Effects of Vitamin D Supplementation on Haematological Values and Muscle Recovery in Elite Male Traditional Rowers

ion: De cient levels of 25-hydroxyvitamin D (25(OH)D) (<30 ng/mL) may compromise health and athletic performance. Supplementation with oral vitamin D can favor the state of iron metabolism, and testosterone and cortisol as an indicator of muscle recovery of the athlete with a de ciency. The main aim of this study was to evaluate the in uence of eight weeks of supplementation with 3000 IU/day of vitamin D on the hematological and iron metabolism pro le, as well as on the analytical values of testosterone and cortisol on elite male traditional rowers. The secondary aim was to examine if serum 25(OH)D is a predictor of testosterone and cortisol levels. Material and Methods: Thirty-six elite male rowers (27 6 years) were assigned to one of the two groups randomly: 1) Control group (CG,n= 18, height: 181.05 3.39 cm and body mass:77.02 7.55 kg ), 2) Group treated with 3,000 IU of vitamin D3/day (VD3G,s= 18, height:179.70 9.07 cm and body mass: 76.19 10.07 kg). The rowers were subjected to blood tests at the beginning of the study (T1) and after eight weeks of treatment (T2), for

two groups randomly: 1) Control group (CG,n= 18, height: 181.05 3.39 cm and body mass:77.02 7.55 kg ), 2) Group treated with 3,000 IU of vitamin D3/day (VD3G,s= 18, height:179.70 9.07 cm and body mass: 76.19 10.07 kg). The rowers were subjected to blood tests at the beginning of the study (T1) and after eight weeks of treatment (T2), for the analysis of hematological and hormonal values. Repeated-measures ANOVA with group factor (GC and GVD3) were used to examine if the interaction of the different values was the same or different between the groups throughout the study (time group) after vitamin D3 treatment. To analyze if 25(OH)D was a good predictor of testosterone, cortisol, and testosterone/cortisol ratio a stepwise regression model was performed. Results: Statistically signi cant and different increases were observed in the group-by-time interaction of 25(OH)D in VD3G in respect to CG during the study (p< 0.001; VD3G (T1: 26.24 8.18 ng/mL vs. T2: 48.12 10.88 ng/mL) vs CG (T1: 30.76 6.95 ng/mL vs. T2: 35.14 7.96 ng/mL ). Likewise, signi cant differences between groups were observed throughout the study in the group-by-time interaction and changes of hemoglobin (GC: 2.89 2.29% vs. VD3G:0.71 1.91% ;p= 0.009), hematocrit (CG: 1.57 2.49% vs. VD3G: 1.16 1.81%;p= 0.019) and transferrin (CG: 0.67 4.88% vs. VD3G: 6.51 4.36%;p= 0.007). However, no differences between groups were observed in the group-by-time interaction of the hormonal parameters (p> 0.05). Regression multivariate analysis showed that cortisol and testosterone levels were associated with 25(OH)D levels (p< 0.05). Conclusion: Oral supplementation with 3000 IU/day of vitamin D3 during eight weeks showed to be suf cient to prevent a decline in hematological levels of hemoglobin and Nutrients2018,10, 1968; doi:10.3390/nu10121968

Nutrients2018,10, 1968 2 of 14 hematocrit, and improve transferrin of 25(OH)D levels. However, although it was not suf cient to enhance muscle recovery observed by testosterone and cortisol responses, it was observed that serum 25(OH)D levels could be a predictor of anabolic and catabolic hormones. Keywords: strength-endurance; vitamin D; hemoglobin; hematocrit; recovery; testosterone; cortisol 1. Introduction Vitamin D is receiving wide attention in athletic communities given that non-optimal 25-hydroxyvitamin D (25(OH)D) values are a prevalent issue among athletes [1,2]. In this sense, it has been observed that de cient values of 25(OH)D may reduce physical performance among others due to inadequate recovery [3]. However, optimal levels of serum 25(OH)D are positively related to strength and potency [4], running performance [5], endurance performance [6], and aerobic capacity [7]. In this way, it has been observed that maximum peak performance coincides with the time of year that athletes have higher serum levels of 25(OH)D [7]. Although vitamin D is found naturally in only a few foods, such as fatty sh (i.e., mackerel, salmon, sardines), egg yolks, certain mushrooms, dairy products, margarine, ready-to-eat cereals, and fruit juices that have been forti ed [8], sun exposure is the main source of vitamin D. Due to the low synthesis of vitamin D during some periods of the year, oral vitamin D supplementation has been proposed as an alternative to obtain adequate levels of 25(OH)D [9]. In this regard, a dose of 3000 IU/day of vitamin D over a period of eight weeks has been shown to adjust the concentration of 25(OH)D to 30–100 ng/mL (optimal range) in a general population with low levels [10,11]. However, optimal sport bene ts occur at 25(OH)D levels above the current de nition of suf ciency (>30 ng/mL) with no reported sports health bene ts above 50 ng/mL [3]. A mechanism by which optimal 25(OH)D levels could enhance athletic performance is improving hematological levels. In this line, vitamin D has been proposed as a suitable factor that facilitates erythropoiesis, given that erythrocyte precursor cells express receptors of the active form of vitamin D, which induce the proliferation and maturation of

