Effect of long-term carnosine/anserine supplementation on iron regulation after a prolonged running session

Nanako Hayashi; Haruna Nagastuka; Mikako Sato; Kazushige Goto

doi:10.20463/pan.2023.0020

Phys Act Nutr > Volume 27(2); 2023 > Article

Hayashi, Nagastuka, Sato, and Goto: Effect of long-term carnosine/anserine supplementation on iron regulation after a prolonged running session

Original Article

Physical Activity and Nutrition 2023;27(2):70-77.

Published online: June 30, 2023

DOI: https://doi.org/10.20463/pan.2023.0020

Effect of long-term carnosine/anserine supplementation on iron regulation after a prolonged running session

Nanako Hayashi¹, Haruna Nagastuka², Mikako Sato³, Kazushige Goto^2,^*

¹Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga, Japan

²Faculty of Sports and Health Science, Ritsumeikan University, Kusatsu, Shiga, Japan

³NH Foods Ltd. R&D Center, Midorigahara, Tsukuba, Ibaraki, Japan

^*Corresponding author : Kazushige Goto Faculty of Sports and Health Science, Ritsumeikan University, Kusatsu, Shiga, Japan. E-mail: kagoto@fc.ritsumei.ac.jp

Received April 24, 2023 Revised June 8, 2023 Accepted June 26, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

[Purpose]

Exercise-induced hemolysis, which is caused by metabolic and/or mechanical stress during exercise, is considered a potential factor for upregulating hepcidin. Intramuscular carnosine has multiple effects including antioxidant activity. Therefore, this study aimed to determine whether long-term carnosine/anserine supplementation modulates exercise-induced hemolysis and subsequent hepcidin elevation.

[Methods]

Seventeen healthy male participants were allocated to two different groups: participants consuming 1,500 mg/day of carnosine/anserine supplements (n = 9, C+A group) and participants consuming placebo powder supplements (n = 8, PLA group). The participants consumed carnosine/anserine or placebo supplements daily for 30.7 ± 0.4 days. They performed an 80-running session at 70% V̇O_2peak pre-and post-supplementation. Iron regulation and inflammation in response to exercise were evaluated.

[Results]

Serum iron concentrations significantly increased after exercise (p < 0.01) and serum haptoglobin concentrations decreased after exercise in both groups (p < 0.01). No significant differences in these variables were observed between pre-and post-supplementation. Serum hepcidin concentration significantly increased 180 min after exercise in both groups (p < 0.01). The integrated area under the curve of hepcidin significantly decreased after supplementation (p = 0.011) but did not vary between the C+A and PLA groups.

[Conclusion]

Long-term carnosine/anserine supplementation does not affect iron metabolism after a single endurance exercise session.

Keywords: hepcidin, imidazole dipeptide, hemolysis, endurance exercise

INTRODUCTION

Iron status is frequently impaired due to iron loss from sweating, hemolysis (i.e., disruption of red blood cells), gastrointestinal bleeding [1-3] during physical training, or inadequate iron intake from the daily diet [4]. Athletes present a higher prevalence of iron deficiency, with 15-35% of female athletes and 3-11% of male athletes [5,6]. Koehler et al. (2012) [7] reported that 30% of male athletes showed a suboptimal iron status (serum ferritin concentration < 35ng/mL) although 81% of male athletes satisfied the recommended dietary allowance criteria for elemental iron. Attenuated iron metabolism due to hepcidin, a liver-derived peptide hormone, contributes to iron deficiency in athletes [8].

Hepcidin regulates iron metabolism by affecting the only known iron-exporter protein, ferroportin [9,10]. Hepcidin hampers iron absorption in the duodenum and iron recycling from the macrophages via the occlusions or endocytosis of ferroportin [11]. Hepcidin gene expression is regulated in response to intracellular and/or extracellular iron levels (e.g., Fe2-transferrin concentration and iron stores in the liver) and interleukin-6 (IL-6) due to systemic inflammation [12]. Oral iron supplementation has been reported to negatively affect iron metabolism by augmenting hepcidin levels, as oral iron supplementation temporarily increases circulating iron levels [13-15]. Moretti et al. (2015) [14] showed that the consumption of oral iron doses ≥ 60 mg (60, 80, 160, 240 mg) significantly increased plasma hepcidin concentration within 24 h, with a 35-45% subsequent reduction in iron absorption. Ishibashi et al. (2017) [13] demonstrated increased basal hepcidin concentrations during three consecutive days of an endurance training block with 24 mg of iron supplementation daily. Altogether, excessive oral iron supplementation appears to attenuate iron absorption via elevated hepcidin concentration. Circulating hepcidin levels also increase after an acute session of prolonged exercise, which is thought to involve elevated IL-6 and hemolysis [8,16]. Serum and urine hepcidin concentrations are generally maximized approximately 3-6 h after completing exercise when a transit window of attenuated iron metabolism exists [17,18]. Therefore, alternative nutritional approaches to iron supplementation are required to prevent iron deficiency in athletes.

Carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-1-meth-yl-L-histidine) are imidazole dipeptides. L-histidine and β-alanine are supplied from carnosine and/or anserine in the diet and are synthesized as carnosine in the skeletal muscle. In previous studies, 4 weeks of β-alanine supplementation significantly increased the muscle carnosine concentration [19,20]. Intramuscular carnosine has multiple effects, such as antioxidant activity, buffering capacity, calcium sensitivity, and metal chelation [21,22], which affect local hemostasis and contribute to homeostasis in other organs by acting on target cells through its release into the blood during muscle contraction (i.e., physical activity and exercise) [23]. Exercise, particularly running, causes hemolysis, leading to increased circulating iron and/or decreased haptoglobin levels [24,25]. Exercise-induced hemolysis is considered a potential upregulating factor for the post-exercise hepcidin response [8], which is attributed to two different mechanisms: mechanical injury (e.g., foot impact, muscle contraction, and vasoconstriction) and metabolic injury (e.g., hyperthermia, lactic acidosis, shear stress, and oxidative damage) [26]. Accumulation of carnosine in skeletal muscle with carnosine/anserine supplementation may attenuate metabolic injury [26] since intramuscular carnosine has an antioxidative capacity and/or buffering capacity, consequently reducing increased erythrocyte fragility and suppressing exercise-induced hemolysis. Moreover, changes in Fe2-transferrin concentrations due to the attenuation of exercise-induced hemolysis may modulate hepcidin secretion [11].

Therefore, the present study investigated the effects of long-term supplementation of carnosine/anserine on iron regulation following a single session of endurance running. We hypothesized that carnosine/anserine supplementation would attenuate exercise-induced hemolysis and the subsequent hepcidin elevation.

METHODS

Participants

The present study included seventeen active males [mean ± standard deviation (SD), age: 21.3 ± 1.0 years, height: 175.0 ± 4.9 cm, body weight (BW): 65.7 ± 9.0 kg, peak oxygen uptake (V̇O_2peak): 48.3 ± 4.0 mL/kg/min)]. All participants were informed of the purpose of the study, the experimental protocol, and the possible risks involved. Written informed consent was obtained from all participants. This study was approved by the Ethics Committee for Human Experiments at Ritsumeikan University (BKC-2018-046) in accordance with the Declaration of Helsinki.

Group Classification

The participants were assigned to two different groups: participants consuming 1,500 mg (500 mg, three times) of carnosine/anserine supplementation (C+A group; n = 9) or participants consuming 1,500 mg of placebo (PLA group; n = 8; Table 1). Participants were instructed to refrain from strenuous activity, consume supplements for at least 48 h before testing, and not consume alcohol for at least 24 h before testing. Consumption of additional supplements was restricted during the experimental period.

Experimental Overview

The participants visited our laboratory four times for the two graded running tests (pre- and post-supplementation) and two experimental trials (pre- and post-supplementation) throughout the experimental period (Figure 1A). At the first visit, the V̇O_2peak was evaluated using a graded running test to determine the relative running velocity (70% V4 O2peak) for the main pre-supplementation trial. On the second visit, an 80-min running task (pre-supplementation experimental trial) was performed. After completion of the experimental trial, participants started consuming either carnosine/anserine or placebo powder supplements three times a day until the day before the secondary experimental trial (the average consumed period was 30.7 ± 0.4 days). Each pack of carnosine/anserine supplements contained 500 mg of imidazole dipeptide (carnosine 384 mg, anserine 116 mg); thus, participants in the carnosine/anserine group consumed a total of 1,500 mg carnosine/anserine per day. Powdered supplements were manufactured by NH Foods Ltd. (Osaka, Japan) (Table 2). On the third visit, V̇O_2peak was reevaluated 1-3 days before the secondary experimental trial after supplementation in both groups. The participants were required to avoid supplement intake 2-h before the graded running test. On the fourth visit, an 80-min running task (post-supplementation experimental trial) was performed. Iron metabolism and inflammation were compared after the running task pre-and post-supplementation.

Graded Exercise Test

A graded exercise test was performed on a treadmill (Valiant, Lode B. V., Groningen, Netherlands) to determine V̇O_2peak. The treadmill gradient was set to 0% during the test. Following a 3 min warm-up at 6 km/h, the initial running velocity was set at 10 km/h and progressively increased by 2 km/h every 2 min. When the running velocity reached 14 km/h, it increased by 0.6 km/h every 1 min until voluntary exhaustion. Respiratory gas was collected throughout the testing session using the breath-by-breath method with an automatic gas analyzer (AE300S; Minato Medical Science, Tokyo, Japan) to determine the V̇O_2peak. Data for respiratory variables (V4 O2 and V4 CO2peak) were measured on average every 30 s.

