Effect of Iron Supplementation on the Modulation of Iron Metabolism, Muscle Damage Biomarkers and Cortisol in Professional Cyclists

ound: The intense efforts made during 3-week stage races may reduce iron metabolism and hematological parameters. These efforts may increase the levels of circulating muscle damage markers and some hormones. All of these physiological changes may have negative consequences not only for the performance of athletes but also for their health. The main aim of this study was to evaluate the effects of supplementation with 80 mg/day of iron on haematological parameters, serum cortisol and biochemical muscle indicators on elite male cyclists during the 3-week stage race theVuelta a España. Our secondary aim was to examine whether the hematological pro le is associated with muscular damage parameters and cortisol. Methods: Eighteen elite male cyclists from two teams were randomly assigned to one of two groups: (1) control group (CG,n= 9; age: 26.1 4.6 years; maximum oxygen uptake per kg: 78.0 5.4 mL/kg/min) or (2) group treated with 80 mg/day iron (800 mg of iron protein succinylate, ITG,n= 9; age: 25.7 6.4 years; maximum oxygen uptake per kg:

and cortisol. Methods: Eighteen elite male cyclists from two teams were randomly assigned to one of two groups: (1) control group (CG,n= 9; age: 26.1 4.6 years; maximum oxygen uptake per kg: 78.0 5.4 mL/kg/min) or (2) group treated with 80 mg/day iron (800 mg of iron protein succinylate, ITG,n= 9; age: 25.7 6.4 years; maximum oxygen uptake per kg: 77.6 6.5 mL/kg/min). The cyclists were subjected to blood tests one week before the start of the race (T1) and after 4 weeks of treatment, coinciding with the end of the competition (T2). Iron metabolism parameters, muscle damage indicators and serum cortisol were assessed. Repeated-measures ANOVA with group as a factor (GC and ITG) were used to examine the differences between groups throughout the study (time group) after iron supplementation treatment. Results: Signi cant differences were observed between groups throughout the study in the group-by-time interaction and changes in serum iron (GC: 8.93 10.35% vs. ITG: 0.60 8.64%;p= 0.018), ferritin (GC: 13.88 23.53% vs. ITG: 91.08 118.30%;p= 0.004), haemoglobin (GC: 10.00 3.32% vs. ITG: 13.04 5.64%;p< 0.001), haematocrit (GC: 1.17 3.78% vs. ITG: 7.32 3.92%;p< 0.001) and cortisol (GC: 24.74 25.84% vs. ITG: –13.54 13.61%;p= 0.005). However, no signi cant group-by-time interaction was observed for the circulating muscle biomarkers. Additionally, signi cant negative correlations of serum iron, haemoglobin and haematocrit with muscle circulating biomarkers and cortisol (p< 0.05) were observed. Conclusions: Oral iron supplementation with 80 mg/day iron (800 mg of iron protein succinylate) effectively prevented a decline in haematological parameters Nutrients2019,11, 500; doi:10.3390/nu11030500

Nutrients2019,11, 500 2 of 13 (serum iron, ferritin, haemoglobin and haematocrit) and maintained optimal levels of recovery in elite cyclists during theVuelta a España. Moreover, the hematological values were shown to have relationship with muscular recovery parameters. Keywords:recovery; iron supplementation; exercise; cyclist; muscular biomarkers; performance 1. Introduction Iron is an essential micronutrient that plays a signi cant role in physical performance, particularly in demanding endurance sports, such as cycling [1–3]. Of special interest are long stage races in which iron metabolism, hematological parameters, muscle enzymes and hormones undergo changes for which impacts on cyclists' health have not been assessed [1,4]. The intense and sustained efforts made during 3-week stage races may reduce blood hemoglobin (Hb), ferritin (FER) and serum iron (sFe) concentrations in endurance cyclists, resulting in marginal or de cient iron states, one of the main causes of fatigue in this discipline [1,5,6]. In addition, sustained and intense exercise may increase the levels of circulating muscle damage markers, such as serum creatine kinase (CK), lactate dehydrogenase (LDH) and myoglobin (Mb) as well as hormones, such as cortisol, which is involved in the activation of catabolic processes and anti-anabolic actions related to protein turnover [7].These parameters are indicative of increased muscle damage and oxidative stress, which negatively affect athletes' performance and increase the duration required for optimal recovery [4,8–10]. As a result, athletes not only may reduce their performance, but also may put their health at risk. Oral iron supplementation is an effective nutritional strategy that may be considered for individuals with suboptimal FER levels and for preventing iron de ciency [11]. In addition to an adequate iron intake from the regular diet, iron supplementation~80 mg/day) prevent decreases in iron levels and related parameters in athletes undergoing a high level of effort [12]. This supplementation has shown improvements in FER and the hematological pro le during training periods in different sports [11–14]. It is well-known that endurance stage races, such as theTour de France[5] or theGiro d'Italia[1] reduce hematological parameters. However, to our knowledge, iron supplementation has not been tested during a 3-week cycling race. Recent studies have determined a

