Elevated Serum Hepcidin Levels during an Intensified Training Period in Well-Trained Female Long-Distance Runners

Iron is essential for providing oxygen to working muscles during exercise, and iron de ciency leads to decreased exercise capacity during endurance events. However, the mechanism of iron de ciency among endurance athletes remains unclear. In this study, we compared iron status between two periods involving different training regimens. Sixteen female long-distance runners participated. Over a seven-month period, fasting blood samples were collected during their regular training period (LOW; middle of February) and during an intensi ed training period (INT; late of August) to determine blood hematological, iron, and in ammatory parameters. Three-day food diaries were also assessed. Body weight and lean body mass did not differ signi cantly between LOW and INT, while body fat and body fat percentage were signi cantly lower in INT (p< 0.05). Blood hemoglobin, serum ferritin, total protein, and iron levels, total iron-binding capacity, and transferrin saturation did not differ signi cantly between the two periods. Serum hepcidin levels were signi cantly higher during INT than LOW (p< 0.05). Carbohydrate and iron intakes from the daily diet were signi cantly higher during INT than LOW (p< 0.05). In conclusion, an elevated hepcidin level was observed during an intensi ed training period in long-distance runners, despite an apparently adequate daily intake of iron. Keywords:iron homeostasis; athletes; training volume 1. Introduction Because iron is essential for oxygen transport to working muscles, it plays an important role in energy production during exercise [1]. Iron de ciency leads to a decreased exercise capacity during endurance events [2,3]. Among endurance athletes, iron de ciency is prevalent as a result of their prolonged training and repetitive

of iron. Keywords:iron homeostasis; athletes; training volume 1. Introduction Because iron is essential for oxygen transport to working muscles, it plays an important role in energy production during exercise [1]. Iron de ciency leads to a decreased exercise capacity during endurance events [2,3]. Among endurance athletes, iron de ciency is prevalent as a result of their prolonged training and repetitive ground impact [4]. Several physiological mechanisms have been proposed to explain the impaired iron status, including gastrointestinal bleeding [5], hemolysis [6], lack of iron in the daily diet [7], and loss via sweat [8]. However, the detailed mechanisms underlying exercise-induced iron de ciency among athletes remain unclear. Hepcidin, a 25-amino-acid peptide hormone, is a key mediator of iron homeostasis [9], and it may represent another mechanism of iron de ciency in response to exercise training [10–12]. Hepcidin regulates iron balance by binding to the iron export protein ferroportin, which inhibits iron ef ux from enterocytes, hepatocytes, and macrophages [13]. Hepcidin expression is upregulated by increased iron stores [14] and in ammation [15]. Because pro-in ammatory cytokines, such as interleukin-6 (IL-6), also stimulate hepcidin production, sustained in ammation may increase hepcidin, resulting in iron de ciency and anemia [16,17]. Nutrients2017,9, 277; doi:10.3390/nu9030277

Nutrients2017,9, 277 2 of 9 Strenuous exercise causes in ammation, and a marked increase in IL-6 levels is observed after prolonged exercise [18], which stimulates hepcidin production. Serum hepcidin levels are elevated around 3–6 h after a single bout of exercise [11,19–21]. However, information about the cumulative effect of daily training on hepcidin levels in female athletes is still limited. Although an increased prevalence of iron depletion was found previously during a competitive season in endurance athletes [22], whether an increased training volume augments hepcidin levels in female athletes has not been fully examined. Thus, the purpose of the present study was to compare serum hepcidin levels and iron metabolism between two periods involving different training volumes in female long-distance runners. We hypothesized that increased training would elevate serum hepcidin levels. 2. Materials and Methods 2.1. Subjects In total, 16 female long-distance runners (top level of university runners in Japan) (mean standard deviation age: 20.5 1.0 years) participated. All subjects belonged to the same track and eld team at a university and lived together in a dormitory for the long-distance running team. They regularly performed training twice per day (1 h in the early morning, 2 h in the afternoon) six days per week. The subjects were informed about the purpose and procedures of the study, and written informed consent was obtained from all subjects. This study was approved by the Ethics Committee for Human Experiments at the Ritsumeikan University (BKC-IRB-2014-025), Japan. 2.2. Experimental Design To evaluate the in uence of different training regimens on iron metabolism, blood drawings and dietary surveys were performed twice over a period of seven months (Figure): during a regular training period (LOW; middle of February 2015) and an intensi ed training period (INT; late of August 2015). We have compared iron metabolism between these periods, because running distances during LOW and INT were apparently different. Moreover, the training menu during each period was maintained over a month, and iron metabolism evaluated during each period was considered to be relatively stable at each time point. The subjects reported average monthly running distances. On the day