Experimental Trials

Experimental trials consisted of an 80-min running exercise at 70% V̇O_2peak followed by a series of blood samplings during post-exercise (Figure 1B). The experimental trials were conducted twice before and after supplementation. On the respective days, the participants arrived at the laboratory (8:00 am) following an overnight fast (> 10 h), and data on body composition and blood samples (baseline) were initially collected. After collecting the initial blood samples, the participants performed 80 min of exercise at 70% V̇O_2peak. The exercise consisted of two 40-minute running sessions and 3-min of passive rest between sessions. During the 3 min passive rest, a blood sample was collected from the fingertip to measure blood glucose and lactate concentrations (Ex40). After completion of the exercise, further blood samples were collected immediately after exercise (Post0), 60 min after exercise (Post60), and 180 min after exercise (Post180). The heart rate (HR) was recorded every 60 s using a wireless HR monitor (RCX5, Polar, Tokyo, Japan).

Blood Sampling and Analysis

Blood samples were collected from the antecubital vein before, immediately after, and 60 and 180 min after exercise using serum-separating tubes and tubes containing Na2-ethylenediaminetetraacetic acid (EDTA). Blood samples were centrifuged at 3,000 rpm and 4℃ for 10 min for serum or plasma separation. Obtained serum and plasma were stored at −60℃ until analysis. Blood glucose and lactate concentrations were measured immediately after blood sampling using a glucose analyzer (Free Style; Nipro Corporation, Osaka, Japan) and a lactate analyzer (Lactate Pro; Arkray, Kyoto, Japan), respectively. The concentrations of ferritin, iron, myoglobin (Mb), haptoglobin (Hp), and hepcidin were determined. Serum ferritin, iron, Mb, and Hp concentrations were measured at a clinical laboratory (SRL, Tokyo, Japan). Serum hepcidin concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) with a commercially available kit (R&D Systems, Minneapolis, MN, USA) The intra-assay coefficient of variation (CV) was 1.9, 2.3, 1.6, 2.3, and 2.2%, respectively. From the obtained plasma samples, IL-6 concentration was also measured using ELISA with a commercially available kit (R&D Systems). The intraassay CV values were 2.2, 1.7, and 1.4%, respectively.

Statistical analysis

Statistical analyses were performed using JASP version 0.16.2 [Computer software] (JASP Team, 2020). An Independent t-test was used to compare participant characteristics between the two groups. A mixed analysis of variance ANOVA was used to analyze a trial effect (pre- and post-intervention), a group effect (C+A, PLA), and an interaction between these two factors on the changes in running velocity, HR, and blood lactate response, baseline iron status (ferritin and iron), and area under the curve (AUC) of iron, haptoglobin, and hepcidin. AUCs of hepcidin, iron, and haptoglobin concentrations were calculated using the trapezoid area method at each time point (Baseline, Post0, Post60, Post180). Two-way repeated measures ANOVA within subjects was applied to confirm a time effect (Baseline, Post0, Post60, Post180), a trial effect (pre, post), and interaction (trial × time) to compare the differences in plasma IL-6, serum Mb, Hp, iron and hepcidin responses before and after supplementation. When significant interactions or main effects were observed, post hoc Bonferroni correction was applied for post hoc comparison to determine where specific differences existed. Effect sizes (ES) were summarized as partial eta squared (η_p²) for ANOVA and as Cohen’s d for post hoc Bonferroni correction. ES of d = 0.2 is classified as “small,” d = 0.5 is as “medium,” and d = 0.8 is “large [27].” All data are presented as means ± standard deviation (SD) and the statistical significance level was accepted at p < 0.05.

RESULTS

Aerobic capacity

No main time effect (p = 0.077, η_p² = 0.194), group (p = 0.787, η_p² = 0.005), or interaction (p = 0.304, η_p² = 0.070) was reported for V̇O_2peak (PLA group: pre 48.3 ± 4.7 ml/kg/min, post 48.6 ± 4.6 ml/kg/min; C+A group: pre 48.4 ± 3.4 ml/kg/min, post 49.6 ± 2.7 ml/kg/min). A main trial effect (p < 0.001, η_p² = 0.665) was observed for running velocity during experimental trials. Running velocity significantly increased post-supplementation compared to pre-supplementation in both groups (PLA group: pre 10.5 ± 0.7 km/h, post 10.9 ± 0.7 km/h; C+A group: pre 10.7 ± 0.7 km/h, post 11.2 ± 0.7 km/h; p < 0.001, d = -0.607).

Iron status

Baseline serum ferritin concentrations did not show any interaction (p = 0.924, η_p² = 0.000), trial (p = 0.141, η_p² = 0.139), or group effects (p = 0.775, η_p² = 0.006, Table 3). Baseline serum iron concentration did not present any interaction (p = 0.376, η_p² = 0.053) or group effects (p = 0.518, η_p² = 0.028), but showed a tendency toward a trial effect (p = 0.087, η_p² = 0.183, Table 3).

