Continuous training in young athletes decreases hepcidin secretion and is positively correlated with serum 25(OH)D and ferritin

Continuous training in young athletes decreases hepcidin secretion and is positively correlated with serum 25(OH)D and ferritin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Continuous training in young athletes decreases hepcidin secretion and is positively correlated with serum 25(OH)D and ferritin Yukiko Kobayashi, Rikako Taniguchi, Emiko Shirasaki, Yuko Yoshimoto-Segawa, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3849457/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Iron deficiency is known to impair muscle function and reduce athletic performance, while vitamin D has been reported to induce iron deficiency. However, the mechanism underlying exercise-induced changes in iron metabolism and the involvement of vitamins in this mechanism are unclear. Methods We examined changes in biological iron metabolism induced by continuous training and the effects of vitamin D on these changes. Diet, physical characteristics, and blood test data were collected from 23 female high school students in a dance club over a 2-month period of continuous training and rest periods. Results Serum hepcidin-25 levels were significantly lower during the training period than the rest period (p = 0.013), as were the red blood cell count, hemoglobin, and hematocrit (all p < 0.001). Serum erythropoietin was significantly higher (p = 0.001) during the training period. Significant positive correlations were observed between 25(OH)D levels and serum iron, serum ferritin, and transferrin saturation during the training period. Conclusion Multiple regression analysis with serum 25(OH)D level as the dependent variable and serum ferritin and iron levels as independent variables during the training period revealed a significant association with serum ferritin. Therefore, the relationship between serum 25(OH)D and iron in vivo may be closely related to metabolic changes induced by the exercise load. hepcidin vitamin D serum 25(OH)D ferritin young athletes continuous training Figures Figure 1 Highlights Young female athletes: two-month continuous training period and a rest period Decreases hepcidin secretion and significant association between serum 25(OH)D and serum ferritin in vivo The relationship between serum 25(OH)D and iron in vivo may be closely related to metabolic changes from the exercise load Introduction Anemia is a condition in which the amount of hemoglobin in the circulating blood decreases and the oxygen-carrying capacity of the blood is reduced, resulting in insufficient oxygen supply to the tissues throughout the body. Moreover, iron deficiency, with or without anemia, can impair muscle function, limit work capacity, and reduce adaptation to training and athletic performance [ 1 ]. Exercise-induced anemia (sports anemia) results from iron deficiency due to increased iron efflux and demand, including exercise-induced hemolysis, increased iron loss through sweat and urine, and increased skeletal muscle growth [ 2 – 4 ]. The prevalence of iron deficiency in athletes is higher than that in the general population. Iron deficiency is reported to be 25–35% in adolescent and adult women and 11–36% in men, with a wide range depending on the sport [ 5 ]. Further, a survey of 13–22-year-old participants in school sports clubs in Japan showed anemia in 16.5% of the participants [ 6 ]. Therefore, it is not difficult to imagine that many athletes, regardless of age, sex, sports discipline, or level of competition, have sports anemia. The mechanisms of iron metabolism have been rapidly elucidated in recent years, and the hepcidin-ferroportin system is understood to be the central regulatory mechanism of iron metabolism in vivo. Ferroportin (FPN) is a membrane protein that pumps iron out of cells, and hepcidin binds to FPN, which then internalizes and degrades the FPN export channels. Decreased FPN in intestinal epithelial cells and macrophages due to increased hepcidin levels inhibits iron absorption in the gastrointestinal tract and iron release from macrophages, and the amount of transferrin-bound iron available in vivo is regulated. Thus, hepcidin serves as an in vivo iron regulator [ 7 , 8 ]. Hepcidin secretion is thought to be regulated by various factors, including iron saturation signals via bone morphogenetic protein (BMP) and inflammatory signals via interleukin-6 (IL-6), which enhance hepcidin expression, whereas erythropoietic and hypoxic signals suppress hepcidin expression [ 9 ]. Increased inflammatory signaling was observed after exercise. For example, many reports show that IL-6 blood levels peak immediately after exercise, and muscle damage from prolonged exercise causes a sustained increase in IL-6, resulting in increased hepcidin secretion [ 10 , 11 ]. Same reports differ, and the mechanism underlying exercise-induced changes in iron metabolism remains inconclusive. Various relationships between iron metabolism and fat-soluble vitamins, particularly vitamin D, have recently been reported. Vitamin D is a fat-soluble prohormone that plays an important role in the endocrine and autocrine processes in vivo, mainly by maintaining adequate blood calcium and phosphate levels. Vitamin D is also involved in the body's inflammatory response through the activation and differentiation of immune and inflammatory cells and has recently been the subject of extensive research on muscle mass, strength, and function [ 12 , 13 ]. Furthermore, it has been reported that vitamin D is associated with the regulation of iron metabolism through its effect on the iron regulator hepcidin [ 14 ], suggesting that reduced vitamin D levels may consequently induce iron deficiency and anemia [ 15 ]. In contrast, a study on the relationship between vitamin D and iron in humans reported a high frequency of iron deficiency in various life stages, including vitamin D-deficient children, the elderly, young adults, and athletes [ 16 , 17 ]. Although iron deficiency is likely to occur during exercise due to increased iron efflux and demand, the mechanism underlying exercise-induced changes in iron metabolism is unknown, and it is unclear whether vitamins and other nutrients are involved in this mechanism. Therefore, in this study, we investigated young athletes during training and non-training periods and observed variations in biological iron metabolism under conditions of repeated training exercises, focusing on hepcidin, and further examined whether vitamin D had an effect. This is the first study to compare the relationship between iron metabolism and vitamin D with and without continuous training in the same adolescent participants, competition category, and practice details. Methods Ethical issues, including informed consent This study was conducted with the approval of the Kyoto Prefectural University Ethics Committee (No. 207). Participants were asked orally and in writing to participate in the study, and written informed consent was obtained from the participants. Informed consent was also obtained from the guardians of paticipants. Participants Twenty-six high school students from a competitive dance club were included in the analysis; of these, 23 were female (mean age 16.4 ± 0.5 years). Data collection The study was divided into two phases: a training phase (6.3 Mets/3 h of training/day, 7 days a week, approximately 2 months passed) and a rest phase (test period without extracurricular activities, approximately 2 weeks passed without training). Participants’ diet, energy consumption, physical characteristics, and blood biochemical tests were assessed. The survey was conducted in 2020 