of August 2015). We have compared iron metabolism between these periods, because running distances during LOW and INT were apparently different. Moreover, the training menu during each period was maintained over a month, and iron metabolism evaluated during each period was considered to be relatively stable at each time point. The subjects reported average monthly running distances. On the day of measurement, recent use of iron and other supplements and menstrual cycle status were noted. In addition to body composition, hematological parameters, iron and in ammatory parameters in blood and dietary intake were assessed. Body composition measurement and blood sampling were taken before breakfast, and all subjects did not consume any uid prior to the measurements (6:30–8:00 a.m.).Nutrients 2017, 9, 277 2 of 9 Strenuous exercise causes inflammation, and a marked increase in IL‐6 levels is observed after prolonged exercise [18], which stimulates hepcidin production. Serum hepcidin levels are elevated around 3–6 h after a single bout of exercise [11,19–21]. However, information about the cumulative effect of daily training on hepcidin levels in female athletes is still limited. Although an increased prevalence of iron depletion was found previously during a competitive season in endurance athletes [22], whether an increased training volume augments hepcidin levels in female athletes has not been fully examined. Thus, the purpose of the present study was to compare serum hepcidin levels and iron metabolism between two periods involving different training volumes in female long‐distance runners. We hypothesized that increased training would elevate serum hepcidin levels. 2. Materials and Methods 2.1. Subjects In total, 16 female long‐distance runners (top level of university runners in Japan) (mean ± standard deviation age: 20.5 ± 1.0 years) participated. All subjects belonged to the same track and field team at a university and lived together in a dormitory for the long‐distance running team. They regularly performed training twice per day (1 h in the early morning, 2 h in the afternoon) six days per week. The subjects were informed about the purpose and procedures of the study, and written informed consent was obtained from all subjects. This study was approved by

university and lived together in a dormitory for the long‐distance running team. They regularly performed training twice per day (1 h in the early morning, 2 h in the afternoon) six days per week. The subjects were informed about the purpose and procedures of the study, and written informed consent was obtained from all subjects. This study was approved by the Ethics Committee for Human Experiments at the Ritsumeikan University (BKC‐IRB‐2014‐025), Japan. 2.2. Experimental Design To evaluate the influence of different training regimens on iron metabolism, blood drawings and dietary surveys were performed twice over a period of seven months (Figure 1): during a regular training period (LOW; middle of February 2015) and an intensified training period (INT; late of August 2015). We have compared iron metabolism between these periods, because running distances during LOW and INT were apparently different. Moreover, the training menu during each period was maintained over a month, and iron metabolism evaluated during each period was considered to be relatively stable at each time point. The subjects reported average monthly running distances. On the day of measurement, recent use of iron and other supplements and menstrual cycle status were noted. In addition to body composition, hematological parameters, iron and inflammatory parameters in blood and dietary intake were assessed. Body composition measurement and blood sampling were taken before breakfast, and all subjects did not consume any fluid prior to the measurements (6:30–8:00 a.m.). Figure 1. Experimental design during LOW and INT. 2.3. Measurement Procedures 2.3.1. Body Composition Body mass and fat mass were evaluated using a multi‐frequency impedance technique (InBody 720, Biospace, Seoul, Korea). Using a range of frequencies from 1 kHz to 1 MHz, the InBody 720 accurately measures the amount of body water and body composition, including fat, free fat, and skeletal muscle masses [23,24]. Subjects emptied their bladders prior to the measurements. Figure 1.Experimental design during LOW and INT. 2.3. Measurement Procedures 2.3.1. Body Composition Body mass and fat mass were evaluated using a multi-frequency impedance technique (InBody 720, Biospace, Seoul, Korea). Using a range of frequencies from 1 kHz to 1 MHz, the

composition, including fat, free fat, and skeletal muscle masses [23,24]. Subjects emptied their bladders prior to the measurements. Figure 1.Experimental design during LOW and INT. 2.3. Measurement Procedures 2.3.1. Body Composition Body mass and fat mass were evaluated using a multi-frequency impedance technique (InBody 720, Biospace, Seoul, Korea). Using a range of frequencies from 1 kHz to 1 MHz, the InBody 720 accurately measures the amount of body water and body composition, including fat, free fat, and skeletal muscle masses [23,24]. Subjects emptied their bladders prior to the measurements.