reported sports health bene ts above 50 ng/mL [3]. A mechanism by which optimal 25(OH)D levels could enhance athletic performance is improving hematological levels. In this line, vitamin D has been proposed as a suitable factor that facilitates erythropoiesis, given that erythrocyte precursor cells express receptors of the active form of vitamin D, which induce the proliferation and maturation of erythrocytes [12]. Therefore, vitamin D de ciency could accelerate the decrease in hemoglobin and increase the incidence of anemia [13]. Thus, an inverse association of 25(OH)D levels with the risk of anemia has been shown [14]. However, there are con icting data regarding the possible improvement of hematological levels with vitamin D supplementation in different populations [15]. Speci cally, in the sports eld, although there is little research, they conclude the existence of a positive relationship between 25(OH)D levels and iron status [16,17]. Another mechanism that promotes sport performance is adequate muscle recovery of athletes [18]. Although the parameters used to know their state are different, analysis of anabolic–catabolic hormones has shown a great relationship with the endogenous regenerative process of athletes [19,20]. Therefore, a low testosterone level and/or a high cortisol value could indicate inadequate muscle recovery [21]. In this sense, in the sports sciences eld, Lombardi et al. [22] observed a positive and close correlation between 25(OH)D levels and testosterone, and inversely with those of cortisol. This detail could indicate that increasing the circulating levels of 25(OH)D in athletes through a supplementation with vitamin D could improve the levels and ratios of these hormones, thus facilitating recovery process. In this regard, supplementation with 3,332 IU/day showed signi cant increases in testosterone levels in non-athletes [23]. However, to our knowledge, no such data have been found in the athlete population. For this reason, the main goal was to evaluate the in uence of eight weeks of supplementation with 3,000 IU/day of vitamin D3 on the hematological and iron metabolism pro le, as well as in the analytical values of testosterone and cortisol in elite traditional rowers. In addition, a secondary aim was to examine if serum 25(OH)D is a

population. For this reason, the main goal was to evaluate the in uence of eight weeks of supplementation with 3,000 IU/day of vitamin D3 on the hematological and iron metabolism pro le, as well as in the analytical values of testosterone and cortisol in elite traditional rowers. In addition, a secondary aim was to examine if serum 25(OH)D is a predictor of testosterone and cortisol levels.

Nutrients2018,10, 1968 3 of 14 2. Materials and Methods 2.1. Participants Thirty-six elite male rowers (27 6 years) who belonged to a rowing club from the First Trainer League in Spain (ACT) participated in this double-blind, placebo-controlled study. The tests were carried out in the spring months (from 8 April to 3 June 2018) with an average solar irradiance of 213.1 W/m 2 . The solar irradiance and outdoor air temperature were recorded every 10 min during the whole study by wireless Vantage Pro2™Plus (Davis Instruments, Hayward, CA, USA). All participants were located in the area of San Sebastian (Guipózcoa, Spain) at a latitude of 43 18 0 46” N and with 20 km between the most distant cities. All rowers followed the same training program conducted and supervised by the same certi cated strength and conditioning coach. The average weekly hours of training were 15 during study (50% strength training, 45% endurance training, and 5% complementary training). Further, the dietitian-nutritionist of the club elaborated an individual diet for each rower. The diets were proposed using previously established energy and macronutrient guidelines for adequate athletic performance [24], and were based on the volume and training load, and personal characteristics of each participant. Importantly, diets were described so that all athletes would meet the micronutrient recommended dietary allowances (RDAs) for men aged 19–30 [25,26]. A medical examination was performed before the study began in order to verify that the participants did not have any disease. In addition, no rower was taking medications that affected body mass, or any supplement or medication that affected analytical or performance values. The experimental procedures, associated risks, and bene ts were explained prior to the medical examination, so each rower signed a written consent form prior to commencing study participation. None of the rowers had pre-existing injuries before or during the intervention period. This study was designed in accordance with the Declaration of Helsinki (2008) and Fortaleza update (2013) and approved by the ethics committee of the University of Basque Country (M10_2017_247). 2.2. Experimental Protocol and Evaluation Program This study was structured with a double-blind, placebo-controlled