HR and Blood Lactate Response

Heart rate as of the end of the exercise did not present any interaction (p = 0.277, η_p² = 0.078), trial (p = 0.168, η_p² = 0.123) and group effects (p = 0.866, η_p² = 0.002; PLA group: pre 169 ± 14 bpm, post 168 ± 15.9 bpm; C+A group: pre 172 ± 10 bpm, post 167.4 ± 10 bpm). Blood lactate concentration immediately after exercise did not present any interaction (p = 0.236; η_p² = 0.092), trial (p = 0.659, η_p² = 0.013) and group effect (p = 0.236; η_p²= 0.092; PLA group: pre 1.9 ± 0.6 mmol/L, post 2.0 ± 1.0 mmol/L; C+A group; pre 2.5 ± 0.7 mmol/L, post 2.2 ± 0.7 mmol/L).

Hemolysis and Iron regulation

Serum Hp concentrations in the PLA group significantly decreased after exercise (Post0, p = 0.001, d = 0.171; Post60, p < 0.001, d = 0.419; Post180, p < 0.001, d = 0.334), with no difference between pre-and post-supplementation (Figure 2). Serum Hp concentrations in the C+A group also significantly decreased after exercise (Post0, p = 0.002, d = 0.226; Post60, p < 0.001, d = 0.559; Post180, p < 0.001, d = 0.56), with no difference pre-and post-supplementation. Serum iron concentrations in the PLA group significantly increased after exercise (Post0, p < 0.001, d = -0.496; Post60, p = 0.002, d = -0.456; Post180, p < 0.001, d = -0.487) with no difference pre- and post-supplementation. Serum iron concentrations in the C+A group also significantly increased after exercise (Post0, p = 0.001, d = -0.503; Post60, p = 0.005, d = -0.443; Post180, p < 0.001, d = -0.556), with no difference pre-and post-supplementation (Figure 2). Serum hepcidin concentrations significantly increased 180 min after exercise in both the PLA and C+A groups (p = 0.004, d = -1.03; p < 0.001, d = -0.811, respectively), and there was no significant difference in hepcidin responses between preand post-supplementation (Figure 2).

The iron AUC and Hp AUC did not present any interaction, trial, or group effects (Figure 3). The hepcidin AUC significantly decreased post-supplementation (p = 0.011, d = 0.696), but did not vary between the groups (Figure 3).

Muscle Damage and Inflammation

Serum Mb concentration showed a time effect in both the PLA (p < 0.01, η_p² = 0.846) and C+A group (p = 0.003, η_p² = 0.654). The serum Mb concentration in the PLA group significantly increased after exercise (Post0: p < 0.001, d = -1.667; Post60: p < 0.001, d = -2.732; Post180: p < 0.001, d = -1.981), but did not differ pre- and post-supplementation (Table 4). Serum Mb concentration in the C+A group also significantly increased after exercise (Post0: p = 0.002, d = -1.180; Post60: p < 0.001, d = -1.850; Post180: p = 0.002, d = -1.201), but did not vary pre- and post-supplementation (Table 4).

Plasma IL-6 concentrations in the PLA group showed a time effect (p = 0.003, η_p² = 0.740). The IL-6 concentration in the PLA group immediately after exercise significantly increased (p = 0.003, d = -1.940) but did not differ between pre- and post-supplementation (Table 4). Plasma IL-6 concentration in the C+A group also showed a time effect (p = 0.002, η_p² = 0.720). Plasma IL-6 concentration in the C+A group also significantly increased immediately after exercise (p = 0.002, d = -1.159) but did not differ between pre- and post-supplementation (Table 4).

DISCUSSION

This study investigated the influence of long-term carnosine/anserine supplementation on post-exercise iron regulation using a single-blind, placebo-controlled test. No significant differences in hemolysis, inflammation, or muscle damage were observed following the 80-min running between pre- and post-supplementation (30.7 ± 0.4 days). Exercise-induced hepcidin elevation appeared to be modulated after supplementation in both groups; however, no apparent effect of carnosine/anserine on exercise-induced hepcidin elevation was observed.

Although recent studies have reported the effects of anti-inflammatory or antioxidant supplements and foods on exercise-induced hepcidin responses, no significant effects on elevated hepcidin concentrations after exercise have been observed regardless of the duration or timing of intake. A study that added vitamin D (5,000 IU) and K (1,000 μg) to mixed carbohydrate and protein intake after high-intensity interval cycling exercise demonstrated that no effect of supplementation on the post-exercise iron status or IL-6 and hepcidin concentrations [28]. Long-term 28-day intake of both vitamin C (500 mg/day) and vitamin E (400 IU/day) did not affect hepcidin concentration after 90 min of running exercise [29]. The supplementation of polyphenols, which have antioxidant properties, is also expected to reduce oxidative stress, leading to improving iron metabolism [12,30]. The consumption of chokeberry juice (50 ml, three times a day) during an 8-week training camp for rowers increased their antioxidant capacity and attenuated an increase in TNF-α concentrations 24 h after a 2,000 min time trial. However, no effect on IL-6 or hepcidin response was observed after the trial [30]. In addition, supplementation with cranberry extract for 6 weeks enhanced the antioxidant capacity associated with high-intensity exercise, while no clear effects on IL-6, Mb, and hepcidin responses were observed12. The results of this study align with those of the aforementioned studies.