Nutrients2017,9, 277 3 of 9 2.3.2. Blood Sampling and Analysis Blood samples were collected following an overnight fast (6:30 p.m. to 8:00 a.m.). All blood samples were obtained via an antecubital vein while in a seated position. After drawing blood, serum and plasma samples were obtained after centrifugation (3000 rpm, 10 min, 4 C) and stored at 80 C until analyzed. The hematological parameters of blood hemoglobin (Hb) levels, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were measured at a clinical laboratory (Falco Holdings, Kyoto, Japan). Serum iron, ferritin, and total protein levels, total iron-binding capacity (TIBC), and creatine kinase (CK) levels were also evaluated at a clinical laboratory (SRL, Tokyo, Japan). Transferrin saturation (TSAT) was calculated as the serum iron level/serum total iron binding capacity 100 [25]. Plasma IL-6 and serum hepcidin level were determined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (R & D Systems Inc., Minneapolis, MN, USA). All samples for ELISA were analyzed in duplicate, and average values were determined. The interassay coef cients of variation were 1.4% (iron), 3.8% (ferritin), 1.3% (total protein), 2.0% (TIBC), 2.7% (CK), 2.3% (IL-6), and 3.1% (hepcidin). 2.3.3. Dietary Assessment Dietary surveys using three-day food diaries (3dFDs) along with verbal and written instructions for accurate recording of all foods and uids consumed were conducted. The subjects were also requested to take photos and weigh their plates before and after a meal. The 3dFD included two week days (training days) and one weekend day (non-training day) to account for variability over a week. Once the subjects submitted their 3dFDs, a dietitian checked the food records and con rmed the contents, clarifying speci c items and/or detailed information as necessary. Dietary analysis of the 3dFD was conducted using specially designed software (Eiyo-kun, Kenpaku-sha, Tokyo, Japan). Information about nutrient supplement use was also collected during recall visits. Nutrient intake from supplements (if any) was included in the present data. 2.3.4. Statistical Analyses All experimental data are shown as means standard deviation. Normal distributions for each variable were assessed using the Kolmogorov-Smirnov test. An independentt-test

the 3dFD was conducted using specially designed software (Eiyo-kun, Kenpaku-sha, Tokyo, Japan). Information about nutrient supplement use was also collected during recall visits. Nutrient intake from supplements (if any) was included in the present data. 2.3.4. Statistical Analyses All experimental data are shown as means standard deviation. Normal distributions for each variable were assessed using the Kolmogorov-Smirnov test. An independentt-test was used to compare variables between LOW and INT. The statistical analyses were performed using SPSS software (ver. 22.0; SPSS Inc., Chicago, IL, USA).p-values < 0.05 were considered to indicate statistical signi cance. 3. Results Monthly running distances were signi cantly longer during INT (622 94 km/month) than during LOW (499 106 km/month,p= 0.029). During each period, 0% (LOW) and 44% (INT) of the subjects used iron supplements. No subject used any other supplement during these periods. Regular menstrual cycles (28 2 days) were observed in 25% (LOW) and 19% (INT) of the subjects. Two subjects were under amenorrhea with irregular menstrual cycles. 3.1. Body Composition Body weight and lean body mass did not differ signi cantly between LOW and INT. The body fat amount and body fat percentage were signi cantly lower in INT (p= 0.015 and 0.023, respectively; Table).