to commencing study participation. None of the rowers had pre-existing injuries before or during the intervention period. This study was designed in accordance with the Declaration of Helsinki (2008) and Fortaleza update (2013) and approved by the ethics committee of the University of Basque Country (M10_2017_247). 2.2. Experimental Protocol and Evaluation Program This study was structured with a double-blind, placebo-controlled design in order to analyze the effects of and 8-week oral supplementation of 3000 IU per day of vitamin D3 (Lindens Health + Nutrition Ltd., Wake eld, WF2) on the hematological, iron metabolism, and muscle recovery studied by catabolic–anabolic hormones. Participants were assigned to groups using a strati ed block design. An independent statistician generated the random allocation sequence: 1) Control group (CG,n= 18, height: 181.05 3.39 cm and body mass: 77.02 7.55 kg), 2) Group treated with 3000 IU/day of vitamin D3 (VD3G,n= 18, height: 179.70 9.07 cm and body mass: 76.19 10.07 kg). All participants attended the laboratory (08:30) for blood collection at two speci c points during the study: 1) at baseline (T1), and 2) post-treatment (T2—the day after 8 weeks of treatment). The VD3G took 3000 IU of vitamin D3 with one capsule per day. While in the GC, the rowers assigned took capsules of similar external appearance lled with 10 mg of maltodextrin. Both groups took their dose every morning with their breakfast from the day following T1 to T2 (during 8 weeks). The control group served as baseline or “standard” condition because this group did not take any vitamin D3. 2.3. Blood Collection Antecubital venous blood samples were collected from all participants to evaluate hematological, iron metabolism, and hormonal parameters in T1 and T2. All samples were examined under basal conditions after a night and after at least 36 h without exercise. At both times the rowers arrived at the laboratory at 08:30 where they were allowed to rest in a seat for 30 min, at which time the blood samples were recollected. In T1 and T2, 22 mL of blood were taken. The rst 13 mL were collected in a tube containing 200

and after at least 36 h without exercise. At both times the rowers arrived at the laboratory at 08:30 where they were allowed to rest in a seat for 30 min, at which time the blood samples were recollected. In T1 and T2, 22 mL of blood were taken. The rst 13 mL were collected in a tube containing 200 L EDTA K anticoagulant (Vacutainer, Becton Dickinson) and used to determine serum iron, ferritin, and transferrin. Serum concentrations of 25(OH)D were determined by HPLC-MS/MS using

Nutrients2018,10, 1968 4 of 14 an AB Sciex 5500 tandem mass spectrometer (AB Sciex UK Ltd., Warrington, UK). The second tube with the remaining 9 mL collected separately was centrifuged for 15 min at 4 C and 3000 rpm. After centrifugation, the serum was separated and stored in aliquots at 20 C until analysis. A STKS (Coulter) analyzer was used to determine the content of hematological parameters. Parameters relative to iron metabolism were measured using a COBAS FARA analyzer (Roche Diagnostics, Basel, Switzerland). Serum iron was determined using the ferrozine colorimetric method but without protein precipitation, while ferritin was measured by immunoturbidimetry. As for the hormonal variables, commercially available enzyme immunoabsorbent assay kits (DRG testosterone ELISA kit ® , DRG Instruments GmbH, Marburg/Lahn, Germany) were used for the measurement of serum total testosterone. The intra-assay coef cient of variation (CV) was 4.3% and the CV among the trials was 9.2%. For the measurement of serum cortisol levels, an enzyme-linked uorescent assay with the aid of a multiparametric analyzer (Minividas ® , Biomerieux, Marcy l'Etoile, France) was used. The substrate, 4-methyl umbelipherone, was used and performed a uorescence emission at 450 nm, after stimulation at 370 nm. The intra-assay CV was 5.7% and the CV of the intermediate assay was 6.2%. Finally, the testosterone/cortisol ratio (TT/C) was calculated from the concentrations of testosterone and cortisol, dividing testosterone between cortisol. 2.4. Dietary Assessment All athletes were informed on proper food tracking by trained and certified dieticians/ nutritionists. They instructed the participants on two methods of dietary recall. The first of them was to complete a food frequency questionnaire (FFQ) [27] following blood collection, which had been previously validated and utilized for an athlete population [28]. The participants completed the FFQ to recall the “frequency” of intake over the previous 8 weeks of 139 different portion sizes of foods and drinks. Frequency categories were based on the number of times and portion sizes, which a food/drink was consumed per day, per week or per month. In addition, daily numbers of portions of different food groups were calculated. Daily consumption of energy (kcal) and