for both the training and rest periods. For the dietary survey, participants were asked to record the contents of their meals for 2 non-consecutive days and capture photographs (meal and measurement together) with a digital camera. From the collected recording forms and photographs, the daily intake of energy and nutrients (energy, protein, fat, carbohydrate, iron, and vitamin D) was calculated using dedicated nutrition calculation software (Excel Eiyoukun Ver. 9, Kenpakusha, Tokyo). For 2 days, on the same day as the dietary record, the paticipants were asked to record their activities at 5-min intervals from the time of waking to the time of going to bed. Exercise intensity and duration were calculated from activity records, referring to the Japanese version of physical activity codes and MET values in the “2011 Compendium” [ 18 ]. The exercise intensity was measured using an activity meter (HJA-750C Active Style Pro; Omron, Japan). The participant's daily energy expenditure was calculated using the following formula; daily energy expenditure (kcal/day) = body weight × 1.05 × Σ (exercise intensity METs × time). Height was measured using a stadiometer, and body weight, lean body mass, skeletal muscle mass, and body fat mass were measured using a impedance body composition analyzer (Inbody 270, Inbody Japan, Tokyo, Japan). Blood samples were drawn once during the training period and once during the rest period. During the training period, blood samples were collected in the morning before the start of training. During the rest period, blood samples were taken in the afternoon after regular examinations in the morning and eating lunch. Blood properties and biochemical tests, such as red blood cell (RBC) count, hemoglobin (Hb) level, hematocrit (Ht), reticulocyte count, and serum erythropoietin (EPO) level, were performed in a laboratory (Kyoto Microbio Laboratory, Kyoto, Japan). The RBC count and Ht were measured by sheath flow DC detection, Hb by the SLS hemoglobin method, reticulocytes by flow cytometry, and EPO by CLEIA. Serum hepcidin-25, iron levels, and unsaturated iron-binding capacity (UIBC) measurements, were commissioned (MC Plot Biotechnology, Kanazawa, Japan) using several methods. Serum hepcidin-25 levels were measured by liquid chromatography with a tandem mass spectrometry assay system (4000 QTRAP; Applied Biosystems, MA, US). Liquid chromatography with tandem mass spectrometry assay system comprised LC (Prominence LC20-ADvp, Shimadzu, Kyoto, Japan); 2.1×150 mm, ZORBAX 300 SB-C8 column (Agilent Technologies, CA, US), and flow velocity of 300 mL/min. Serum iron and UIBC were measured using the nitrosoPSAP method. Total iron-binding capacity (TIBC) and serum transferrin saturation were calculated as follows: TIBC = serum iron + UIBC; serum transferrin saturation = serum iron/TIBC×100. Serum IL-6 levels were measured using an enzyme-linked immunosorbent assay kit (KE10007; Protein Tech Japan, Tokyo, Japan). Serum 25-hydroxyvitamin D (25(OH)D) levels were measured using enzyme-linked immunosorbent assay kits (25-OH Vitamin D Total ELISA Kit, KA6138, Abnova, Taipei, Taiwan). Statistical analyses The Shapiro–Wilk test was used to test for normality. For comparisons between the training and rest periods, a paired t-test was used for normally distributed data and a Wilcoxon signed-rank sum test for non-normally distributed data. A single correlation analysis was performed using Spearman’s rank correlation coefficient. A multiple regression analysis was performed using the forced entry method. IBM SPSS Statistics 25 (Armonk, NY, US) was used for all statistical analyses. The significance level for all analyses was 5%. Results Physical characteristics of the participants The physical characteristics of the participants are shown in Table 1. The percentage of participants with a BMI less than 18.5 was 35% in the training phase and 26% in the rest phase. BMI (p=0.009) and body fat mass (p<0.001) were significantly lower during the training phase than during the rest phase, whereas lean body mass and skeletal muscle mass were significantly higher (both p<0.001). Energy and nutrients intake Table 1 shows the nutrient and other intakes of the participants. There was no significant difference in energy intake between the two groups; however, intake/consumption was significantly lower during the training period than during the rest period (p<0.001), with 87% of the participants having an energy intake less than their consumption. Carbohydrate intake was higher during the training period (p=0.057) and fat intake was significantly lower (p<0.05) than during the rest period. Vitamin D and iron intakes were not significantly different between the two periods. Blood and biochemical test data The participants’ blood properties and biochemical data of the participants are shown in Table 1 and Figure 1. The erythrocyte count, hemoglobin concentration, and hematocrit values were significantly lower during the training period than during the rest period (all p<0.001). The reticulocyte count was not significantly different between the two periods but was slightly higher during the training period, and the hematopoietic factor EPO was significantly higher during the training period than during the rest period (p=0.001). Serum iron and transferrin saturation did not differ significantly between the two periods, but the median values tended to be lower and varied more during the training period. TIBC was significantly lower during the training period than during the rest period. Ferritin levels were not significantly different between the two periods. Serum hepcidin-25 levels were significantly lower during the training period than those during the rest period (p=0.013). Serum 25(OH)D levels were not significantly different between the two periods, and IL-6 was detected in only three participants in both groups, while it was below the detection limit in the remaining 20 participants. When iron-deficiency anemia was defined as a hemoglobin concentration less than 12 g/dl and serum ferritin less than 12 ng/dl, two (8.7%) were applicable in the training period, but none were applicable in the rest period. Correlations between serum 25(OH)D and iron metabolism factors during the training period Correlations between blood cell parameters, iron metabolism-related parameters, physical characteristics, dietary factors, and serum 25(OH)D are shown in Table 2. Significant positive correlations were found between the lipid energy ratio (r=0.470, p=0.024) and dietary vitamin D intake (r=0.432, p=0.040) during the rest period. However, no significant correlations were observed with blood cells or iron metabolism-related items. In contrast, significant positive correlations were observed between serum 25(OH)D levels and serum iron (r=0.447, p=0.032), ferritin (r=0.520, p=0.011), and transferrin saturation (r=0.554, p=0.006) levels during the training phase. Therefore, we performed a multiple regression analysis to determine which factors were more strongly associated with serum 25(OH)D levels. A preliminary observation of the correlation between serum iron, ferritin, and transferrin saturation showed that the correlation between serum iron and transferrin saturation was r>0.8, and that between serum iron and ferritin was r<0.8; no issues were identified with multicollinearity. Therefore, multiple regression analysis was conducted using the forced entry method with serum iron and ferritin as bivariate independent variables and 25(OH)D as the dependent variable. The results in Table 3 show that the association between serum 25(OH)D and ferritin was stronger and more significant than that between serum iron and