An increased serum iron concentration with a concomitant reduction in serum Hp concentration was observed in this study, which is a typical symptom of exercise-induced hemolysis [24,25]. Accordingly, hemolysis would occur after the endurance running session; however, the magnitude of exercise-induced hemolysis was independent of carnosine/anserine or placebo supplementation because of similar responses in serum iron and Hp concentrations. The baseline iron status (i.e., serum iron and ferritin concentrations) was slightly different between pre-and post-supplementation in the C+A group; however, these differences did not reach statistical difference. A slight decrease in baseline serum iron (PLA group -9.1%; C+A group -27.7%) and ferritin (PLA group, -17.0%, C+A group -17.8%) may affect the post-exercise hepcidin response. Peeling et al. (2017) [31] also showed that both basal serum iron and ferritin concentrations significantly correlated with serum hepcidin concentration 3 h after exercise. Therefore, changes in baseline iron status would modulate iron regulation in response to the running exercise and, as a result, would have lowered the AUC of the hepcidin concentration post-supplementation. L-Histidine, a component of carnosine and anserine, may also be involved in the reduction of the AUC of serum hepcidin concentrations; it is involved in erythropoiesis and is thought to be related to iron absorption [32,33]. It is an essential amino acid for erythrocyte production [34]. Therefore, extra- and intracellular iron may be mobilized for hematopoiesis, leading to a slight decrease in ferritin and serum iron levels and a subsequent attenuated hepcidin response.

The increase in IL-6 concentration immediately after exercise is thought to be a potential factor that induces 3h post-exercise hepcidin elevation via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway [35]. Running sessions for 80 min significantly increased the plasma IL-6 concentration, and this response did not change post-supplementation. The hepcidin response post-supplementation was lower than that pre-supplementation in both groups, indicating that modification of the IL-6- hepcidin axis was not a primary factor for the attenuation of hepcidin concentration. A previous clinical study reported lower IL-6 concentrations after 12 weeks of l-carnosine supplementation in patients with type 2 diabetes [36]. In addition, carnosine supplementation attenuated IL-6 secretion by activated murine macrophages in vitro [37]. Increased IL-6 concentration immediately after exercise can be differentiated as muscle-derived IL-6 [38], indicating that although carnosine/anserine intake may downregulate the increase in macrophage-derived IL-6 production due to chronic inflammation, it does not influence muscle-derived IL-6. Although the relative load (70%V4 O2peak) was standardized pre- and post-supplementation in this study, the absolute load (i.e., running speed) post-supplementation was not equivalent to that pre-supplementation because of the slight increase in V̇O_2peak. Since exercise-induced IL-6 is influenced by exercise modality, duration, and intensity [39,40], caution is needed when interpreting the results when absolute running speed differs. However, the difference in speed pre- and post-supplementation in this study was ~0.4-0.5 km/h, suggesting that it is unlikely to affect IL-6 production.

A major limitation of the present study was the lack of detailed data on energy intake and macro- and micronutrient intake, as no dietary survey was conducted during the supplementation period; therefore, dietary data would be helpful to understand the effects of carnosine/anserine supplementation more fully. All participants were instructed to maintain their daily activities and regular diets during the supplementation period, and no significant changes in body composition were observed pre- and post-supplementation. A previous study demonstrated that the 30-day ingestion of chicken breast extract (CEBX) containing 1,500 mg of carnosine/anserine significantly increased intramuscular carnosine concentration in the vastus lateralis muscle in healthy men [41]. Because the C+A group consumed the same amount of carnosine/anserine as Sato et al. (2003) [41] for approximately 30 days, apart from their diet, it was assumed that the intramuscular carnosine concentration also increased in this study as well.

The participants, in this study, did not conduct any structured exercise training during the supplementation period; therefore, the effects of carnosine/anserine in combination with exercise training on iron metabolism are unclear. Zügel et al. (2020) [42] showed an increase in hepcidin concentrations with increasing amounts of high-intensity training, indicating that the regulation of hepcidin secretion is sensitive to changes in training load. In athletes, exercise stimuli may affect iron regulation [43], and metabolic stress-induced hemolysis during exercise has been reported to vary with subject characteristics (fitness level) and exercise intensity [44,45]. In addition, intramuscular carnosine levels are affected by muscle fiber type composition (types I and II) and training status [23]. Therefore, the effect of carnosine/anserine supplementation may differ between athletes and nonathletes (sedentary or active men), and further studies are warranted.

In summary, these results found that while hemolysis and increased hepcidin concentration occurred after an 80 min-running session, long-term carnosine/anserine supplementation does not affect iron regulation post-exercise in active males.

Acknowledgments

We thank all participants in this study for completing the long-term carnosine/anserine supplementation and the four exercise sessions. We also thank the laboratory members and NH Foods Ltd. for their technical support. This study was financially supported by NH Foods Ltd.