the “frequency” of intake over the previous 8 weeks of 139 different portion sizes of foods and drinks. Frequency categories were based on the number of times and portion sizes, which a food/drink was consumed per day, per week or per month. In addition, daily numbers of portions of different food groups were calculated. Daily consumption of energy (kcal) and each macronutrient and micronutrient were determined by dividing the reported intake by the frequency in days [27]. The second method was for athletes to complete a 7-day dietary recall at T1 and T2 of the previous 7 days to compare if these results were similar to that of the FFQ. If participants could weigh food, then this data was included in the dietary recall; however, if weighing food was not possible serving sizes consumed were estimated from the standard weight of food items or by determining portion size via looking at a book with 500 photographs of foods. Food values were then converted into intakes of total energy and micronutrients by a validated software package (Easy diet © , online version). This software package was developed by the Spanish Centre for Higher Studies in Nutrition and Dietetics (CESNID), which is based on Spanish tables of food composition [8]. 2.5. Body Composition and Anthropometric Measures Anthropometric measurements were taken following “The International Society for the Advancement of Kinanthropometry” (ISAK) protocol [29]. Additionally, the same internationally certi ed anthropometrist (ISAK level 3) took measurements for all participants. All measurements were undertaken in duplicate to establish within-day retest reliability. If the difference between the duplicate measures exceeded 5% for an individual skinfold, a third measurement was taken. The mean of duplicate or median of triplicate anthropometric measurements were used for all analysis. Height (cm) was measured using a SECA measuring rod, with a precision of 1 mm, while BM (kg.) was assessed by a SECA model scale, with a precision of 0.1 kg. Body mass index (BMI) was calculated using the formula BM height 2 (kg/ m 2 ). The sum of 4 skinfolds (mm) (Triceps, subscapular, suprailiac, and abdominal) was calculated, previously

(cm) was measured using a SECA measuring rod, with a precision of 1 mm, while BM (kg.) was assessed by a SECA model scale, with a precision of 0.1 kg. Body mass index (BMI) was calculated using the formula BM height 2 (kg/ m 2 ). The sum of 4 skinfolds (mm) (Triceps, subscapular, suprailiac, and abdominal) was calculated, previously analyzed with a Harpenden ® skinfold caliber, with a precision of 0.2 mm. Absolute fat mass and muscle mass (absolute and percentage) were predicted using the Faulkner equation [30]. Lastly, absolute fat free mass was calculated as body mass (kg) fat mass (kg).

Nutrients2018,10, 1968 5 of 14 2.6. Statistical Analysis Data are presented as means and standard deviations. Analyses were performed using SPSS software version 24.0 (SPSS, Inc, Chicago, IL, USA). Statistical significance was indicated whenp< 0.05. Firstly, Kolmogorov–Smirnov tests were performed on the values of the parameters studied (n< 50) to decide parametric or non-parametric data. Secondly, the homoscedasticity of the variables analyzed by the Levene test was checked. As the distribution of all the parameters was normal, the results are presented as means standard deviation. Differences from T1 to T2 were assessed by a non-paired Student'st-test. Likewise, an independent Student´st-test was performed to calculate the differences in different parameters studied in CG and VD3G between baseline (T1) and after 8 weeks (T2). Repeated-measures ANOVA with group factor (GC and GVD3) were used to examine the existence of an interaction effect of vitamin D3 treatment throughout the study (time group) on hematological parameters, iron metabolism, and hormonal, that is, if the group-by-time interaction of the different values was the same or different between the groups throughout the study. Effect sizes among participants were calculated using partial eta square ( 2p). Since this measure is likely to overestimate effect sizes, values were interpreted according to Ferguson [31] which indicates that there has been no effect if 0 2p < 0.05; a minimum effect if 0.05 2p < 0.26; a moderate effect if 0.26 2p < 0.64; and a strong effect if 2 p 0.64. A McNemar test was performed to compare differences in vitamin D status between groups at baseline (T1) and after 8 weeks of treatment (T2). In order to compare the differences in vitamin D status, several groups were identi ed in which non-optimal level of 25(OH)D for athletic performance enhancement were considered below 50 ng/mL and within the optimal values between 50 and 100 ng/mL [3]. Finally, to analyze if 25(OH)D was a good predictor of testosterone, cortisol, and TT/C ratio, a stepwise regression model was performed using anabolic–catabolic hormones as the dependent variables and 25(OH)D serum level as predictors. 3. Results In Figure, a statistically signi cant increase

The study assesses the effects of vitamin D on health and performance in elite rowers.