ferritin. The variance inflation factor was less than 10, and there were no problems with multicollinearity. Discussion This study aimed to clarify the changes in biological iron metabolism due to continuous training in young athletes by comparing the status of the same participants with and without training and examining the effect of vitamin D on these changes. The results showed that hemolysis and erythropoiesis were enhanced during the training phase, and serum levels of hepcidin, a regulator of iron metabolism, were altered. The correlation between serum 25(OH)D levels and iron metabolism factors was observed only in the training phase, with ferritin showing the strongest and most significant relationship. Therefore, based on the present results, we discuss how continuous training affects blood cell characteristics and iron metabolism and whether vitamin D is involved. First, we focused on hepcidin, a regulator of iron metabolism. Serum hepcidin-25 levels were significantly lower during the training period than during the rest period. Hepcidin is a regulator of iron metabolism released from the liver, and the suppression of hepcidin expression increases iron absorption from the gastrointestinal tract and tissue release. Factors that regulate hepcidin expression include (1) iron stores in the body, (2) acute or chronic inflammation, and (3) increased red blood cell production [ 9 ]. Serum ferritin levels, an indicator of body iron stores, were not significantly different between the two periods, suggesting that their effects on hepcidin expression were small. In the present study, serum iron, transferrin saturation, and ferritin levels were not significantly different between the two periods. However, the median serum iron and transferrin saturation during the training period were lower than during the rest period, and the data distribution was wider. Lower hepcidin levels in the training phase may have increased iron absorption from the gastrointestinal tract and iron release from macrophages, thereby preventing iron deficiency. TIBC during the training period was significantly lower than that during the rest period to lower serum iron and apo-transferrin levels, suggesting that transferrin expression does not increase because the organism does not recognize it as being iron-deficient. Second, IL-6, one of the indicators of acute or chronic inflammation, depends on the signal transducer and activator of the transcription 3 (STAT3) signaling pathway to promote hepcidin expression. IL-6 is a cytokine produced by the liver, muscle, and adipocytes. It can be muscle-derived, which increases immediately after exercise, or immune cell-derived, which increases several hours after exercise in response to cell injury or infection [ 19 ]. IL-6 levels increase immediately after exercise and return to baseline 3–6 h later [ 20 ]. In the present study, IL-6 was detectable in both the training and rest periods in three of the 23 parrticipants, and the average of these three participants was similar in both periods. Thus, the promotion of hepcidin expression by IL-6 production may not occur during continuous training. Third, the RBC, Hb, and Ht levels were significantly lower during the training period than during the rest period. Ferritin level, serum iron level, and transferrin saturation, which are indicators of body iron content, were not significantly different between the two groups, suggesting that iron deficiency did not occur. Although not shown in the results, haptoglobin levels in all participants were lower during the training period than during the rest period for all types, suggesting that hemolysis may have occurred due to continuous training over the 2 months. The reticulocyte count and serum EPO concentration were higher during the training phase than during the resting phase. Reticular erythrocytes are an indicator of increased erythropoiesis in the bone marrow immediately after the maturation and denucleation of erythroblasts [ 21 ]. EPO, a regulator of erythropoiesis, enhances hematopoiesis by acting on erythroid progenitor cells and erythroblasts before Hb synthesis [ 22 ]. Therefore, it can be inferred that hemolysis and erythropoiesis are converted into hyperproduction during the training phase, which may suppress hepcidin expression. Thus, continuous training suppressed hepcidin expression, which may be partly due to the increased erythropoiesis caused by hemolysis. Next, to examine the influence of vitamin D on exercise, we observed correlations between serum 25(OH)D levels and blood properties, physical characteristics, iron metabolism-related items, and nutrient intake during the training and rest periods. Significant positive correlations were observed between serum iron, ferritin, and transferrin saturation levels during the training period. However, no correlation was observed between these items during the rest period, suggesting that this phenomenon occurred only during the training period. Furthermore, multiple regression analysis with serum 25(OH)D as the dependent variable and serum iron and ferritin levels as the independent variables during the training period showed that ferritin levels were significantly associated with serum 25(OH)D levels. The relationship between serum 25(OH)D and serum ferritin levels is debatable with only a few reports. For example, a significant positive correlation was observed between serum 25(OH)D and ferritin in adolescents [ 23 ]. Serum 25(OH)D levels are inversely correlated with serum ferritin levels in males but positively correlated in premenopausal women [ 24 ]; however, consistent results have not been obtained [ 25 ]. Only a few reports have mentioned the effects of exercise load. In vitro validation has shown that iron deficiency reduces the activity of heme-containing vitamin D-activating enzymes, such as 25- and 1α-hydroxylase [ 26 ], and induces vitamin D deficiency by promoting the transcription of fibroblast growth factor 23 (FGF23), which inhibits vitamin D 1α-hydroxylation [ 27 ]. Iron deficiency has also been recently reported to affect hydroxylase activity by decreasing iron levels in the liver and kidneys and reducing serum 25(OH)D3 and 1,25(OH)2D levels [ 28 ]. However, some studies have shown that high doses of vitamin D 3 (VD 3 ) reduce hepcidin levels [ 29 ]. In vitro experiments have reported that the 1,25(OH)2D -vitamin D receptor complex binds to the vitamin D response element (VDRE) on the hepcidin gene (HAMP) and represses its transcription [ 14 ], suggesting a negative feedback-like regulatory mechanism by which VD3 increases body iron via hepcidin repression. Futhermore, a notable phenomenon in this study was that serum 25(OH)D was significantly positively correlated with dietary vitamin D intake and the lipid energy ratio during the resting period; however, this correlation was lost during the training period. These findings suggest a change in VD 3 consumption and metabolism during exercise. This may be due to the effects of FGF23 and the parathyroid hormone PTH. FGF23 is reportedly stimulated by exercise for secretion [ 30 ]. FGF23 has been implicated in vitamin D activity and negatively regulates erythropoiesis and iron metabolism [ 31 ]. High concentrations of FGF23 have been reported to suppress 1α-hydroxylase activity, resulting in lower 1,25(OH)2D concentrations [ 27 ]. PTH is known to increase in an exercise intensity- and time-dependent manner [ 32 ], and its concentration is inversely correlated with the serum 25(OH)D concentration [ 33 ]. A limitation of this study is that it is an observational study with a small number of participants. However, the fact that metabolic changes could be observed