Single high-dose vitamin D supplementation impacts ultramarathon-induced changes in serum levels of bone turnover markers: a double-blind randomized controlled trial

ABSTRACT

Background

Recent studies indicate a protective role of vitamin D supplementation against sports performance-induced dysregulation of body homeostasis. However, the effects of a single high dose of vitamin D on changes in bone formation and resorption markers due to ultramarathon running have yet to be explored. This study aimed to analyze the effect of a single high-dose vitamin D supplementation on serum levels of bone turnover markers after a mountain ultramarathon run.

Methods

In this clinical trial (reg. number NCT03417700), 35 semiprofessional male ultramarathon runners were assigned into two groups: supplemented group, administered a single high dose of vitamin D₃ (cholecalciferol, 150,000 IU) in vegetable oil 24 h before the start of the run (n = 16), and placebo group (n = 19), administered placebo solution 24 h before the start of the run. Blood samples were collected for analysis at three timepoints: 24 h before, immediately after, and 24 h after the run.

Results

Serum 25(OH)D₃ level significantly increased (p ≤ 0.05.) after the ultramarathon in both groups. The increase was more pronounced in the supplemented population, especially 24 h after the run (147.01% vs 84.71%). According to post-hoc and other analyses, the levels of N-terminal propeptides of type I collagen, a PINP marker, were increased immediately after the run. The increase was significantly higher in the supplemented group than in the control group. CTX, PTH, sclerostin, and procalcitonin levels were significantly higher 24 h after the run in the control group.

Conclusions

The observed attenuation of post-exercise bone resorption and enhancement of bone formation suggest that vitamin D supplementation may modulate bone metabolism in response to extreme physical exertion, potentially through effects on calcium – PTH homeostasis.

1. Introduction

Prolonged physical exercise impacts nearly every tissue in the body, leading to significant metabolic changes and influencing inflammatory responses [1–4]. One of the tissues particularly affected by physical activity is the osseous tissue. Therein, physical activity induces dynamic changes in the local mechanical conditions, stimulating resident osteocytes through fluid shifts in the canalicular network [5]. Mechanical load during physical activity is imposed on the osseous tissue, it induces an osteogenic response and activation of specific bone cell types, i.e. osteoclasts, osteoblasts, and osteocytes [6]. Osteocytes are located in small cavities, the lacunas, in the mineralized bone matrix, and effectively recognize mechanical signals. While transmitting signals to other cells, osteocytes initiate the bone tissue rebuilding process [7,8]. The mechanism by which mechanical energy is transformed into electrical stimuli, with subsequent biochemical responses, is called mechanotransduction and plays a key role in skeletal adaptation to the actual mechanical load [9,10].

Many markers of bone formation and resorption have been established. The former includes bone-specific alkaline phosphatase, osteocalcin, sclerostin, and the C- and N-terminal propeptides of type I collagen (PICP and PINP, respectively) [10,11]. The latter include pyridinoline, deoxypyridinoline, and N- and C-terminal telopeptides of type I collagen (NTX and CTX, respectively) [10,11]. PINP and CTX are of particular interest because it was previously shown, that strenuous exercise like ultramarathon run induces a great inflammatory response [1–3]. In addition, many other proteins [e.g. fibroblast growth factor-23 (FGF23)] and hormones [e.g. parathyroid hormone (PTH)] affect bone remodeling [12–14].

Further, many external factors regulate bone formation and/or resorption (nutrition ex. vitamin D, calcium, proteins, Magnesium, phosphorus, vitamin K, and zinc; Physical Activity; Medications ex. Glucocorticoids, bisphosphonates; Lifestyle, Sunlight Exposure) [15–20] Without a doubt, exercise, i.e. its nature and intensity, in conjunction with dietary factors, determines the bone marker response and regulates bone remodeling [16]. Of note, many athletes who practice certain sports are vitamin D-deficient. Vitamin D plays an important role in bone metabolism. The endocrine effects of vitamin D mainly control serum calcium homeostasis. Vitamin D function is also related to nuclear receptors of vitamin D [11], which are abundantly present in, among others, muscle tissue. That is why vitamin D also influences skeletal muscle activity (strength), reduces muscle atrophy, impacts the secretion of many hormones and proteins, and exerts many other effects [21,22].

Many studies suggest that vitamin D plays an anti-inflammatory role [4]. This specific activity may also affect bone remodeling homeostasis through calcium regulation and other factors. Moreover, numerous researchers have explored the interplay between vitamin D supplementation, prolonged physical exercise, and post-exercise functional changes in tissues and organs. For instance, [23] investigated the effects of high-dose vitamin D3 supplementation on heart damage and iron metabolism in ultramarathon runners, suggesting that vitamin D3 could reduce exercise-induced cardiac stress. Another study demonstrated that ultra-marathon running mobilizes vitamin D release into the bloodstream, enhancing its metabolism, which could be beneficial for athletes’ health, as its increased concentration may affect the inflammation process [24]. Additionally, [25] examined the effects of vitamin D3 supplementation on iron metabolism and inflammatory post-exercise markers, highlighting its potential role in modulating exercise-induced physiological responses and iron metabolism.