This study was financially supported by NH Foods Ltd. An author (M.S.), is affiliated with NH Foods Ltd. R&D Center, involved in the development of powdery imidazole dipeptide (carnosine/anserine) supplements. M.S. contributed to this study (e.g., preparing the carnosine/anserine and placebo supplement, development of methodology), but was not involved in data collection, manuscript preparation, discussion, and drawing the conclusion, in terms of conflict of interest.

Figure 1.

Diagrammatic representation of an experimental overview.

Figure 2.

Changes in serum haptoglobin, iron, and hepcidin response pre-and post-supplementation. Values are presented as means ± SD. Pre, pre-supplementation; Post, post-supplementation; PLA, placebo group; C+A, carnosine/anserine group, Post0, a point immediately after exercise; Post60, a point of 60 min after exercise, Post180, a point of 180 min after exercise. a Significant difference compared to baseline, b Significant difference compared to P0, c Significant difference compared to Post60

Figure 3.

AUC of serum iron, haptoglobin, and hepcidin concentrations. Values are presented as means ± SD. AUC, the area under the curve, Pre, pre-supplementation; Post, post-supplementation; PLA, a placebo group; C+A, a carnosine/anserine group. * Significant difference compared with pre-supplementation

Table 1.

Characteristics of participants.

	PLA (n=8)	C+A (n=9)	t-test
Age (years)	21.0 ± 1.2	21.6 ± 0.7	p = 0.259
Height (cm)	174.2 ± 3.8	175.7 ± 5.9	p = 0.536
Body weight (kg)	67.5 ± 11.1	64.1 ± 6.9	p = 0.443
%Body fat (%)	16.0 ± 6.8	12.9 ± 4.9	p = 0.293
V̇O_2peak (ml/kg/min)	48.3 ± 4.7	48.4 ± 3.4	p = 0.969
Serum ferritin (ng/mL)	90.8 ± 58.9	83.9 ± 43.6	p = 0.789

Values are presented as means ± SD. C+A, a carnosine/anserine group; PLA, a placebo group.

Table 2.

Ingredient composition of carnosine/anserine and placebo supplements (3g/a pack).

	PLA	C+A
Energy (kcal)	11.34	11.07
Protein (g)	0.00	1.30
Fat (g)	0.01	0.00
Carbohydrate (g)	2.81	1.46
Water (g)	0.15	0.17
Ash (g)	0.03	0.07
Imidazole dipeptide (g)	0.00	0.50
carnosine/anserine (mg)	-	116/384

C+A, carnosine/anserine group; PLA, placebo group.

Table 3.

Iron status pre- and post-supplementation.

		Pre	Post
Ferritin (ng/mL)	PLA	83.9 ± 43.6	69.6 ± 40.2
	C+A	90.8 ± 58.9	74.6 ± 40.6
Iron (µg/dL)	PLA	105 ± 54	96 ± 39
	C+A	104 ± 41	75 ± 25

Values are presented as means ± SD. Pre, pre-supplementation; Post, postsupplementation; C+A, carnosine/anserine group; PLA, placebo group.

Table 4.

Muscle damage and inflammatory responses to exercise pre-and post-supplementation.

		Pre				Post
		Baseline	Post0	Post60	Post180	Baseline	Post0	Post60	Post180
Mb _(ng/mL)	PLA	35.7 ± 11.0	170.0 ± 61.9^a	241.5 ± 87.3^a^b	197.5 ± 95.3^a	31.6 ± 6.7	125.7 ± 42.5^a^b	200.1 ± 95.0^a	141.2 ± 77.4^a
	C+A	41.1 ± 34.7	183.6 ± 94.3^a	262.7 ± 185.3^a	203.9 ± 160.0^a	36.9 ± 16.0	161.9 ± 82.6^a	234.7 ± 136.7^a	146.4 ± 83.7^a
IL-6 _(pg/mL)	PLA	0.99 ± 0.74	4.43 ± 1.51^a	-	-	1.15 ± 1.02	4.64 ± 2.98^a	-	-
	C+A	1.84 ± 2.10	7.54 ± 8.12^a	-	-	1.16 ± 0.75	5.44 ± 1.81^a	-	-

Values are presented as means ± SD. Mb, myoglobin; IL-6, interleukin-6, Pre, pre-supplementation; Post, post-supplementation; C+A, carnosine/anserine group; PLA, placebo group; Post0, a point immediately after exercise; Post60, a point of 60 min after exercise, Post180, a point of 180 min after exercise.

^a Significant difference compared to baseline,

^b Significant difference compared to P0,

^c Significant difference compared to Post60.

REFERENCES

1. Babic Z, Papa B, Sikirika-Bosnjakovic M, Prkacin I, Misigoj-Durakovic M, Katicic M. Occult gastrointestinal bleeding in rugby player. J Sports Med Phys Fitness 2001;41:399-402.