in the same participants with and without exercise is uniquely valuable data. Moreover, as the dietary and physical activity surveys are self-reported responses by the participants, the possibility of psychological selection bias and differences from the actual content cannot be ruled out. The influence of the food environment, menstruation, physical development rate, and food preferences was not investigated; therefore, their relevance to anemia is unknown. As the present study did not examine different sexes and age groups, further studies are needed. Conclusions In conclusion, the present study examined the changes in biological iron metabolism during continuous training load and the effects of vitamin D on these changes, suggesting that continuous training enhances hemolysis and erythropoiesis and may be a factor in the suppression of hepcidin expression. Multiple regression analysis revealed a significant relationship between serum 25(OH)D and ferritin levels only during the training period, suggesting that the relationship between 25(OH)D and iron levels may be closely related to metabolic changes in the body due to exercise load. In the future, we would like to focus on the relationship between iron and vitamin D during exercise, including the observation of iron metabolic changes with vitamin D supplementation, to clarify the role of iron and vitamin D in maintaining athletes' health. Declarations Acknowledgements We would like to thank Hiroshi Kawabata, M.D., Ph.D., Kyoto National Hospital Organization Kyoto Medical Center, for supporting this research. We would like to thank the director of the high school dance club, Daishi Kobayashi, and the members of the club for their cooperation in conducting this study. Funding This work was supported by the JSPS KAKENHI Grant-in-Aid for Scientific Research (C) (grant number 19K11695), Japan Society for the Promotion of Science. Competing Interests All authors have no conflicts of interest. Informed Consent Statement Informed consent was obtained from all participants involved in the study. Data Availability Statement All data will be available on request to the corresponding author. Author Contribution Statement YK, RT and ES conceived and designed research. YK, RT and ES conducted experiments. YK, RT, ES and YYS analyzed and interpreted the data. YK wrote the manuscript with input from other authors. 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J Clin Endocrinol Metab. 2001 Mar;86(3):1212-21. doi: 10.1210/jcem.86.3.7327. Tables Tables 1 to 3 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3849457","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266466002,"identity":"7fe00fd1-f157-436d-ac01-dd48919d4cd1","order_by":0,"name":"Yukiko Kobayashi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYFACNoYDDEDEzwPlMzYQq0WyhxQtDCAtBmeIdZbBjbTEAz8q7sgbnzl7TIKhxo6BeTYBa4BaDhzsOfPMcNvZvjQJhmPJDIxzDhDSkt5wmLHtMOO28zxmEgxsBxgYZyQQp8V+cz9Iyz+itKQdAGlJ3MDbYybB2EaEFskzzxJAfkmeceZcskViXzIPQb/wHU8z/gAMMdv+ntyDNz58s5MzJBRiCggjgdEPdBKP4Qz8OhjkEUZCU4y8BAEto2AUjIJRMOIAAGhqTMqdYUNkAAAAAElFTkSuQmCC","orcid":"","institution":"Kyoto Prefectural University","correspondingAuthor":true,"prefix":"","firstName":"Yukiko","middleName":"","lastName":"Kobayashi","suffix":""},{"id":266466003,"identity":"133b12fe-ce94-416c-906f-b69b4343eb90","order_by":1,"name":"Rikako Taniguchi","email":"","orcid":"","institution":"Kyoto Prefectural University","correspondingAuthor":false,"prefix":"","firstName":"Rikako","middleName":"","lastName":"Taniguchi","suffix":""},{"id":266466004,"identity":"2e6c4109-bde8-40c6-aecd-411ac680d58f","order_by":2,"name":"Emiko Shirasaki","email":"","orcid":"","institution":"Kyoto Prefectural University","correspondingAuthor":false,"prefix":"","firstName":"Emiko","middleName":"","lastName":"Shirasaki","suffix":""},{"id":266466005,"identity":"31071cde-551e-433b-a02a-d5bf35eb7b55","order_by":3,"name":"Yuko Yoshimoto-Segawa","email":"","orcid":"","institution":"Kyoto Prefectural University","correspondingAuthor":false,"prefix":"","firstName":"Yuko","middleName":"","lastName":"Yoshimoto-Segawa","suffix":""},{"id":266466006,"identity":"069c3cca-d5d4-4d0a-9a51-df7f1660237a","order_by":4,"name":"Wataru Aoi","email":"","orcid":"","institution":"Kyoto Prefectural University","correspondingAuthor":false,"prefix":"","firstName":"Wataru","middleName":"","lastName":"Aoi","suffix":""},{"id":266466007,"identity":"74c19a07-1a40-41eb-b4f3-37eade7c3f08","order_by":5,"name":"Masashi Kuwahata","email":"","orcid":"","institution":"Kyoto Prefectural University","correspondingAuthor":false,"prefix":"","firstName":"Masashi","middleName":"","lastName":"Kuwahata","suffix":""}],"badges":[],"createdAt":"2024-01-10 04:59:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3849457/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3849457/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49631726,"identity":"1834d508-9d11-4d00-8adf-af9974ca4238","added_by":"auto","created_at":"2024-01-15 15:48:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38904,"visible":true,"origin":"","legend":"\u003cp\u003eThe blood properties and biochemical data of the participants during training and resting periods. Dark gray boxes indicate training period; light gray boxes, resting period. All groups are n=23. Paired t-test or Wilcoxson signed-rank test. The center line of the boxplot indicates the median, the crosses indicate the mean, the top of the box indicates the 75th percentile, and the bottom of the box indicates the 25th percentile. RBC, Red blood cell count; Hb, Hemoglobin; Ht, Hematocrit; EPO, erythropoietin; Tf-Sat, Transferrin saturation; TIBC, Total iron-binding capacity.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3849457/v1/20b4a31ac961ade7b4100572.png"},{"id":49706402,"identity":"2b43af82-9694-46dd-a92f-90ed833b9c84","added_by":"auto","created_at":"2024-01-16 18:22:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":352986,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3849457/v1/9975c32e-0dac-4ec8-b991-5276612242c0.pdf"},{"id":49631727,"identity":"b128df80-917c-4845-9023-cbcced7c9b8e","added_by":"auto","created_at":"2024-01-15 15:48:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":360209,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3849457/v1/aaf669fde8c1552d30693269.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Continuous training in young athletes decreases hepcidin secretion and is positively correlated with serum 25(OH)D and ferritin","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eYoung female athletes: two-month continuous training period and a rest period\u003c/li\u003e\n \u003cli\u003eDecreases hepcidin secretion and\u0026nbsp;significant association between\u0026nbsp;serum 25(OH)D and\u0026nbsp;serum ferritin in vivo\u003c/li\u003e\n \u003cli\u003eThe relationship between serum 25(OH)D and iron in vivo may be closely related to metabolic changes from the exercise load\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eAnemia is a condition in which the amount of hemoglobin in the circulating blood decreases and the oxygen-carrying capacity of the blood is reduced, resulting in insufficient oxygen supply to the tissues throughout the body. Moreover, iron deficiency, with or without anemia, can impair muscle function, limit work capacity, and reduce adaptation to training and athletic performance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Exercise-induced anemia (sports anemia) results from iron deficiency due to increased iron efflux and demand, including exercise-induced hemolysis, increased iron loss through sweat and urine, and increased skeletal muscle growth [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The prevalence of iron deficiency in athletes is higher than that in the general population. Iron deficiency is reported to be 25\u0026ndash;35% in adolescent and adult women and 11\u0026ndash;36% in men, with a wide range depending on the sport [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Further, a survey of 13\u0026ndash;22-year-old participants in school sports clubs in Japan showed anemia in 16.5% of the participants [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, it is not difficult to imagine that many athletes, regardless of age, sex, sports discipline, or level of competition, have sports anemia.