However, the effects of prolonged strenuous effort combined with vitamin D supplementation on bone remodeling especially in such demanding endurance exercises like mountain running remain to be explored. Mountain long-distance running is biochemically demanding due to several factors that place unique stresses on the body, requiring complex physiological adaptations and responses ex. increases metabolic demands, changes in muscle metabolism, increased caloric burn, necessitating efficient metabolic pathways for energy production, excessive muscle fatigue and damage (due to downhill running and significant eccentric contractions, oxidative stress and inflammatory response hormonal changes. These factors combined together make mountain long-distance running a highly demanding sport, requiring optimal training, nutrition, and recovery strategies to support the biochemical and physiological demands placed on the body.

In this study, we hypothesis that single high-dose vitamin D as a nutrition and recovery strategy will modify the changes in serum levels of bone turnover markers induced by excessive physical exercise, thereby improving bone metabolism homeostasis in endurance runners. The results aim to inform the application of vitamin D supplementation protocols in endurance runners to decrease exercise-associated dysregulation of bone metabolism homeostasis.

2. Materials and methods

2.1. Experimental overview

The study is a double-blind, randomized, controlled trial with parallel groups, namely, the supplemented and placebo (control) group. Supplementation involved administration of a single high dose of vitamin D. For the study, venous blood was sampled 24 h before the ultramarathon (pre-supplementation), immediately after the run, and 24 h after the run. Vitamin D status and serum bone turnover marker levels were assessed at Gdańsk University of Physical Education (Gdańsk, Poland). The study is a continuation of a previous investigation [24] aimed at assessing the impact of a single high dose of vitamin D on vitamin D metabolites in ultramarathon runners and is a part of the project funded by the National Science Center, Poland (number 2020/37/B/NZ7/01794).

The Bioethics Committee for Clinical Research at the Regional Medical Chamber in Gdańsk approved the study protocol (decision no. KB-24/16), which was implemented in compliance with the Declaration of Helsinki. The study subjects gave informed written consent before enrolling in the study. The study has been registered as a clinical trial NCT03417700.

2.2. Participants

A group of 40 semiprofessional ultramarathon runners (men) took part in the study. All participants started in the Lower Silesian Mountain Run Festival 2018 ultramarathon race. Week before the run all runners performed second time online health questionnaire (according to the Inclusion/Exclusion criteria) and confirmed again all information about the previous ultramarathon experience (min. 5 starts), not taking any medications or supplements and following dietary guidelines in the specified period before starting the race (in accordance with the mentioned participation criteria). Additionally, subjects filled the information about their Cooper test in a 12-min running version with the maximum possible intensity that was performed about 2 weeks before the start of the competition. Obtained results allowed to calculate (indirect method) the VO2 max indicator using the formula: VO2 max = (22.351 × distance covered in kilometers) − 11.288). Afterward they were randomly assigned to two groups: experimental (supplemented, S; n = 20) or placebo (control, C; n = 20) groups.

All 40 participants included in the study cohort had declared their intent to complete the 240 km ultramarathon and were monitored prior to the event. Four participants in the supplemented (supplemented, S; n = 16) group and one in the control group (control, C; n = 19) did not complete the trial. These dropouts were unrelated to adverse effects or supplement tolerability and resulted from disqualification due to race rules (e.g. failure to reach time-controlled checkpoints) or personal logistical issues. No side effects were reported in either group. The participants in the supplemented group were 42.40 ± 7.59 years old, 175.20 ± 4.34 cm high, and weighed 72.51 ± 6.71 kg. The participants in the control group were 39.48 ± 6.89 years old, 179.67 ± 4.64 cm high, and weighed 76.19 ± 5.25 kg. The participants’ inclusion and exclusion criteria are presented in Figure 1.

Figure 1.

Eligibility criteria during project.

The enrolled participants completed a survey to assess the methods and loads used during the training period (subdivided into a period of general preparation and pre-start preparation). During the initial visit (19–20 July 2018), information on the subjects’ age, body composition, and height was obtained. A professional physician examined all the runners. Details of the participants’ physical characteristics, training loads, and performance in the Cooper test [26] are reported elsewhere [4].

Similar eating patterns, based on a randomized diet for a corresponding age group and the intensity of physical activity, were devised for the participants, who were then asked to adopt them on the measurement days. One week before the experiment and during the testing periods, competition and post-competition period. participants refrained from the intake of stimulants, such as alcohol, caffeine, chocolate, guarana, tea, or theine.

Before participating in the study, the subjects were informed about the study procedures. However, they were not aware of the rationale or study aim and thus remained unaware of the potential effects of vitamin D supplementation.

