured using the AngioExpert device (Angionica, âd´z, Poland, 2016) based on the ow mediated skin uorescence (FMSF) method. FMSF enables recording of the changes in NADH uorescence as a function of time in response to ischemia and reperfusion in forearm skin cells. During the measurement, AngioExpert emits light at the wavelength of 460 nm [6,7]. NADH molecules have auto uorescence capability at a wavelength of 460 nm [9]. The changes in uorescence intensity observed during the examination are produced in the most super cial skin cells (epidermis) [6,29], which is due to very shallow skin penetration by excitation light at the wavelength of 340 nm. About 90% of the recorded signal comes from the skin depth up to 0.5 mm. The activated skin region is not directly supplied with blood, but is supplied with oxygen by deeper blood vessels [6,7,29]. During the examination, each participant sat on a chair with his/her arm resting on the measuring device. Immediately before examination, systolic (SBP) and diastolic (DBP) blood pressure was measured using the Omron 3 (Omron, Kyoto, Japan) device. At the start of the FMSF examination, basal uorescence was registered for 2 min. Then, an occlusion cu was in ated up to the pressure of 50 mmHg above the SBP for 200 s. After this time, blood ow in the forearm was restored (cu de ated) and the changes in NAD uorescence were recorded for a further 3 min [7]. The following parameters related to NAD uorescence were measured or calculated (Figure): Bmean—Basal uorescence at the wavelength of 460 nm, recorded at rest at the beginning of the measurement; FImax—The maximal increase in uorescence above the baseline observed during forearm ischemia; FRmin—The maximal drop in uorescence below the baseline observed during reperfusion; Imax—The

recorded for a further 3 min [7]. The following parameters related to NAD uorescence were measured or calculated (Figure): Bmean—Basal uorescence at the wavelength of 460 nm, recorded at rest at the beginning of the measurement; FImax—The maximal increase in uorescence above the baseline observed during forearm ischemia; FRmin—The maximal drop in uorescence below the baseline observed during reperfusion; Imax—The relative increase in uorescence=the di erence between Imaxand Bmean; Rmin—The relative drop in uorescence=the di erence between Bmeanand FRmean; IRampl—The maximal range of changes in uorescence=the sum of Rminand Imax; and CImax—The relative (percentage) contribution of Imaxto IRampl[7].Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 12 Figure 1. Parameters describing the Flow Mediated Skin Fluorescence. B mean—Mean value of the basal fluorescence; FI max—Maximal fluorescence during ischemia; FR min—The first minimal fluorescence value during reperfusion; I max—The net increase in fluorescence over the baseline during ischemia; IR ampl—The amplitude of fluorescence change during ischemia and reperfusion; R min—The net reduction in fluorescence below the baseline. Reprinted from Bugaj et al. [5]. The second measurement was made according to the same methodology, 3 min after the end of the treadmill test. A sample measurement of the NADH fluorescence from a 23-year-old male sprinter before and after training was shown in Figure 2. Figure 2. A sample Flow Mediated Skin Fluorescence measurement in a 23-year-old male sprinter. Changes in nicotinamide adenine dinucleotide fluorescence are shown before and after 7-weeks of training, at rest, and after cardiopulmonary exercise test until exhaustion. The first 2 minutes serve to determine the baseline fluorescence level. This was followed by a 200-s ischemia (increase in fluorescence) and a 290-s reperfusion (decrease in fluorescence). Figure 1. Parameters describing the Flow Mediated Skin Fluorescence. Bmean—Mean value of the basal uorescence; FImax—Maximal uorescence during ischemia; FR min—The rst minimal uorescence value during reperfusion; Imax—The net increase in uorescence over the baseline during ischemia; IR ampl—The amplitude of uorescence change during ischemia and reperfusion; R min—The net reduction in uorescence below the baseline. Reprinted from Bugaj et al. [5].

the basal uorescence; FImax—Maximal uorescence during ischemia; FR min—The rst minimal uorescence value during reperfusion; Imax—The net increase in uorescence over the baseline during ischemia; IR ampl—The amplitude of uorescence change during ischemia and reperfusion; R min—The net reduction in uorescence below the baseline. Reprinted from Bugaj et al. [5].