of effort [12]. This supplementation has shown improvements in FER and the hematological pro le during training periods in different sports [11–14]. It is well-known that endurance stage races, such as theTour de France[5] or theGiro d'Italia[1] reduce hematological parameters. However, to our knowledge, iron supplementation has not been tested during a 3-week cycling race. Recent studies have determined a relationship between skeletal muscle and body iron metabolism [15]. In fact, increased iron availability triggered by low hepcidin levels may provide iron levels that contribute to muscle growth [15]. However, no authors have demonstrated iron supplementation to be a successful nutritional strategy to reduce muscle damage and stress. We hypothesize that oral iron supplementation will be a useful ergogenic aid to improve hematological pro les and reduce muscle damage and stress in non-anemic athletes. The aim of this study was to evaluate the effects of 80 mg/day of oral iron supplementation (800 mg of iron protein succinylate) on the hematological pro le (sFe, FER, transferrin saturation index (TSI), hematocrit (Htc), and Hb), muscle damage intracellular enzymes (CK, LDH, Mb) and cortisol in elite male cyclists over a time-span of 4 weeks during theVuelta a España. Our secondary aim was to examine the relationship between the hematological pro le and biochemical muscular damage parameters and cortisol. We hypothesize that iron supplementation will prevent any decline in the hematological pro le and subsequently, decrease muscle damage. 2. Materials and Methods 2.1. Sample Eighteen professional male cyclists from two different elite teams participated in the study. Both teams had similar objectives and competed in the 3-week cycling stage race known as theVuelta a España, which covers around 3300 km. Medical examinations were conducted by the Union Cyclist

Nutrients2019,11, 500 3 of 13 International (UCI) to ensure participants were free of disease prior to the beginning of the study. To the authors' knowledge, no banned drugs or medications were taken by the riders. None of the athletes tested positive for the routine doping tests performed before and during the race according to the World Anti-Doping Agency (WADA). All participants had analogous dietetic and training programs to prepare the race and were supervised by a team physician in collaboration with one of the authors of the study. Physiotherapeutic and nutritional post-exercise recovery strategies were also pretty similar. All participants were informed of the experimental procedures, risks and bene ts and voluntarily signed a written consent. The study was designed according to the Declaration of Helsinki for experiments with human beings and was approved by the local Ethics Committee of University of Valladolid. 2.2. Experimental Protocol and Assessment Plan A randomized and non-controlled by placebo design was applied to analyse the effects of oral iron supplementation on the hematological pro le, biomarkers of muscular damage and cortisol levels. Participants were assigned to groups using a strati ed block design. An independent statistician generated the following random allocation sequence: (a) a control group (CG,n= 9, age: 26.1 4.6 years; maximum oxygen uptake per kg: 78.0 5.4 mL/kg/min) that did not receive oral iron, (b) group treated with 80 mg/day iron (ITG,n= 9, age: 25.7 6.4 years; maximum oxygen uptake per kg: 77.6 6.5 mL/kg/min). The cyclists were tested at two speci c points during the study: at baseline one week before the start of the race (T1) for diet/supplementation homogenization and at the beginning of the last stage (T2) (3rd week of race). Therefore, the total time between T1 and T2 was 4 weeks, including the rst week being for homogenization and the remaining 3 weeks for the race. The ITG received 80 mg/day oral iron in the form of 800 mg of iron protein succinylate (Ferplex 40 ® , Italfarmaco S.A., Madrid, Spain). Iron supplementation was provided in doses of 40 mg twice per day according to the laboratory´s instructions