Nutrients2017,9, 277 4 of 9 Table 1.Comparisons of physical characteristics over experiment period. Variables LOW INT p Height (cm) 160.5 3.9 - - Body weight (kg) 49.7 5.1 49.1 4.0 N.S. Lean body mass (kg) 41.4 3.4 41.7 2.0 N.S. Body fat (kg) 8.0 2.9 7.4 2.3 0.015 % Body fat (%) 15.8 4.9 15.0 4.3 0.023 Values are means SD. 3.2. Iron Status Assessment Comparisons of hematological variables between the two periods are shown in Table. Blood Hb, MCV, MCH, MCHC, serum ferritin, total protein, iron, TIBC, and TSAT did not differ signi cantly (p> 0.05 for all variables). Serum CK levels tended to be higher during INT (p= 0.09). Plasma IL-6 levels did not differ signi cantly between the two periods. None of subjects were regarded as iron-de cient with anemia. However, among the 16 subjects, 31% (LOW) and 37% (INT) of subjects were found to be iron-de cient (serum ferritin levels < 20 ng/mL) [26]. Table 2.Comparisons of blood variables over experiment period. Variables LOW INT p Hb (g/dL) 12.9 0.8 13.4 0.1 N.S. MCV ( m 3 ) 90.0 2.8 92.7 3.0 N.S. MCH (pg) 30.6 2.6 30.7 1.0 N.S. MCHC (g/dL) 32.1 0.7 33.1 0.7 N.S. Ferritin (ng/mL) 30.9 22.2 28.1 11.8 N.S. Total Protein (g/dL) 7.2 0.4 7.1 0.3 N.S. Iron ( g/dL) 55 24 65 8 N.S. TIBC ( g/dL) 340.7 44.6 323.0 6.9 N.S. TSAT (%) 16.4 7.5 20.1 2.4 N.S. CK (IU) 227 110 369 66 0.09 IL-6 (pg/mL) 0.35 0.23 0.33 0.23 N.S. Values are means SD. Abbreviations presents as follow; Hemoglobin (Hb), mean corpuscular volume (MVC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total iron-binding capacity (TIBC), transferrin saturation (TSAT), creatine kinase (CK), interleukin (IL). 3.3. Serum Hepcidin Levels Serum hepcidin levels were signi cantly higher during INT than during LOW (LOW 8.8 6.1 ng/mL, INT 16.3 6.5 ng/mL,p= 0.027; Figure). Several subjects revealed an irregular menstrual cycle, but it did not markedly affect serum hepcidin levels in either LOW or INT. A positive correlation was found between serum hepcidin and ferritin levels during both LOW (r=

Hepcidin Levels Serum hepcidin levels were signi cantly higher during INT than during LOW (LOW 8.8 6.1 ng/mL, INT 16.3 6.5 ng/mL,p= 0.027; Figure). Several subjects revealed an irregular menstrual cycle, but it did not markedly affect serum hepcidin levels in either LOW or INT. A positive correlation was found between serum hepcidin and ferritin levels during both LOW (r= 0.498,p= 0.049) and INT (r= 0.681,p= 0.027; Figure). When the in uence of the iron supplement on the serum hepcidin levels was determined, there was no signi cant difference in serum hepcidin levels between the iron-supplemented subjects and the iron–non-supplemented subjects (p= 0.10). 3.4. Energy and Macronutrient Intake Comparisons of energy and macronutrient intakes during the three days between the two periods are shown in Table. Carbohydrate and iron intakes from the daily diet were signi cantly higher during INT (p= 0.002 and 0.047, respectively). Iron intake was calculated, including iron supplements. Among the subjects with iron supplements, the average amount of the iron supplements was 9.5 3.0 mg (n= 7). The Energy intake tended to be higher during INT than LOW (p= 0.052). In contrast, protein and fat intakes were signi cantly lowered during INT (p= 0.002 and 0.010, respectively).