2.3. Ultramarathon run

All participants participated in the Lower Silesian Mountain Run Festival 2018, organized at Lądek Zdrój on 19–21 July 2018 (Lower Silesian Voivodeship, Poland). The race and race track characteristics were as follows: maximum course length, 240 km; maximum altitude, ca. 1425 m a.s.l.; minimum altitude, ca. 261 m a.s.l.; entire altitude range, ca. 1164 m; total ascent and descent, 7670 m; run start time, 18:00 h; temperature range during the run, from 18 °C (at the starting point) to 4 °C (on top of the Śnieżnik Mountain). No intense rain or wind was registered during the run.

2.4. Vitamin D supplementation

The study participants were randomly assigned to the S (supplemented) or C (control) groups. All participants in the S group were given a single high dose (150,000 IU) of vitamin D (cholecalciferol), as a solution in 10 mL of vegetable oil 24 h before starting in the ultramarathon. The placebo group received an equivalent volume of a placebo solution. The taste (anise), consistency, and color of the placebo solution matched those of the vitamin D oil solution. The participants and researchers were unaware of the group allocations, as the supplementation and placebo solutions were presented in carefully sealed sintered glass bottles marked with randomly assigned numbers.

2.5. Sample collection, and 25(OH)D₃ and bone turnover marker measurements

The blood was sampled by a medical diagnostic professional, according to the experimental protocol, i.e. at three times points: 24 h before the run, immediately after the run (within 5 min of the run finishing), and 24 h after the run. The blood (9 mL) was collected into Sarstedt S-Monovette tubes (S-CrossLaps® Sarstedt AG&Co, Nümbrecht, Germany) containing a coagulation accelerator for serum separation. The serum was obtained using standard laboratory procedures, aliquoted into 500 µl portions, and frozen at − 80 °C until analysis (up to 6 months). Samples had only been thawed once, i.e. before the analysis. All analyses were performed immediately after sample thawing. Assay performance was verified by using the manufacturer-supplied controls. Selected markers of bone remodeling were measured (Figure 2).

Figure 2.

Selected and measured markers of bone turnover and procalcitonine after the ultramarathon in runners who received a single high dose of vitamin D.

For vitamin D analysis, serum proteins were first precipitated and derivatized. Quantitative analysis was done using liquid chromatography-tandem mass spectrometry (Shimadzu Nexera X2 UHPLC, Shimadzu, Japan) coupled with an 8050 triple quadrupole detector (Shimadzu, Japan). The raw data were collected, processed, and quantified using LabSolutions LCGC software (Shimadzu, Japan). The concentration of the vitamin D metabolite 25(OH)D₃ was determined at all sampling timepoints (24 h before the start, immediately after, and 24 h after the race).

CTX and PINP levels were determined by using IDS-iSYS CTX (CrossLaps®) (Immunodiagnostic Systems, Tyne and Wear, UK), a chemiluminescence immunoassay, and iSYS analyzer (Immunodiagnostic Systems), according to the manufacturer’s protocol. PTH levels were measured using a commercially available enzyme-linked immunosorbent assay kit – Parathyroid Hormone ELISA Kit (Sigma-Aldrich, USA) with high sensitivity on Elisa Analyzer (Thermo Fisher Scientific Waltham, MA, USA).

FGF23 and sclerostin levels were determined using a MAGPIX fluorescence detection system (Luminex Corp., Austin, TX, USA) and relevant Luminex assays [Luminex Corp. Luminex Human Magnetic Assay (13-Plex) LXSAHM-13].

Procalcitonin (PCT) concentration was determined using the ELFA enzyme immunofluorescence method. (Enzyme-Linked Fluorescent Assay) in the VIDAS® system (bioMerieux).

2.6. Statistical analysis

Descriptive statistics for all measured variables involved the mean ± standard deviation (SD). Two-way ANOVA with repeated measures (2 × 3) was used to determine the impact of ultramarathon (ultramarathon: 24 h before, immediately after, and 24 h after the run) on the 25(OH)D₃ levels vs. vitamin D supplementation (group: S, C). Another set of two-way ANOVA with repeated measures (group: S, C; ultramarathon: 24 h before, immediately after, and 24 h after the run) was used to evaluate the impact of ultramarathon running on the levels of bone formation markers vs. vitamin D supplementation. If significant interactions were detected, Tukey’s post-hoc test was used to determine the differences in specific subgroups. The assumption of normality and homogeneity of variances was checked by Shapiro – Wilk’s and Levene’s tests.

Pearson’s (r) correlation analysis of changes in the serum 25(OH)D₃ levels with changes in the bone formation marker levels induced by the ultramarathon was also performed. Eta-squared statistics (η²) were used to determine the effect size. Values equal to or exceeding 0.01, 0.06, and 0.14 indicated a small, moderate, and large effect, respectively. All calculations were done, and graphics were generated, in Statistica 12 (StatSoft, Tulsa, OK, USA). Statistical significance was set at p ≤0.05. Power analysis for the interactions between effects to determine the appropriate sample size was performed in GPower ver. 3.1.9.2 [27]. The minimal total sample size for a medium effect size at the power of 0.8 and significance level of 0.05 was calculated as 28 subjects.