\u003c/p\u003e \u003cp\u003eThe mechanisms of iron metabolism have been rapidly elucidated in recent years, and the hepcidin-ferroportin system is understood to be the central regulatory mechanism of iron metabolism in vivo. Ferroportin (FPN) is a membrane protein that pumps iron out of cells, and hepcidin binds to FPN, which then internalizes and degrades the FPN export channels. Decreased FPN in intestinal epithelial cells and macrophages due to increased hepcidin levels inhibits iron absorption in the gastrointestinal tract and iron release from macrophages, and the amount of transferrin-bound iron available in vivo is regulated. Thus, hepcidin serves as an in vivo iron regulator [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHepcidin secretion is thought to be regulated by various factors, including iron saturation signals via bone morphogenetic protein (BMP) and inflammatory signals via interleukin-6 (IL-6), which enhance hepcidin expression, whereas erythropoietic and hypoxic signals suppress hepcidin expression [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Increased inflammatory signaling was observed after exercise. For example, many reports show that IL-6 blood levels peak immediately after exercise, and muscle damage from prolonged exercise causes a sustained increase in IL-6, resulting in increased hepcidin secretion [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Same reports differ, and the mechanism underlying exercise-induced changes in iron metabolism remains inconclusive.\u003c/p\u003e \u003cp\u003eVarious relationships between iron metabolism and fat-soluble vitamins, particularly vitamin D, have recently been reported. Vitamin D is a fat-soluble prohormone that plays an important role in the endocrine and autocrine processes in vivo, mainly by maintaining adequate blood calcium and phosphate levels. Vitamin D is also involved in the body's inflammatory response through the activation and differentiation of immune and inflammatory cells and has recently been the subject of extensive research on muscle mass, strength, and function [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, it has been reported that vitamin D is associated with the regulation of iron metabolism through its effect on the iron regulator hepcidin [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], suggesting that reduced vitamin D levels may consequently induce iron deficiency and anemia [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In contrast, a study on the relationship between vitamin D and iron in humans reported a high frequency of iron deficiency in various life stages, including vitamin D-deficient children, the elderly, young adults, and athletes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough iron deficiency is likely to occur during exercise due to increased iron efflux and demand, the mechanism underlying exercise-induced changes in iron metabolism is unknown, and it is unclear whether vitamins and other nutrients are involved in this mechanism. Therefore, in this study, we investigated young athletes during training and non-training periods and observed variations in biological iron metabolism under conditions of repeated training exercises, focusing on hepcidin, and further examined whether vitamin D had an effect. This is the first study to compare the relationship between iron metabolism and vitamin D with and without continuous training in the same adolescent participants, competition category, and practice details.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthical issues, including informed consent\u003c/h2\u003e \u003cp\u003e This study was conducted with the approval of the Kyoto Prefectural University Ethics Committee (No. 207). Participants were asked orally and in writing to participate in the study, and written informed consent was obtained from the participants. Informed consent was also obtained from the guardians of paticipants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eTwenty-six high school students from a competitive dance club were included in the analysis; of these, 23 were female (mean age 16.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 years).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData collection\u003c/h2\u003e \u003cp\u003eThe study was divided into two phases: a training phase (6.3 Mets/3 h of training/day, 7 days a week, approximately 2 months passed) and a rest phase (test period without extracurricular activities, approximately 2 weeks passed without training). Participants\u0026rsquo; diet, energy consumption, physical characteristics, and blood biochemical tests were assessed. The survey was conducted in 2020 for both the training and rest periods.\u003c/p\u003e \u003cp\u003eFor the dietary survey, participants were asked to record the contents of their meals for 2 non-consecutive days and capture photographs (meal and measurement together) with a digital camera. From the collected recording forms and photographs, the daily intake of energy and nutrients (energy, protein, fat, carbohydrate, iron, and vitamin D) was calculated using dedicated nutrition calculation software (Excel Eiyoukun Ver. 9, Kenpakusha, Tokyo).\u003c/p\u003e \u003cp\u003eFor 2 days, on the same day as the dietary record, the paticipants were asked to record their activities at 5-min intervals from the time of waking to the time of going to bed. Exercise intensity and duration were calculated from activity records, referring to the Japanese version of physical activity codes and MET values in the \u0026ldquo;2011 Compendium\u0026rdquo; [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The exercise intensity was measured using an activity meter (HJA-750C Active Style Pro; Omron, Japan). The participant's daily energy expenditure was calculated using the following formula; daily energy expenditure (kcal/day)\u0026thinsp;=\u0026thinsp;body weight \u0026times; 1.05\u0026thinsp;\u0026times;\u0026thinsp;Σ (exercise intensity METs \u0026times; time).