Nutrients2017,9, 277 5 of 9Nutrients 2017, 9, 277 5 of 9 (p = 0.052). In contrast, protein and fat intakes were significantly lowered during INT (p = 0.002 and 0.010, respectively). Figure 2. Comparison of serum hepcidin levels between LOW and INT. Values are means ± SD. * p < 0.05 between the periods. Figure 3. Correlation between serum hepcidin and ferritin levels. Table 3. Comparisons of energy and macronutrient intakes during the three‐day training period. Variables LOW INT p Energy (kcal) 2140 ± 130 2318 ± 343 0.052 (KJ) 8958 ± 544 9703 ± 1437 0.052 (kcal/BWkg) 44 ± 5 48 ± 4 0.034 Protein (g) 115.8 ± 9.7 103.5 ± 17.9 0.002 (g/BWkg) 2.4 ± 0.4 2.1 ± 0.5 <0.001 Fat (g) 64.2 ± 13.5 54.2 ± 0.6 0.010 Carbohydrate (g) 275.8 ± 31.2 353.0 ± 75.4 0.002 (g/BWkg) 5.6 ± 1.0 7.2 ± 1.5 <0.001 Iron (mg) 14.4 ± 1.6 17.3 ± 5.4 0.047 Vitamin C (mg) 228 ± 53 243 ± 94 N.S. Values are means ± SD. 4. Discussion The major finding in this study was that serum hepcidin levels were elevated significantly with an increase in the monthly running distance (INT) in female long‐distance runners. Several physiological mechanisms have been suggested to explain exercise‐induced impairment of iron status. In addition to the typically reported factors, excessive physical activity Figure 2. Comparison of serum hepcidin levels between LOW and INT. Values are means SD. *p< 0.05 between the periods.Nutrients 2017, 9, 277 5 of 9 (p = 0.052). In contrast, protein and fat intakes were significantly lowered during INT (p = 0.002 and 0.010, respectively). Figure 2. Comparison of serum hepcidin levels between LOW and INT. Values are means ± SD. * p < 0.05 between the periods. Figure 3. Correlation between serum hepcidin and ferritin levels. Table 3. Comparisons of energy and macronutrient intakes during the three‐day training period. Variables LOW INT p Energy (kcal) 2140 ± 130 2318 ± 343 0.052 (KJ) 8958 ± 544 9703 ± 1437 0.052 (kcal/BWkg) 44 ± 5 48 ± 4 0.034 Protein (g) 115.8 ± 9.7 103.5

between the periods. Figure 3. Correlation between serum hepcidin and ferritin levels. Table 3. Comparisons of energy and macronutrient intakes during the three‐day training period. Variables LOW INT p Energy (kcal) 2140 ± 130 2318 ± 343 0.052 (KJ) 8958 ± 544 9703 ± 1437 0.052 (kcal/BWkg) 44 ± 5 48 ± 4 0.034 Protein (g) 115.8 ± 9.7 103.5 ± 17.9 0.002 (g/BWkg) 2.4 ± 0.4 2.1 ± 0.5 <0.001 Fat (g) 64.2 ± 13.5 54.2 ± 0.6 0.010 Carbohydrate (g) 275.8 ± 31.2 353.0 ± 75.4 0.002 (g/BWkg) 5.6 ± 1.0 7.2 ± 1.5 <0.001 Iron (mg) 14.4 ± 1.6 17.3 ± 5.4 0.047 Vitamin C (mg) 228 ± 53 243 ± 94 N.S. Values are means ± SD. 4. Discussion The major finding in this study was that serum hepcidin levels were elevated significantly with an increase in the monthly running distance (INT) in female long‐distance runners. Several physiological mechanisms have been suggested to explain exercise‐induced impairment of iron status. In addition to the typically reported factors, excessive physical activity Figure 3.Correlation between serum hepcidin and ferritin levels. Table 3.Comparisons of energy and macronutrient intakes during the three-day training period. Variables LOW INT p Energy (kcal) 2140 130 2318 343 0.052 (KJ) 8958 544 9703 1437 0.052 (kcal/BWkg) 44 5 48 4 0.034 Protein (g) 115.8 9.7 103.5 17.9 0.002 (g/BWkg) 2.4 0.4 2.1 0.5 <0.001 Fat (g) 64.2 13.5 54.2 0.6 0.010 Carbohydrate (g) 275.8 31.2 353.0 75.4 0.002 (g/BWkg) 5.6 1.0 7.2 1.5 <0.001 Iron (mg) 14.4 1.6 17.3 5.4 0.047 Vitamin C (mg) 228 53 243 94 N.S. Values are means SD. 4. Discussion The major nding in this study was that serum hepcidin levels were elevated signi cantly with an increase in the monthly running distance (INT) in female long-distance runners. Several physiological mechanisms have been suggested to explain exercise-induced impairment of iron status. In addition to the typically reported factors, excessive physical activity may aggravate the iron status via increased hepcidin levels [12,27]. In the present study, we observed

This study compares iron status between two training periods in female long-distance runners.