3. Results

The percent changes in vitamin D3 metabolite levels induced by the ultramarathon run and single high-dose vitamin D supplementation are shown in Table 1. A significant group × time interaction (F_{2, 60} = 7.43, p <0.01, η² = 0.21) indicated that the increase in serum 25(OH)D₃ concentrations was significantly greater in the supplemented group compared to the control group, both immediately (p <0.01) and 24 hours following the ultramarathon (p <0.01). Table 1. Near here

Table 1.

Percent changes in vitamin D₃ metabolite levels induced by vitamin D supplementation and ultramarathon running.

Variable	Group	24 h before the run (mean ± SD)	% Change immediately after the run	% Change 24 h after the run
25(OH)D3 [ng/mL]	Supplemented (n = 16) Control (n = 19)	27.50 ± 7.01 26.82 ± 5.22	111.38% †# 53.09% †	147.01% †# 84.71% †

†, significant change vs. 24 h before the run; #, significant difference vs. control group immediately and 24 after the run, as indicated. The significance level was set at p <0.01.

The results of analyzed biomarkers in particular groups before and after the ultramarathon are depicted in Figure 3.

Figure 3.

Changes in bone turnover marker and PCT levels after the ultramarathon in runners who received a single high dose of vitamin D (supplemented group, green) and runners who received the placebo (control group, black). Note: I, 24 h before the run; II, immediately after the run; and III, 24 h after the run. CTX, terminal telopeptide of type I collagen (A); FGF23, fibroblast growth factor 23 (B); PCT, procalcitonin (C); PINP, N-terminal propeptide of type I collagen (D); PTH, parathyroid hormone (E); sclerostin (F). * significant difference vs. supplemented group 24 h before the run; # significant difference vs. control group 24 h before the run; † significant difference vs. supplemented group. The significance threshold was set at p <0.01.

Two-way ANOVA with repeated measures of changes in bone turnover marker and procalcitonin levels induced by ultramarathon run is shown in Table 2. A significant effect of ultramarathon on CTX levels was noted, with a significant decrease of serum CTX levels immediately after the run (−19.30%, p <0.01), regardless the groups. However significant interaction of both factors showed, that while there was a significant decrease in CTX concentration (Figure 3(A)) immediately after (−26.9%, p <0.01) and 24 h after the run (−18.9%, p <0.01) in the supplemented group, no significant changes were observed in the control group.

Table 2.

Two-way ANOVA (2 groups × 3 repeated measures) of changes in bone turnover marker levels and PCT levels induced by ultramarathon run.

Variable	Effect	F	Df	p	Effect size (η2)	Post-hoc outcome
CTX	GR UM GR × UM	0.86 29.67 13.11	1, 30 2, 60 2, 60	0.36 0.01** 0.01**	0.02 0.49 0.30	II <I, III SI > SII, SIII
FGF23	GR UM GR × UM	1.31 232.63 1.15	1, 30 2, 60 2, 60	0.26 0.01** 0.32	0.05 0.91 0.05	I <II > III
PCT	GR UM GR × UM	3.64 4.93 4.42	1, 30 2, 60 2, 60	0.06 0.01* 0.01*	0.10 0.14 0.13	I, II <III CIII, CII > CI; CIII > SIII
PINP	GR UM GR × UM	1.93 21.64 10.69	1, 30 2, 60 2, 60	0.17 0.01** 0.01**	0.06 0.41 0.26	I <II, III SI <SII, SIII
PTH	GR UM GR × UM	15.98 95.74 15.46	1, 30 2, 60 2, 60	0.01** 0.01** 0.01**	0.34 0.76 0.34	S <C I <II, III CII > SII; CIII > SIII
Sclerostin	GR UM GR × UM	0.26 288.77 37.59	1, 30 2, 60 2, 60	0.61 0.01** 0.01**	0.01 0.90 0.55	I <II, III CIII > SIII

CTX, terminal telopeptide of type 1 collagen; FGF23, fibroblast growth factor 23; PCT, procalcitonin; PINP, N-terminal propeptide of type 1 collagen; PTH, parathyroid hormone; GR, group; UM, ultramarathon; S, supplemented group; C, control group; I, 24 h before the run; II, immediately after the run; and III, 24 h after the run. Significant difference detected at *p <0.01.

The analysis of FGF23 showed a significant ultramarathon effect. Only immediately after the run was the FGF23 concentration elevated, regardless of the groups.

The analysis of PCT results showed both main ultramarathon and interaction effects. PCT levels significantly increased 24 h after the ultramarathon (171.97%, p <0.01), regardless of the groups. However, as the interaction showed, that was only due to elevated PCT levels in the control group in both time points after the run (Figure 3(C)). Moreover, the control group had significantly higher PCT concentration 24 h after the run in comparison to supplemented group (Figure 3(C)).