Appl. Sci.2020,10, 5133 5 of 11 The second measurement was made according to the same methodology, 3 min after the end of the treadmill test. A sample measurement of the NADH uorescence from a 23-year-old male sprinter before and after training was shown in Figure.Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 12 Figure 1. Parameters describing the Flow Mediated Skin Fluorescence. B mean—Mean value of the basal fluorescence; FI max—Maximal fluorescence during ischemia; FR min—The first minimal fluorescence value during reperfusion; I max—The net increase in fluorescence over the baseline during ischemia; IR ampl—The amplitude of fluorescence change during ischemia and reperfusion; R min—The net reduction in fluorescence below the baseline. Reprinted from Bugaj et al. [5]. The second measurement was made according to the same methodology, 3 min after the end of the treadmill test. A sample measurement of the NADH fluorescence from a 23-year-old male sprinter before and after training was shown in Figure 2. Figure 2. A sample Flow Mediated Skin Fluorescence measurement in a 23-year-old male sprinter. Changes in nicotinamide adenine dinucleotide fluorescence are shown before and after 7-weeks of training, at rest, and after cardiopulmonary exercise test until exhaustion. The first 2 minutes serve to determine the baseline fluorescence level. This was followed by a 200-s ischemia (increase in fluorescence) and a 290-s reperfusion (decrease in fluorescence). Figure 2. A sample Flow Mediated Skin Fluorescence measurement in a 23-year-old male sprinter. Changes in nicotinamide adenine dinucleotide uorescence are shown before and after 7-weeks of training, at rest, and after cardiopulmonary exercise test until exhaustion. The rst 2 min serve to determine the baseline uorescence level. This was followed by a 200-s ischemia (increase in uorescence) and a 290-s reperfusion (decrease in uorescence). 3. Results 3.1. Basic Characteristics The resting DBP, SBP, and BMI were within normal ranges. Other descriptive characteristics are presented in Table. Table 1.Basic characteristics of the studied athletes. Parameter Before Training After Training Age (years) 22.4 4 Training experience (years) 8 2.3 Height (cm) 178.1 7.3 178.1 7.3 Weight (kg) 69.1 10.3 69 10.3 BMI (kg/m 2 ) 21.6

3. Results 3.1. Basic Characteristics The resting DBP, SBP, and BMI were within normal ranges. Other descriptive characteristics are presented in Table. Table 1.Basic characteristics of the studied athletes. Parameter Before Training After Training Age (years) 22.4 4 Training experience (years) 8 2.3 Height (cm) 178.1 7.3 178.1 7.3 Weight (kg) 69.1 10.3 69 10.3 BMI (kg/m 2 ) 21.6 2.3 21.6 2.3 SBPrest(mmHg) 127.6 14.3 119.3 10.8 *** DBPrest(mmHg) 69.9 7.3 72.9 9.3 * SBPexerc(mmHg) 148 18.3 139.2 16.3 ** DBPexerc(mmHg) 74.5 8.1 78.2 8.1 * VO 2max (mL/min/kg) 58.8 8.6 59.5 8.6 HR peak(beats/min) 191.7 8 191.9 8.9 LArest(mmol/L) 1.2 0.5 1.0 0.3 ** LAmax(mmol/L) 9.9 1.5 10.2 1.9 Averaged data are presented as mean standard deviation (SD), and results of thet-test for dependent samples, *p<0.05 , **p<0.01, ***p<0.001 signi cantly di erent pre-training. BMI body mass index; SBP systolic blood pressure; DBP diastolic blood pressure; rest before cardiopulmonary exercise test; exerc after cardiopulmonary exercise test; VO2max (mL/min/kg) maximal oxygen uptake; HRpeak peak heart rate; LArest resting lactate concentration; LAmax maximal lactate concentration.

This study investigates the impact of a 7-week training period on skin NADH fluorescence in athletes.