\u003c/p\u003e \u003cp\u003eHeight was measured using a stadiometer, and body weight, lean body mass, skeletal muscle mass, and body fat mass were measured using a impedance body composition analyzer (Inbody 270, Inbody Japan, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eBlood samples were drawn once during the training period and once during the rest period. During the training period, blood samples were collected in the morning before the start of training. During the rest period, blood samples were taken in the afternoon after regular examinations in the morning and eating lunch. Blood properties and biochemical tests, such as red blood cell (RBC) count, hemoglobin (Hb) level, hematocrit (Ht), reticulocyte count, and serum erythropoietin (EPO) level, were performed in a laboratory (Kyoto Microbio Laboratory, Kyoto, Japan). The RBC count and Ht were measured by sheath flow DC detection, Hb by the SLS hemoglobin method, reticulocytes by flow cytometry, and EPO by CLEIA. Serum hepcidin-25, iron levels, and unsaturated iron-binding capacity (UIBC) measurements, were commissioned (MC Plot Biotechnology, Kanazawa, Japan) using several methods. Serum hepcidin-25 levels were measured by liquid chromatography with a tandem mass spectrometry assay system (4000 QTRAP; Applied Biosystems, MA, US). Liquid chromatography with tandem mass spectrometry assay system comprised LC (Prominence LC20-ADvp, Shimadzu, Kyoto, Japan); 2.1\u0026times;150 mm, ZORBAX 300 SB-C8 column (Agilent Technologies, CA, US), and flow velocity of 300 mL/min. Serum iron and UIBC were measured using the nitrosoPSAP method. Total iron-binding capacity (TIBC) and serum transferrin saturation were calculated as follows: TIBC\u0026thinsp;=\u0026thinsp;serum iron\u0026thinsp;+\u0026thinsp;UIBC; serum transferrin saturation\u0026thinsp;=\u0026thinsp;serum iron/TIBC\u0026times;100. Serum IL-6 levels were measured using an enzyme-linked immunosorbent assay kit (KE10007; Protein Tech Japan, Tokyo, Japan). Serum 25-hydroxyvitamin D (25(OH)D) levels were measured using enzyme-linked immunosorbent assay kits (25-OH Vitamin D Total ELISA Kit, KA6138, Abnova, Taipei, Taiwan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eThe Shapiro\u0026ndash;Wilk test was used to test for normality. For comparisons between the training and rest periods, a paired t-test was used for normally distributed data and a Wilcoxon signed-rank sum test for non-normally distributed data. A single correlation analysis was performed using Spearman\u0026rsquo;s rank correlation coefficient. A multiple regression analysis was performed using the forced entry method. IBM SPSS Statistics 25 (Armonk, NY, US) was used for all statistical analyses. The significance level for all analyses was 5%.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhysical characteristics of the participants\u003c/em\u003e\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The physical characteristics of the participants are shown in Table 1. The percentage of participants with\u0026nbsp;a\u0026nbsp;BMI less than 18.5 was 35% in the\u0026nbsp;training\u0026nbsp;phase and 26% in the rest phase. BMI (p=0.009) and body fat mass (p\u0026lt;0.001) were significantly lower during the\u0026nbsp;training\u0026nbsp;phase than during the rest phase, whereas lean body mass and skeletal muscle mass were significantly higher (both p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEnergy and nutrients intake\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 1 shows the nutrient and other intakes of the participants. There was no significant difference in energy intake between the two groups; however, intake/consumption was significantly lower during the training period than during the rest period (p\u0026lt;0.001), with 87% of the participants having\u0026nbsp;an energy intake less than their consumption.\u0026nbsp;Carbohydrate intake was higher during the training period (p=0.057) and fat intake was significantly lower (p\u0026lt;0.05) than during the rest period. Vitamin D and iron intakes were not significantly different between the two periods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBlood and biochemical test data\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe participants’ blood properties and biochemical data of the participants are shown in Table 1 and Figure 1. The erythrocyte count, hemoglobin concentration, and hematocrit values were significantly lower\u0026nbsp;during the training period than during the rest period (all p\u0026lt;0.001). The reticulocyte count was not significantly different between the two periods\u0026nbsp;but was slightly higher\u0026nbsp;during the training period, and the hematopoietic factor EPO was significantly higher during the training period than during the rest period (p=0.001). Serum iron and transferrin saturation did not differ significantly between the two periods, but the median values tended to be lower and varied more during the training period. TIBC was significantly lower during the training period than during the rest period. Ferritin levels were not significantly different between the two periods. Serum hepcidin-25 levels were significantly lower\u0026nbsp;during the training period than those during the rest period (p=0.013). Serum 25(OH)D\u0026nbsp;levels were not significantly different between the two periods, and IL-6 was detected in only three participants in both groups, while it was below the detection limit in the remaining 20 participants. When iron-deficiency anemia was defined as\u0026nbsp;a\u0026nbsp;hemoglobin concentration less than 12\u0026nbsp;g/dl and serum ferritin less than 12 ng/dl, two (8.7%) were applicable in the training period, but none\u0026nbsp;were applicable in the rest period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCorrelations between serum 25(OH)D and iron metabolism factors during the training period\u003c/em\u003e\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Correlations between blood cell parameters, iron metabolism-related parameters, physical characteristics, dietary factors, and serum 25(OH)D are shown in Table 2. Significant positive correlations were found between\u0026nbsp;the\u0026nbsp;lipid energy ratio (r=0.470, p=0.024) and dietary vitamin D intake (r=0.432, p=0.040) during the rest period. However, no significant correlations were observed with blood cells or iron metabolism-related items. In contrast, significant positive correlations\u0026nbsp;were observed between serum 25(OH)D\u0026nbsp;levels and serum iron (r=0.447, p=0.032), ferritin (r=0.520, p=0.011), and transferrin saturation (r=0.554, p=0.006) levels during the training phase. Therefore, we performed a multiple regression analysis to determine which factors were more strongly associated with serum 25(OH)D levels.\u0026nbsp;A preliminary observation of the correlation between serum iron, ferritin, and transferrin saturation showed that the correlation between serum iron and transferrin saturation was r\u0026gt;0.8, and that between serum iron and ferritin was r\u0026lt;0.8; no issues were identified with multicollinearity. Therefore, multiple regression analysis was conducted using the forced entry method with serum iron and ferritin as bivariate independent variables and 25(OH)D as\u0026nbsp;the dependent variable. The results in Table 3 show that the association between serum 25(OH)D and ferritin was stronger and more significant than\u0026nbsp;that between serum iron and ferritin. The variance inflation factor was less than 10, and there were no problems with multicollinearity.\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study aimed to clarify the changes in biological iron metabolism due to continuous training in young athletes by comparing the status of the same participants with and without training and examining the effect of vitamin D on these changes. The results showed that hemolysis and erythropoiesis were enhanced during the training phase, and serum levels of hepcidin, a regulator of iron metabolism, were altered. The correlation between serum 25(OH)D levels and iron metabolism factors was observed only in the training phase, with ferritin showing the strongest and most significant relationship. Therefore, based on the present results, we discuss how continuous training affects blood cell characteristics and iron metabolism and whether vitamin D is involved.