PINP, PTH, and sclerostin levels increased significantly immediately after ultramarathon run (p <0.01), by 15.65%, 482.33%, and 23.96%, respectively, and remained significantly (p <0.01), elevated 24 h after the run, regardless the groups. Interaction of ultramarathon and group factors (Figure 3) showed that while the significant increase of PTH (Figure 3(E)) and Sclerostin (Figure 3(F)) was observed in both groups, in case of PINP, only the supplemented group showed significant increase (Figure 3(D)). Sclerostin concentration was also significantly higher in control group 24 h after the run in comparison with supplemented group (Figure 3(F)). Moreover, a significant group effect on PTH levels was noted, with higher PTH levels in the control group than those in supplemented group. However, this was only due to higher PTH levels immediately and 24 h after the ultramarathon run (Figure 3(E)).

Table 3 presents the results of correlation analysis of the levels of vitamin D3 with those of bone turnover markers and procalcitonin 24 h before, immediately after, and 24 h after the ultramarathon. The CTX and PCT baseline concentrations were significantly (p <0.01) negatively correlated with the vitamin D₃ concentration before the run in both groups. In the case of PINP, the correlation was positive, and in addition, the supplemented group showed a significant (p <0.01) positive correlation between vitamin D₃ and PINP concentrations 24 h after the ultramarathon run.

Table 3.

Pearson’s correlation coefficient between vitamin D₃ levels, and bone turnover marker and procalcitonin levels 24 h before, immediately after, and 24 h after the run.

Variable	Blood simple	Control			Supplemented
		I	II	III	I	II	III
CTX	I	−0.66**			−0.71**
	II		0.41			0.48
	III			−0.10			−0.42
FGF23	I	−0.42			−0.08
	II		0.23			−0.81**
	III			0.22			−0.88**
PCT	I	−0.76**			−0.62**
	II		−0.28			−0.52*
	III			−0.40			−0.35
PINP	I	0.53*			0.51*
	II		−0.22			−0.44
	III			0.08			0.79**
PTH	I	−0.65**			−0.69**
	II		−0.59*			−0.68**
	III			−0.53*			−0.66**
Sclerostin	I	−0.37			−0.68**
	II		−0.35			−0.77**
	III			−0.09			−0.81**

CTX, terminal telopeptide of type 1 collagen; FGF23, fibroblast growth factor 23; PCT, procalcitonin; PINP, N-terminal propeptide of type collagen; PTH, parathyroid hormone; GR, group; UM, ultramarathon; S, supplemented group; C, control group; I, 24 h before the run; II, immediately after the run; and III, 24 h after the run. Significant difference detected at * p <0.05 or ** p <0.01.

The sclerostin was the only marker that had a significant (p <0.01) negative correlation with vitamin D₃ at baseline, which was observed only in the supplemented group. Moreover, the sclerostin concentrations after the ultramarathon run were also significantly (p <0.01) negatively correlated with vitamin D₃ only in the supplemented group.

The PTH levels at each analyzed timepoints showed a significant (p ≤ 0.05) negative correlation with vitamin D₃ levels in both groups.

FGF23, while there was no correlation with baseline values of the marker, there was a significant (p <0.01) negative correlation between FGF23 and vitamin D₃ concentration, immediately and 24 h after the ultramarathon.

4. Discussion

In the current study, we investigated the effect of a single high dose of vitamin D₃ supplementation on bone modeling processes (formation and resorption) induced by an ultramarathon run. We have observed that a single high dose of vitamin D3 supplementation effectively increased serum 25(OH)D3 levels, decreasing post-run CTX secretion and increasing PINP content. It clearly indicates that such long lasting and demanding exercise can dysregulate bone homeostasis whether the vitamin D supplementation can potentially modify this process. Moreover, our observations suggest that vitamin D induces a protective effect against exercise-induced PTH dependent bone resorption process The results suggest that prolonged physical activity, like ultramarathon running, can adversely affect bone remodeling and increase inflammatory markers. Vitamin D supplementation appears to mitigate these effects, offering a protective role against exercise-induced bone and inflammation changes.

Many factors affect the secretion of bone formation and resorption markers. Physical activity is one such factor that may affect the activity of the osseous cells [28]. It has been established that regular physical activity positively affects human bones, protecting them from mass loss during aging [28]. On the other hand, excessive high-intensity exercise can affect bone homeostasis, causing its dysregulation [29]. Supplements, drugs, macro- and microelements, and vitamins may play a key role in post-exercise regulation of bone [16]. However, to date, few studies have explored the effect of demanding and exhausting physical activity, such as ultramarathon run, combined with specific supplements on human bone homeostasis and specific secreted bone remodeling factors.