was 4 weeks, including the rst week being for homogenization and the remaining 3 weeks for the race. The ITG received 80 mg/day oral iron in the form of 800 mg of iron protein succinylate (Ferplex 40 ® , Italfarmaco S.A., Madrid, Spain). Iron supplementation was provided in doses of 40 mg twice per day according to the laboratory´s instructions during breakfast and dinner in the 4-week period. Protein succinylate was chosen in order to avoid problems related to tolerance, gastrointestinal discomfort, bioavailability and food/supplement compatibility [16]. During the competition, the research team ensured that constant circadian rhythms were maintained in terms of nutrition, hydration, timing of food intake and sleep, which is a normal practice in cycling competitions. 2.3. Dietary Assessment A professional dietitian strictly recorded the daily food and uid intake of the cyclists during the study. The EasyDiet © package, which is available online (https://www.easydiet.es/), was used to calculate the nutrient composition and energy intake from the foods and drinks consumed by the cyclists. This software package was developed by the Spanish Centre for Higher Studies in Nutrition and Dietetics (CESNID) and is based on the Spanish tables of food composition [17]. In addition, all participants received multivitamin pills that included folic acid (5 mg/day), vitamin C (1 g/day), B12 vitamin (1000 g/day), branched amino acids and glutamine. 2.4. Body Composition and Anthropometric Measures Anthropometric measurements were taken by an ISAK (International Society for Advancement in Kinanthropometry) level 3 anthropometric device, following the standard procedures in T1. The height and body weight technical error of measurement was less than 0.02%, and it was less than 2.6% in the case of skinfolds. The height (cm) was measured with a SECA © measuring rod (Barcelona, Spain), with a precision of 1 mm (range: 130–210 cm), and body weight (kg) was assessed by a SECA © scale, with a precision of 0.1 kg (range: 2–130 kg). All skinfolds (triceps, biceps, abdominal, supraspinal, subscapular, chest, front thigh and medial calf) were taken using a Harpenden skinfold caliper (CMS instruments, London, UK), with a precision of 0.2 mm. The sum of 6

of 1 mm (range: 130–210 cm), and body weight (kg) was assessed by a SECA © scale, with a precision of 0.1 kg (range: 2–130 kg). All skinfolds (triceps, biceps, abdominal, supraspinal, subscapular, chest, front thigh and medial calf) were taken using a Harpenden skinfold caliper (CMS instruments, London, UK), with a precision of 0.2 mm. The sum of 6 skinfolds (triceps, abdominal, supraspinal, subscapular, front thigh and medial calf) was calculated as a reference for fat content.

Nutrients2019,11, 500 4 of 13 2.5. Blood Collection and Analysis Blood extraction and transportation were performed according the UCI and WADA guidelines (www.ama-wada.org). All samples were collected under basal conditions after a 10–12 h overnight fast at T1 and T2. The blood samples (15 mL) were obtained at 8:30 a.m. from the antecubital vein with the subject seated in a comfortable position using Vacutainer tubes. Blood was distributed in one tube with gel and clot activator (10 mL) to obtain serum, and other EDTA tubes (3–5 mL) were used to obtain plasma. Immediately after lling, EDTA tubes containing blood were inverted 10 times and stored in a sealed box at 4 C. Temperature control was assured during transportation; the speci c tag Libero Ti1-Elpro (Buchs, Switzerland) was used for temperature measurement and recording. The EDTA anticoagulated blood was processed according to UCI and WADA recommendations [18]. Blood in EDTA tubes was centrifuged at 2000 rpm for 15 min. Plasma (upper layer) was extracted using a Pasteur pipette, transferred to a sterile tube and stored at 20 C until the moment of analysis. Red blood cells (RBC), Hb and Hct were determined in the Hematology Analyser Sysmex XE-2100 (Barcelona, Spain). Serum from blood samples was separated by low-speed centrifugation and analysed according to Wilkinson et al. [19]. Total serum protein was determined by a colorimetric method using an autoanalyser Hitachi 917 (Tokyo, Japan). Serum Fe and total iron-binding capacity were measured in an automated chemistry analyser Synchron CX (Beckman Coulter Diagnosis, Madrid, Spain). FER was determined in duplicate using the IRMA kit (Bio-Rad, Madrid, Spain). Transferrin (TRF) was measured by nephelometry (Boehringer–Mannheim, Barcelona, Spain). Finally, the transferrin saturation index (TSI) (%) was calculated as the ratio of sFe to the total iron binding capacity. Serum levels of CK and LDH were measured using coupled enzyme reactions on an automatic autoanalyser Hitachi 917. Mb assessment was performed using a chemiluminescence immunoassay. Cortisol levels were measured by an enzyme-linked uorescent assay in a multiparametric analyser Minividas ® (Biomerieux, Marcy l'Etoile, France) using 4 methyl umbelipherone as a substrate with stimulation at 370 nm