2. DeRuisseau KC, Cheuvront SN, Haymes EM, Sharp RG. Sweat iron and zinc losses during prolonged exercise. Int J Sport Nutr Exerc Metab 2002;12:428-37.

3. Telford RD, Sly GJ, Hahn AG, Cunningham RB, Bryant C, Smith JA. Footstrike is the major cause of hemolysis during running. J Appl Physiol 2003;94:38-42.

4. Lampe JW, Slavin JL, Apple FS. Poor iron status of women runners training for a marathon. Int J Sports Med 1986;7:111-4.

5. Badenhorst CE, Goto K, O’Brien WJ, Sims S. Iron status in athletic females, a shift in perspective on an old paradigm. J Sports Sci 2021;39:1565-75.

6. Kuwabara AM, Tenforde AS, Finnoff JT, Fredericson M. Iron deficiency in athletes: a narrative review. PM R 2022;14:620-42.

7. Koehler K, Braun H, Achtzehn S, Hildebrand U, Predel HG, Mester J, Schanzer W. Iron status in elite young athletes: gender-dependent influences of diet and exercise. Eur J Appl Physiol 2012;112:513-23.

8. Peeling P. Exercise as a mediator of hepcidin activity in athletes. Eur J Appl Physiol 2010;110:877-83.

9. Nemeth E, Ganz T. Regulation of iron metabolism by hepcidin. Annu Rev Nutr 2006;26:323-42.

10. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004;306:2090-93.

11. Nemeth E. Ganz T. Hepcidin-ferroportin interaction controls systemic iron homeostasis. Int J Mol Sci 2021;22:6493.

12. Skarpańska-Stejnborn A, Basta P, Trzeciak J, Michalska A, Kafkas ME, Woitas-Slubowska D. Effects of cranberry (Vaccinum macrocarpon) supplementation on iron status and inflammatory markers in rowers. J Int Soc Sports Nutr 2017;14:7.

13. Ishibashi A, Maeda N, Kamei A, Goto K. Iron supplementation during three consecutive days of endurance training augmented hepcidin levels. Nutrients 2017;9:820.

14. Moretti D, Goede JS, Zeder C, Jiskra M, Chatzinakou V, Tjalsma H, Melse-Boonstra A, Brittenham G, Swinkels DW, Zimmermann MB. Oral iron supplements increase hepcidin and decrease iron absorption from daily or twice-daily doses in iron-depleted young women. Blood 2015;126:1981-9.

15. Stoffel NU, Cercamondi CI, Brittenham G, Zeder C, Geurts-Moespot AJ, Swinkels DW, Moretti D, Zimmermann MB. Iron absorption from oral iron supplements given on consecutive versus alternate days and as single morning dos-es versus twice-daily split dosing in iron-depleted women: two open-label, randomised controlled trials. Lancet Haematol 2017;4:E524-33.

16. Banzet S, Sanchez H, Chapot R, Bigard X, Vaulont S, Koulmann N. Interleukin-6 contributes to hepcidin mRNA increase in response to exercise. Cytokine 2012;58:158-61.

17. Newlin MK, Williams S, McNamara T, Tjalsma H, Swinkels DW, Haymes EM. The effects of acute exercise bouts on hepcidin in women. Int J Sport Nutr Exerc Metab 2012;22:79-88.

18. Peeling P, Dawson B, Goodman C, Landers G, Wiegerinck ET, Swinkels DW, Trinder D. Effects of exercise on hepcidin response and iron metabolism during recovery. Int J Sport Nutr Exerc Metab 2009;19:583-97.

19. Harris RC, Tallon MJ, Dunnett M, Boobis L, Coakley J, Kim HJ, Fallowfield JL, Hill CA, Sale C, Wise JA. The absorption of orally supplied β-alanine and its effect on muscle carnosine synthesis in human vastus lateralis. Amino Acids 2006;30:279-89.

20. Hill CA, Harris RC, Kim HJ, Harris BD, Sale C, Boobis LH, Kim CK, Wise JA. Influence of beta-alanine supplementation on skeletal muscle carnosine concentrations and high intensity cycling capacity. Amino Acids 2007;32:225-33.

21. Artioli GG, Gualano B, Smith A, Stout J, Lancha AH Jr. Role of beta-alanine supplementation on muscle carnosine and exercise performance. Med Sci Sports Exerc 2010;42:1162-73.

22. Boldyrev AA, Aldini G, Derave W. Physiology and pathophysiology of carnosine. Physiol Rev 2013;93:1803-45.

23. Derave W, Everaert I, Beeckman S, Baguet A. Muscle carnosine metabolism and beta-alanine supplementation in relation to exercise and training. Sports Med 2010;40:247-63.

24. Buchman AL, Keen C, Commisso J, Killip D, Ou CN, Rognerud CL, Dennis K, Dunn JK. The effect of a marathon run on plasma and urine mineral and metal concentrations. J Am Coll Nutr 1998;17:124-7.