\u003c/p\u003e \u003cp\u003eFirst, we focused on hepcidin, a regulator of iron metabolism. Serum hepcidin-25 levels were significantly lower during the training period than during the rest period. Hepcidin is a regulator of iron metabolism released from the liver, and the suppression of hepcidin expression increases iron absorption from the gastrointestinal tract and tissue release. Factors that regulate hepcidin expression include (1) iron stores in the body, (2) acute or chronic inflammation, and (3) increased red blood cell production [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSerum ferritin levels, an indicator of body iron stores, were not significantly different between the two periods, suggesting that their effects on hepcidin expression were small. In the present study, serum iron, transferrin saturation, and ferritin levels were not significantly different between the two periods. However, the median serum iron and transferrin saturation during the training period were lower than during the rest period, and the data distribution was wider. Lower hepcidin levels in the training phase may have increased iron absorption from the gastrointestinal tract and iron release from macrophages, thereby preventing iron deficiency. TIBC during the training period was significantly lower than that during the rest period to lower serum iron and apo-transferrin levels, suggesting that transferrin expression does not increase because the organism does not recognize it as being iron-deficient.\u003c/p\u003e \u003cp\u003eSecond, IL-6, one of the indicators of acute or chronic inflammation, depends on the signal transducer and activator of the transcription 3 (STAT3) signaling pathway to promote hepcidin expression. IL-6 is a cytokine produced by the liver, muscle, and adipocytes. It can be muscle-derived, which increases immediately after exercise, or immune cell-derived, which increases several hours after exercise in response to cell injury or infection [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. IL-6 levels increase immediately after exercise and return to baseline 3\u0026ndash;6 h later [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the present study, IL-6 was detectable in both the training and rest periods in three of the 23 parrticipants, and the average of these three participants was similar in both periods. Thus, the promotion of hepcidin expression by IL-6 production may not occur during continuous training.\u003c/p\u003e \u003cp\u003eThird, the RBC, Hb, and Ht levels were significantly lower during the training period than during the rest period. Ferritin level, serum iron level, and transferrin saturation, which are indicators of body iron content, were not significantly different between the two groups, suggesting that iron deficiency did not occur. Although not shown in the results, haptoglobin levels in all participants were lower during the training period than during the rest period for all types, suggesting that hemolysis may have occurred due to continuous training over the 2 months. The reticulocyte count and serum EPO concentration were higher during the training phase than during the resting phase. Reticular erythrocytes are an indicator of increased erythropoiesis in the bone marrow immediately after the maturation and denucleation of erythroblasts [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. EPO, a regulator of erythropoiesis, enhances hematopoiesis by acting on erythroid progenitor cells and erythroblasts before Hb synthesis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, it can be inferred that hemolysis and erythropoiesis are converted into hyperproduction during the training phase, which may suppress hepcidin expression. Thus, continuous training suppressed hepcidin expression, which may be partly due to the increased erythropoiesis caused by hemolysis.\u003c/p\u003e \u003cp\u003eNext, to examine the influence of vitamin D on exercise, we observed correlations between serum 25(OH)D levels and blood properties, physical characteristics, iron metabolism-related items, and nutrient intake during the training and rest periods. Significant positive correlations were observed between serum iron, ferritin, and transferrin saturation levels during the training period. However, no correlation was observed between these items during the rest period, suggesting that this phenomenon occurred only during the training period.\u003c/p\u003e \u003cp\u003eFurthermore, multiple regression analysis with serum 25(OH)D as the dependent variable and serum iron and ferritin levels as the independent variables during the training period showed that ferritin levels were significantly associated with serum 25(OH)D levels. The relationship between serum 25(OH)D and serum ferritin levels is debatable with only a few reports. For example, a significant positive correlation was observed between serum 25(OH)D and ferritin in adolescents [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Serum 25(OH)D levels are inversely correlated with serum ferritin levels in males but positively correlated in premenopausal women [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]; however, consistent results have not been obtained [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOnly a few reports have mentioned the effects of exercise load. In vitro validation has shown that iron deficiency reduces the activity of heme-containing vitamin D-activating enzymes, such as 25- and 1α-hydroxylase [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and induces vitamin D deficiency by promoting the transcription of fibroblast growth factor 23 (FGF23), which inhibits vitamin D 1α-hydroxylation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Iron deficiency has also been recently reported to affect hydroxylase activity by decreasing iron levels in the liver and kidneys and reducing serum 25(OH)D3 and 1,25(OH)2D levels [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, some studies have shown that high doses of vitamin D\u003csub\u003e3\u003c/sub\u003e (VD\u003csub\u003e3\u003c/sub\u003e) reduce hepcidin levels [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In vitro experiments have reported that the 1,25(OH)2D -vitamin D receptor complex binds to the vitamin D response element (VDRE) on the hepcidin gene (HAMP) and represses its transcription [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], suggesting a negative feedback-like regulatory mechanism by which VD3 increases body iron via hepcidin repression.\u003c/p\u003e \u003cp\u003eFuthermore, a notable phenomenon in this study was that serum 25(OH)D was significantly positively correlated with dietary vitamin D intake and the lipid energy ratio during the resting period; however, this correlation was lost during the training period. These findings suggest a change in VD\u003csub\u003e3\u003c/sub\u003e consumption and metabolism during exercise. This may be due to the effects of FGF23 and the parathyroid hormone PTH. FGF23 is reportedly stimulated by exercise for secretion [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. FGF23 has been implicated in vitamin D activity and negatively regulates erythropoiesis and iron metabolism [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. High concentrations of FGF23 have been reported to suppress 1α-hydroxylase activity, resulting in lower 1,25(OH)2D concentrations [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. PTH is known to increase in an exercise intensity- and time-dependent manner [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and its concentration is inversely correlated with the serum 25(OH)D concentration [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA limitation of this study is that it is an observational study with a small number of participants. However, the fact that metabolic changes could be observed in the same participants with and without exercise is uniquely valuable data. Moreover, as the dietary and physical activity surveys are self-reported responses by the participants, the possibility of psychological selection bias and differences from the actual content cannot be ruled out. The influence of the food environment, menstruation, physical development rate, and food preferences was not investigated; therefore, their relevance to anemia is unknown. As the present study did not examine different sexes and age groups, further studies are needed.