iron binding capacity. Serum levels of CK and LDH were measured using coupled enzyme reactions on an automatic autoanalyser Hitachi 917. Mb assessment was performed using a chemiluminescence immunoassay. Cortisol levels were measured by an enzyme-linked uorescent assay in a multiparametric analyser Minividas ® (Biomerieux, Marcy l'Etoile, France) using 4 methyl umbelipherone as a substrate with stimulation at 370 nm and emission at 450 nm. 2.6. Statistical Data Analyses Statistical analyses were performed using the using SPSS Statistics software (SPSS: An IBM Company, version 24.0, IBM Corporation, Armonk, NY, USA) and Graphpad Prism (Graphpad Software version 6, San Diego, CA, USA). Data were expressed as mean standard (SD) deviation. Signi cant differences were established atp< 0.05. Differences between groups for hematological parameters, muscle damage and cortisol parameters were assessed by independent Student'sttests after normality had been con rmed with the Shapiro–Wilk test, opting for a parametric analysis. Likewise, parametric pairedt-tests were used with the blood sample parameters to identify signi cant differences between T1 and T2 in each group independently. Additionally, a two-way repeated measure analysis of variance (ANOVA) was carried out using the Greenhouse–Geisser test to con rm the existence of an interaction effect (time group) between CG and ITG for hematological parameters, muscle damage and cortisol parameters throughout the study (T1 and T2). Effect sizes among participants were calculated using partial eta square (h 2p). Since this measure is likely to overestimate effect sizes, values were interpreted in accordance with Ferguson [20], whereby no effect is indicated by 0 h 2p< 0.05; a minimum effect is indicated if 0.05 h 2p< 0.26; a moderate effect is indicated if0.26 h 2 p< 0.64; and a strong effect is indicated ifh 2p 0.64. Lastly, correlations between hematological parameters and muscular biomarkers were examined using Pearson´s correlation coef cient in order to estimate the association between two variables.

Nutrients2019,11, 500 5 of 13 3. Results Table No signi cant differences between groups were found (p> 0.05). The average daily energy and micronutrient intake in CG and ITG during the 4 weeks of study can be observed in Table. No signi cant differences between groups for total caloric intake and micronutrients, including iron, were found (p> 0.05). Table 1. Physical and anthropometric characteristics in the control group (CG) and iron treatment group (ITG) at baseline (T1). CG ITG p Age (years) 26.1 4.6 25.7 6.4 0.880 Weight (kg) 69.3 5.6 69.9 7.9 0.854 Height (cm) 179.1 12.8 178.2 16.3 0.898 S6 skinfolds (mm) 35.2 7.5 35.4 8.2 0.956 Maximum oxygen uptake (mL/kg/min) 78.0 5.4 77.6 6.5 0.889 Data are expressed as mean standard deviation.p: Differences between groups as determined by independent t-tests (CG vs. ITG). Table 2. Average daily energy and micronutrient intakes in the control group (CG) and iron treatment group (ITG) during the 4 weeks of study. GC ITG p Energy (kcal/kg) 76.4 5.8 75.4 4.7 0.693 Calcium (mg) 2153 222 2336 257 0.126 Magnesium (mg) 1030 109 1042 72 0.786 Phosphorus (mg) 3928 84 4032 291 0.318 Iron (mg) 43.2 5.6 43.7 4.0 0.830 Zinc (mg) 26.5 0.8 27.0 2.2 0.531 Vitamin A ( g) 3512 1270 3186 585 0.494 Vitamin E (mg) 32.4 3.5 30.6 2.9 0.252 Thiamine (mg) 4.86 0.20 4.64 0.54 0.269 Ribo avin (mg) 5.08 0.22 5.31 0.54 0.254 Niacin (mg) 70.2 6.8 69.1 7.3 0.745 Vitamin B6 (mg) 7.18 0.62 7.04 0.97 0.720 Folic Acid ( g) 1177 176 1175 158 0.980 Vitamin B12( g) 17.62 3.89 16.61 4.38 0.611 Vitamin C (mg) 650 148 702 158 0.482 Data expressed as mean standard deviation.p: Differences between groups as determined by independentt-tests (CG vs. ITG). Table SD of certain hematological parameters directly or indirectly related to iron homeostasis. These include TRF, TSI, total serum proteins and RBC in CG and ITG at T1 and T2. Statistically signi cant differences in the group-by-time interaction of TSI (p= 0.025;h 2p= 0.248), TP (p= 0.017;h 2p= 0.290), and RBC (p< 0.001;h 2p= 0.815) between the