Vitamin D has been shown to significantly impact bone metabolism; thus, it is essential to assess its impact on selected markers of skeletal management in light of the response to extreme physical exertion. In the current study, observed percent changes in vitamin D₃ metabolite levels induced by vitamin D supplementation and ultramarathon running indicated the effectiveness of supplementation (increased serum 25(OH)D₃ concentration. Previous studies [30] in the absence of physical activity (25(OH]D₃ below the median value of 22.50 ng/mL was associated with higher CTX levels at baseline). Accordingly, we can suggest that ultramarathon-Induced changes in vitamin D levels may modify the nature of the post-exercise serum CTX levels: these decreased immediately after the run in both study groups, but reached statistical significance only in the supplemented group. Moreover, in the supplemented group, CTX levels 24 h after the run were statistically lower than those before the run. At the same time, we observed a slight increase in the levels at that timepoint in the control group. According to most studies [15,31], CTX levels increase after physical activity. Nonetheless, the type of physical effort investigated in those studies (cycloergometer testing lasting no more than 60 min) may not have been exhausting enough to affect the serum vitamin D status (by increasing its liberation from fatty tissue), in contrast to the current study, where we observed changes in serum vitamin D metabolite levels even in the control group.

PCT is one such factor whose levels may be associated with inflammation and bone metabolism. It is a 116-amino acid peptide that is a precursor in calcium homeostasis and has been used as a marker to differentiate between specific systemic inflammatory processes, i.e. bacterial vs. non-infection – related. Plasma levels of PCT in healthy individuals typically range from 0.0 to 0.5 ng/mL. According to many studies, PCT levels of 0.5 to 2.0 ng/mL are associated with a possible bacterial infection or other noninfectious inflammatory conditions [32], while PCT levels over 2.0 ng/mL may indicate the possibility of a severe bacterial infection [32,33]. In the current study, PCT levels significantly increased in the control group 24 h after the run compared to the levels 24 h before (356.65%, p <0.01) and immediately after the run (174.05%, p <0.01), and were significantly higher than those in the supplemented group. We did not observe significant changes in PCT levels after the ultramarathon in the group supplemented with 150,000 IU of vitamin D. These results indicate that such demanding physical activity as prolonged distance running (where the duration of the activity typically exceeds 40 h) may induce inflammation, leading to the activation of acute-phase reactants, even those which are mainly indicative of other causes of inflammation [34]. Furthermore, the lack of changes in PCT levels in the supplemented group may indicate the anti-inflammatory effect of vitamin D, as demonstrated previously [4,35]. Although we observed a significant increase in 25(OH)D₃ levels 24 h after the ultramarathon in both groups (147.01% in the supplemented group and 84.71% in the control group vs. baseline), only the change in the supplemented group was sufficiently high to affect PCT levels, limiting their increase. The overall increase may be associated with the low initial vitamin D status in the tested population: 27.50 ± 7.01 ng/mL in the supplemented group and 26.82 ± 5.22 ng/mL in the control group

It was previously shown that an ultramarathon run decreases the PINP concentration [36]. Nevertheless, PINP levels were still higher in athletes in comparison to moderate active persons (aerobic activity performed for 30 min, 5 times a week). In the current study, PINP levels did not significantly change over time in the control group, based on the readings at the two timepoints after the run. This agrees with previous reports that bone formation markers are less responsive to exercise than resorption markers [37,38]. As was mentioned earlier, vitamin D is liberated from fatty tissue during metabolic activity and physical effort, which could explain the lack of decrease in PINP levels observed in the current study. Moreover, PINP levels were significantly higher in the supplemented group immediately after the run by 15.57% (p <0.01) and 24 h after the run by 19.75% (p <0.01) than in the control group. It may be further evidence of this specific role of vitamin D. The results may be associated with the effect of the observed 147.01% increase in vitamin D levels in the supplemented group on PINP secretion as confirmed by correlation analysis. This is consistent with the previous observations [20] that only vitamin D serum levels below 30 nmol/L are associated with a visible decrease in serum PINP levels.

Long-distance running is likely associated with changes in the levels of PTH, another bone remodeling – resorption factor [14]. In the current study, we noted a significant increase of about 93% in the levels of this hormone at both timepoints after the run in the control group. Similar trends were observed in ultramarathon runners supplemented with a single high dose of vitamin D, where PTH levels increased by 47.96% immediately after the run. Lippi et al. [14] have suggested that the observed PTH level increase after an ultramarathon run is likely associated with a transient suppression of osteoblast function. Of note, vitamin D plays a major role in the regulation of bone metabolism as osteoblasts express CYP27B1, an enzyme that converts 25(OH)D₃ to 1,25(OH)₂D₃, the bioactive form of vitamin D associated, among others, with the body calcium homeostasis in which PTH plays a critical role [39].

It is important to note that the modulation of PTH and CTX levels following vitamin D supplementation may not reflect solely the direct anti-resorptive action of vitamin D on bone cells. Rather, a significant portion of the observed effects may be mediated by changes in calcium homeostasis. Previous studies have shown that maintaining serum ionized calcium through dietary intake or supplementation can attenuate exercise-induced PTH release and bone resorption [40,41]. Moreover, vitamin D supplementation enhances intestinal calcium absorption, which may reduce the compensatory PTH response and downstream effects on bone turnover markers such as CTX. Although we did not assess calcium status or dietary intake during the study period, the possibility that vitamin D-mediated calcium retention contributed to the observed attenuation of PTH and CTX responses should be considered. These interactions highlight the complexity of calcium – PTH – vitamin D signaling in bone remodeling under extreme physical stress, as described in the framework proposed by [42] and reviewed by [43].