25. Telford RD, Sly GJ, Hahn AG, Cunningham RB, Bryant C, Smith JA. Footstrike is the major cause of hemolysis during running. J Appl Physiol 2003;94:38-42.

26. Lippi G, Sanchis-Gomar F. Epidemiological, biological and clinical update on exercise-induced hemolysis. Ann Transl Med 2019;7:270.

27. Cohen J. tistical Power Analysis for the Behavioral Sciences (2nd ed.). New York, USA: Routledge. 1988.

28. Dahlquist DT, Stellingwerff T, Dieter BP, McKenzie DC, Koehle MS. Effects of macro- and micronutrients on exercise-induced hepcidin response in highly trained endurance athletes. Appl Physiol Nutr Metab 2017;42:1036-43.

29. Díaz V, Peinado AB, Barba-Moreno L, Altamura S, Butragueño J, González-Gross M, Alteheld B, Stehle P, Zapico AG, Muckenthaler MU, Gassmann M. Elevated hepcidin serum level in response to inflammatory and iron signals in exercising athletes is independent of moderate supplementation with vitamin C and E. Physiol Rep 2015;3:e12475.

30. Skarpańska-Stejnborn A, Basta P, Sadowska J, Pilaczynska-Szczesniak L. Effect of supplementation with chokeberry juice on the inflammatory status and markers of iron metabolism in rowers. J Int Soc Sports Nutr 2014;11:48.

31. Peeling P, McKay AKA, Pyne DB, Guelfi KJ, McCormick RH, Laarakkers CM, Swinkels DW, Garvican-Lewis LA, Ross MLR, Sharma AP, Leckey JJ, Burke LM. Factors influencing the post-exercise hepcidin-25 response in elite athletes. Eur J Appl Physiol 2017;117:1233-9.

32. Holeček M. Histidine in health and disease: metabolism, physiological importance, and use as a supplement. Nutrients 2020;12:848.

33. Vera-Aviles M, Vantana E, Kardinasari E, Koh NL, Latunde-Dada GO. Protective role of histidine supplementation against oxidative stress damage in the management of anemia of chronic kidney disease. Pharmaceuticals (Basel) 2018;11:111.

34. Kopple JD, Swendseid ME. Evidence that histidine is an essential amino acid in normal and chronically uremic man. J Clin Invest 1975;55:881-91.

35. Ganz T, Nemeth E. Hepcidin and iron homeostasis. Biochim Biophys Acta 2012;1823:1434-43.

36. Houjeghani S, Kheirouri S, Faraji E, Jafarabadi MA. l-Carnosine supplementation attenuated fasting glucose, triglycerides, advanced glycation end products, and tumor necrosis factor-alpha levels in patients with type 2 diabetes: a double-blind placebo-controlled randomized clinical trial. Nutr Res 2018;49:96-106.

37. Caruso G, Fresta CG, Martinez-Becerra F, Antonio L, Johnson RT, de Campos RPS, Siegel JM, Wijesinghe MB, Lazzarino G, Lunte SM. Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages. Mol Cell Biochem 2017;431:197-210.

38. Hennigar SR, McClung JP, Pasiakos SM. Nutritional interventions and the IL-6 response to exercise. FASEB J 2017;31:3719-28.

39. Febbraio MA, Pedersen BK. Muscle-derived interleukin-6: mechanisms for activation and possible biological roles. FASEB J 2002;16:1335-47.

40. Pedersen BK, Steensberg A, Schjerling P. Muscle-derived interleukin-6: possible biological effects. J Physiol 2001;536:329-37.

41. Sato M, Suzuki Y, Morimatsu F, Takamatsu K. Effect of carnosine concentration in muscle and improvement of exercise performances due to long-term intake of chicken breast extract. Jpn J Phys Fit Sport 2003;52:255-63.

42. Zügel M, Treff G, Steinacker JM, Mayer B, Winkert K, Schumann U. Increased hepcidin levels during a period of high training load do not alter iron status in male elite junior rowers. Front Physiol 2019;10:1577.

43. Moretti D, Mettler S, Zeder C, Lundby C, Geurts-Moetspot A, Monnard A, Swinkels DW, Brittenham GM, Zimmermann MB. An intensified training schedule in recreational male runners is associated with increases in erythropoiesis and inflammation and a net reduction in plasma hepcidin. Am J Clin Nutr 2018;108:1324-33.

44. Senturk UK, Gunduz F, Kuru O, Kocer G, Ozkaya YG, Yesilkaya A, Bor-Kucukatay M, Uyuklu M, Yalcin O, Baskurt OK. Exercise-induced oxidative stress leads hemolysis in sedentary but not trained humans. J Appl Physiol 2005;99:1434-41.

45. Smith JA, Kolbuch-Braddon M, Gillam I, Telford RD, Weidemann MJ. Changes in the susceptibility of red blood cells to oxidative and osmotic stress following submaximal exercise. Eur J Appl Physiol Occup Physiol 1995;70:427-36.