(CG vs. ITG). Table SD of certain hematological parameters directly or indirectly related to iron homeostasis. These include TRF, TSI, total serum proteins and RBC in CG and ITG at T1 and T2. Statistically signi cant differences in the group-by-time interaction of TSI (p= 0.025;h 2p= 0.248), TP (p= 0.017;h 2p= 0.290), and RBC (p< 0.001;h 2p= 0.815) between the two groups through the study were observed in favour of ITG. Along this line, ITG presented signi cant increases between T1 and T2 (p< 0.05) in TSI (T1: 35.5 10.0 vs. T2: 31.1 12.9%), TP (T1: 6.9 0.3 vs. T2: 7.1 0.3 g/L) and RBC (T1: 4.8 0.3 vs. T2: 5.3 0.2 10 6 / L). However, CG did not present signi cant differences between T1 and T2 for any hematological parameters (p> 0.05). Figure SD of other hematological parameters that are related to iron homeostasis. These include sFe, FER, Hb and Hct in CG and ITG at T1 and T2. The results show statistical differences in the group-by-time interaction for sFe (p= 0.018;h 2p= 0.271), FER (p= 0.004; h 2p= 0.395), Hb (p< 0.001;h 2p= 0.723) and Hct (p< 0.001;h 2p= 0.586) in favor of ITG. As a result, signi cant increases were observed in ITG with respect to CG at T2 (sFe: CG: 98.9 15.8 vs. ITG: 116.7 14.4 g/dL; FER: CG: 104.7 41.2 vs. ITG: 231.0 62.0 ng/mL; Hb: CG: 15.0 1.2 vs. ITG:

Nutrients2019,11, 500 6 of 13 16.6 0.5 g/L; Hct: CG: 44.1 3.0 vs. ITG: 49.7 2.0 %; allp< 0.05). The absence of signi cant differences at T1 reinforces the homogeneity of the participants at the beginning of intervention T2 (sFe: CG: 108.6 39.6 vs. ITG: 116.0 21.6 g/dL; FER: CG: 128.3 55.7 vs. ITG: 153.3 68.8 ng/mL; Hb: CG: 15.1 0.8 vs. ITG: 14.7 0.9 g/L; Hct: CG: 44.6 2.5 vs. ITG: 46.3 2.6%; allp> 0.05). On the other hand, while CG only presented signi cant differences in FER between T1 and T2 (p< 0.05), ITG presented signi cant increase (p< 0.05) in FER, Hb and Hct. Table 3. Hematological parameters and total protein in the control group (CG) and iron treatment group (ITG) at baseline (T1) and after 3 weeks (T2). Group T1 T2 p h 2 p Transferrin (mg/dL) CG 243.3 25.4 234.4 24.2 0.195 0.097 ITG 232.0 18.6 242.3 44.4 Transferrin Saturation Index (%) CG 31.7 9.6 29.9 8.9 0.025 0.248 ITG 35.5 10.031.1 12.9 & Total Protein (g/dL) CG 7.0 0.3 7.0 0.6 0.017 0.290 ITG 6.9 0.3 7.1 0.3 & Red Blood Cells ( 10 6 cells/ L) CG 4.9 0.4 4.9 0.4 <0.001 0.815 ITG 4.8 0.3 5.0 0.2 & Data are expressed as mean standard deviation.p: group-by-time interaction (p< 0.05, all such occurrences). Two-factor repeated-measures ANOVA. No signi cant differences between groups (CG vs. ITG) were found by independentt-tests. & Signi cant differences (p< 0.05) over time (T1 vs. T2) within the same group as determined by dependentt-tests.Nutrients 2019, 11, x FOR PEER REVIEW 6 of 13 Table 3. Hematological parameters and total protein in the control group (CG) and iron treatment group (ITG) at baseline (T1) and after 3 weeks (T2). Group T1 T2 p η 2 p Transferrin (mg/dL) CG 243.3 ± 25.4 234.4 ± 24.2 0.195 0.097 ITG 232.0 ± 18.6 242.3 ± 44.4 Transferrin Saturation Index (%) CG 31.7 ± 9.6 29.9 ± 8.9 0.025 0.248 ITG 35.5 ± 10.0 31.1 ± 12.9 & Total Protein (g/dL) CG 7.0 ± 0.3 7.0 ± 0.6 0.017 0.290 ITG 6.9 ±

Iron supplementation prevents declines in hematological parameters and supports recovery in elite cyclists.