1,25(OH)₂D is one of the most important factors regulating the activity of FGF23. This explains why supplementation with vitamin D leads to an increase in FGF23 levels and why, by contrast, a decrease in vitamin D concentration may lead to a downregulation of FGF23 level [44]. Increased 1,25(OH)₂D levels stimulate an increase in gastrointestinal absorption of calcium and phosphate, which leads to an increase in blood calcium levels. This results in an inhibition of PTH secretion and increased urinary calcium excretion. The concentration of calcium is thus maintained at an optimal level [19].

Decreased PTH secretion also leads to decreased urinary phosphate excretion. However, an increased FGF23 activity in response to high vitamin D levels prevents phosphorus dysregulation. Of note, an increase in FGF23 levels leads to a decrease in 1,25(OH)₂ D synthesis [44], also shown in the presented correlation analysis. This is a typical feedback regulation; however, according to some experimental studies, FGF23 expression is controlled not only by vitamin D receptor (VDR)-dependent regulation but also by some other, VDR-independent mechanisms, such as the normalization of calcium and phosphorus serum levels achieved through diet [12,13]. In the current study, we observed a significant increase in FGF23 levels immediately after the run in all subjects. This outcome is in accordance with a previous study [45], where authors suggest that the reason for such an increase may be associated with inflammation, renal dysfunction, gastrointestinal bleeding, electrolyte imbalances and rather not the change in serum phosphate levels. This may be directly associated also with VDR-dependent regulation by the significant increase in vitamin D serum levels in all subjects. What is interesting is that the previous study [45] predicted that 1,25-dihydroxyvitamin D would have affected the excretion of FGF23, but not within the first 30 h after the run, which was confirmed in the current study. The 24 h post-run return to the initial values in FGF23 levels was probably mainly associated with the calcium and PTH dysregulation directly linked to post-exercise impairment of glomerular filtration [46].

Importantly, in the current study, we observed a significant increase in sclerostin levels in both groups after the run at all timepoints investigated. However, the increase was much higher in the control group than in the supplemented group. Sclerostin is an endogenous inhibitor of Wnt signaling, secreted by osteocytes. Its levels impact bone mass and bone strength. For instance, increased bone mass and bone strength were reported in sclerostin-deficient mice [47]. Further, overexpression of the human sclerostin gene (SOST alleles) in mice leads to osteopenia [48], indicating an important role of sclerostin in maintaining the appropriate bone mass. Typically, sclerostin levels are negatively correlated with bone formation markers, especially in individuals immobilized for long periods of time [18]. In the current study, sclerostin levels were also negatively correlated with vitamin D metabolite. Vitamin D levels are associated with a decrease in PTH levels, which may affect sclerostin levels. Nonetheless, similar to Acıbucu et al. [49], we did not observe a direct correlation between sclerostin and vitamin D. This could be explained by the proposed role of several systemic and local factors as regulators of sclerostin expression by the osteocyte [50,51]. The optimal vitamin D status may be only one of these factors. We did not focus on the other regulators in the current study.

Collectively, the data presented herein indicate that prolonged physical activity may modulate the levels of bone remodeling markers. Theoretically, physical activity and professional sports should protect against the risk of osteoporosis and bone loss. However, as we observed herein, that is not the case for a prolonged exercise. One-off high-intensity physical activity does not appear to provide additional benefits for bone turnover, and may not protect against bone loss [52]. Prolonged exercise is linked to the rise of inflammatory factor concentrations, and may lead to an increase in the levels of bone resorption markers [4]. Furthermore, prolonged physical activity may be associated with additional bone loss and calcium homeostasis dysregulation, and may itself lead to bone loss. Similar conclusions have been reached before [52–55]. Hence, vitamin D supplementation and its appropriate serum levels appear essential for protecting against changes in bone remodeling and inflammation associated with prolonged physical activity [4]. In conclusion, we here showed that ultramarathon running affects bone remodeling and that this effect is significantly blunted in runners who receive a single high dose of vitamin D before the run. This novel outcome is important from the perspective of pre-run protection and post-exercise regeneration in situations of prolonged physical activity.

According to the changes in biomarker levels and serum vitamin D metabolite levels observed in the control group, the amount of vitamin D that is released into the bloodstream during exercise is not sufficient to protect from increased bone resorption although it may be one of the factors that could reduce it.

The observed vitamin D levels were significantly increased and remained higher after the ultramarathon in the supplemented group than those in the control group, which may have influenced bone remodeling by attenuating the exercise-induced elevations in PTH, CTX, and PCT potentially via calcium – PTH regulatory mechanisms. Hence, elevated vitamin D levels play a key role in modulating the serum levels of bone turnover (formation and resorption) markers. This realization is an important step in understanding the role of vitamin D in professional sports.