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, the present study examined the changes in biological iron metabolism during continuous training load and the effects of vitamin D on these changes, suggesting that continuous training enhances hemolysis and erythropoiesis and may be a factor in the suppression of hepcidin expression. Multiple regression analysis revealed a significant relationship between serum 25(OH)D and ferritin levels only during the training period, suggesting that the relationship between 25(OH)D and iron levels may be closely related to metabolic changes in the body due to exercise load. In the future, we would like to focus on the relationship between iron and vitamin D during exercise, including the observation of iron metabolic changes with vitamin D supplementation, to clarify the role of iron and vitamin D in maintaining athletes' health.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Hiroshi Kawabata, M.D., Ph.D., Kyoto National Hospital Organization Kyoto Medical Center, for supporting this research. We would like to thank the director of the high school dance club, Daishi Kobayashi, and the members of the club for their cooperation in conducting this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the JSPS KAKENHI Grant-in-Aid for Scientific Research (C) (grant number 19K11695), Japan Society for the Promotion of Science.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all participants involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data will be available on request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYK, RT and ES conceived and designed research. YK, RT and ES conducted experiments. YK, RT, ES and YYS analyzed and interpreted the data. YK wrote the manuscript with input from other authors. YK, RT, ES, YYS, WA and MK conceived the study, researched the date, and reviewed the manuscript. All authors read and approved the final version of manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGIOVANNI L, GIUSEPPE L, GIUSEPPE B. Chapter 19:Iron Requirements and Iron Status of Athletes. Sports Nutrition, 1st Edition. Edited by Ronald J. Maughan. International Olympic Committee. Published 2014 by John Wiley \u0026amp; Sons, Ltd; 2014: 229-241.\u003c/li\u003e\n \u003cli\u003eSiegel AJ, Hennekens CH, Solomon HS, Van Boeckel B. Exercise-related hematuria. Findings in a group of marathon runners. JAMA. 1979 Jan 26;241(4):391-2. doi: 10.1001/jama.241.4.391.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eStewart JG, Ahlquist DA, McGill DB, Ilstrup DM, Schwartz S, Owen RA. 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Membranes (Basel). 2022 Mar 10;12(3):311. doi: 10.3390/membranes12030311.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHeeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, Mildner-Rihm C, Martin H, Zeiher AM, Dimmeler S. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood. 2003 Aug 15;102(4):1340-6. doi: 10.1182/blood-2003-01-0223. Epub 2003 Apr 17. Erratum in: Blood. 2004 Jun 15;103(12):4388.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAndıran N, \u0026Ccedil;elik N, Ak\u0026ccedil;a H, Doğan G. Vitamin D deficiency in children and adolescents. J Clin Res Pediatr Endocrinol. 2012 Mar;4(1):25-9. doi: 10.4274/jcrpe.574.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSeong JM, Yoon YS, Lee KS, Bae NY, Gi MY, Yoon H. Gender difference in relationship between serum ferritin and 25-hydroxyvitamin D in Korean adults. 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Metabolism. 2016 May;65(5):747-756. doi: 10.1016/j.metabol.2016.02.009.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCoe LM, Madathil SV, Casu C, Lanske B, Rivella S, Sitara D. FGF-23 is a negative regulator of prenatal and postnatal erythropoiesis. J Biol Chem. 2014 Apr 4;289(14):9795-810. doi: 10.1074/jbc.M113.527150. Epub 2014 Feb 7.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eScott JP, Sale C, Greeves JP, Casey A, Dutton J, Fraser WD. The role of exercise intensity in the bone metabolic response to an acute bout of weight-bearing exercise. J Appl Physiol (1985). 2011 Feb;110(2):423-32. doi: 10.1152/japplphysiol.00764.2010.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLips P, Duong T, Oleksik A, Black D, Cummings S, Cox D, Nickelsen T. A global study of vitamin D status and parathyroid function in postmenopausal women with osteoporosis: baseline data from the multiple outcomes of raloxifene evaluation clinical trial. J Clin Endocrinol Metab. 2001 Mar;86(3):1212-21. doi: 10.1210/jcem.86.3.7327.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"hepcidin, vitamin D, serum 25(OH)D, ferritin, young athletes, continuous training","lastPublishedDoi":"10.21203/rs.3.rs-3849457/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3849457/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003ePurpose\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIron deficiency is known to impair muscle function and reduce athletic performance, while vitamin D has been reported to induce iron deficiency. However, the mechanism underlying exercise-induced changes in iron metabolism and the involvement of vitamins in this mechanism are unclear.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe examined changes in biological iron metabolism induced by continuous training and the effects of vitamin D on these changes. Diet, physical characteristics, and blood test data were collected from 23 female high school students in a dance club over a 2-month period of continuous training and rest periods.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSerum hepcidin-25 levels were significantly lower during the training period than the rest period (p\u0026thinsp;=\u0026thinsp;0.013), as were the red blood cell count, hemoglobin, and hematocrit (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Serum erythropoietin was significantly higher (p\u0026thinsp;=\u0026thinsp;0.001) during the training period. Significant positive correlations were observed between 25(OH)D levels and serum iron, serum ferritin, and transferrin saturation during the training period.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMultiple regression analysis with serum 25(OH)D level as the dependent variable and serum ferritin and iron levels as independent variables during the training period revealed a significant association with serum ferritin. Therefore, the relationship between serum 25(OH)D and iron in vivo may be closely related to metabolic changes induced by the exercise load.\u003c/p\u003e","manuscriptTitle":"Continuous training in young athletes decreases hepcidin secretion and is positively correlated with serum 25(OH)D and ferritin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-15 15:48:40","doi":"10.21203/rs.3.rs-3849457/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ca3b048e-ef9c-401c-aadd-87eee4ec5c46","owner":[],"postedDate":"January 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-19T03:14:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-15 15:48:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3849457","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3849457","identity":"rs-3849457","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}