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5. Conclusion

This study demonstrates that a single high dose of vitamin D₃ administered prior to an ultramarathon attenuated post-exercise increases in bone resorption markers and promoted a more favorable bone formation profile in male ultramarathon runners. These effects were accompanied by a significant suppression of PTH and PCT responses, suggesting a potentially protective role of vitamin D₃ against exercise-induced bone and inflammatory stress. However, as calcium intake and serum calcium concentrations were not assessed, the observed hormonal and bone marker responses may have been mediated indirectly through vitamin D – induced alterations in calcium availability, rather than a direct anti-resorptive action. Further studies incorporating detailed calcium kinetics and extending to female populations are needed to confirm and extend these findings.

6. Limitations and future research directions

Although the obtained results are valuable, the presented work certainly has several limitations. Firstly, it would seem appropriate to examine and qualify for the experiment also people with extremely low or extremely high concentration of serum vitamin D. Such type of analyzes would show the populational effect of presented study protocol. In addition, used dose could cause a slightly different effect if its supply was spread over a time (longer period with lower used doses). This could be associated with a different effect (possible different rates of changes in vitamin D serum concentration). However, it should be remembered that such type of effort (running on a distance of 240 km) is in itself a factor that greatly differentiates the populations taking part in the study, hence the obtained results seem to be fully representative of the analyzed population of runners. However, the study cohort consisted exclusively of male ultramarathon runners, which limits the generalizability of the findings to female athletes due to known sex-based differences in bone metabolism and hormonal regulation.

Additionally, we did not analyze prolonged effects of presented nutritional manipulation on bone remodeling process. There is a need to investigate individuals over an extended post-exercises period to explore the long-term implications of vitamin D supplementation on bone health and inflammatory responses in endurance athletes. Furthermore, in the present study, only serum vitamin D levels were measured. In context of muscle activity and physical performance measurement of vitamin D within muscle cells can provide deeper mechanistic insights into how vitamin D functions at a cellular level, contributing to broader understanding and potential therapeutic applications. Also we have to have in mind that muscle cells contain vitamin D receptors, which suggests that vitamin D can exert direct effects on muscle tissue. This localized action might influence muscle strength, function, and repair more directly than serum levels alone. Additionally, vitamin D influences calcium levels, which are crucial for muscle contraction and function. Measuring intramuscular vitamin D could provide insights into how well muscles can utilize calcium and perform contractions efficiently.

Moreover, our study did not assess serum calcium levels or dietary calcium intake before, during, or after the ultramarathon. This omission limits our ability to distinguish between the direct skeletal effects of vitamin D and its indirect actions mediated via calcium absorption and retention. Moreover, hydration status and potential hemoconcentration effects were not evaluated, which may have influenced the concentrations of PTH, CTX, and PCT immediately after exercise, particularly due to fluid shifts and changes in plasma volume associated with prolonged physical exertion.

Future studies should incorporate assessments of ionized and total calcium, dietary calcium intake, urinary calcium excretion, and hydration status to comprehensively evaluate the calcium – vitamin D – PTH axis in the regulation of exercise-induced bone turnover. Taken together, these limitations highlight the complexity of vitamin D’s role in exercise-induced physiological responses and underscore the need for multifactorial approaches in future research. Despite these constraints, the current findings emphasize the importance of vitamin D status in managing bone health and inflammatory responses in endurance athletes exposed to prolonged and intense physical exertion.

Funding Statement

This research was funded by the National Science Centre, Poland [2020/37/B/NZ7/01794].

Abbreviation list

CTX

terminal telopeptide of type 1 collagen

NTX

N-terminal telopeptides of type I collagen

FGF23

fibroblast growth factor 23

PCT

procalcitonin

PINP

N-terminal propeptide of type 1 collagen

PTH

parathyroid hormone

VDR

vitamin D receptor

GR

group

UM

ultramarathon

Author contributions

Conceptualization, J.M. and J.A.; Methodology, B.S., J.M., P.B, A.K., B.N., T.W., E.W., J.R., J.A.; Software, J.M., P.B., B.S., A.K., B.N., E.W., T.W.; Validation, J.M., P.B., B.S., B.N., A.K.; Formal analysis, J.M. and J.A.; Investigation, B.S., J.M., P.B., A.K., T.W., T.K., E.W., J.A.; Resources, J.A., J.M.; Data curation, J.M., B.S., J A.K..A.; Writing – original draft preparation, B.S., J.M., P.B., A.K., B.N., E.W., J.R., J.A.; Writing – review and editing, B.S., J.M., P.B., A.K., B.N., J.R., T.W., J.A.; Visualization, A.K., B.N., J.R.; Supervision, J.M., J.A. B.S.; Project administration, J.M., J.A.; Funding acquisition, J.M. and J.A. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Data availability statement

The data that support the findings of this study are available on request from the corresponding authors J.M. and J.A.