Seasonal changes in free 25-(OH)D and vitamin D metabolite ratios and their relationship with psychophysical stress markers in male professional football players

Introduction: Novel markers of vitamin D status are currently being investigated, including free 25-(OH)D (25-(OH)DF) and the vitamin D metabolite ratio (24,25-(OH)2D3:25-(OH)D3; VMR). The VMR may provide additional functional information on vitamin D metabolism in athletes. Therefore, the main objective of the current study was to evaluate 25-(OH)DF, bioavailable 25-(OH)D (25-(OH)DB), VMR, and psychophysical stress markers during different training periods over a half-season. The second aim was to assess the association between vitamin D binding protein (VDBP), total and free 25-(OH)D, VMRs, and psychophysical stress markers in professional football players. Moreover, we examined the relationship between 25-(OH)D3 and vitamin D metabolites (24,25-(OH)2D3, 3-epi-25-(OH)D3) to determine if training loads in different training periods influenced the vitamin D metabolome. Methods: Twenty professional football players were tested at six different time points across half a year (V1—June; V2—July; V3—August; V4—October; V5—December; V6—January). Results: Analyses indicated a significant seasonal rhythm for VDBP, and total 25-(OH)D (25-(OH)DT), 25-(OH)DB, 24,25-(OH)2D3, 3-epi-25-(OH)D3, 25-(OH)D3:24,25-(OH)2D3, and 24,25-(OH)2D3:25-(OH)D3 VMRs throughout the training period. No correlation was detected between 25-(OH)DT, 25-(OH)DB, 25-(OH)DF, vitamin D metabolites, VMRs, VDBP, and ferritin, liver enzymes (aspartate transaminase [AST] and alanine transaminase [ALT]), creatine kinase (CK), cortisol, testosterone, and testosterone-to-cortisol ratio (T/C) in each period (V1-V6). However, there was a strong statistically significant correlation between 25-(OH)D3 and 24,25-(OH)D3 in each training period. Conclusion: In conclusion, a seasonal rhythm was present for VDBP, 25-(OH)DT, 25-(OH)DB, vitamin D metabolites (24,25-(OH)2D3, 3-epi-25-(OH)D3), and VMRs (25-(OH)D3:24,25-(OH)2D3, 25-(OH)D3:3-epi-25-(OH)D3). However, no rhythm was detected for 25-(OH)DF and markers of psychophysical stress (ferritin, liver enzymes, CK, testosterone, cortisol, and T/C ratio). Moreover, the relationships between free and total 25-(OH)D with psychophysical stress markers did not demonstrate the superiority of free over total measurements. Furthermore, training loads in different training periods did not affect resting vitamin D metabolite concentrations in football players.


Introduction
Vitamin D is an important compound related to many aspects of athletic performance and recovery, with the most studied functions of vitamin D concerning bone (Herrmann et al., 2017) and skeletal muscle health (Dzik and Kaczor, 2019).Furthermore, vitamin D plays a vital role in modulating the functions of many other tissues that are important in a sports context, including those impacting immune (Martens et al., 2020) and cardiac function (Messa et al., 2014).Vitamin D status is a common topic in sports science due to the high prevalence of vitamin D insufficiency in athletes (Farrokhyar et al., 2015;Harju et al., 2022), which can negatively influence musculoskeletal function, power, force production, and recovery from fractures (Owens et al., 2018;Ribbans et al., 2021).All of these factors are pivotal for athletes as they may have impact on sport performance and general health.
In most athlete trials, serum 25-(OH)D T level is a biomarker of vitamin D status due to its relatively long half-life.Consistent with the free hormone hypothesis (Chun et al., 2014), only the non-bounded fraction (the free fraction, 25-(OH)D F ) can enter cells and exert its biological effects (Bikle, 2021).Some studies have shown that some vitamin D functions may be closely related to its free fraction than to VDBP bound 25-(OH)D T concentrations (Powe et al., 2011;Shieh et al., 2018).VDBP levels and activities influence vitamin D bioavailability, altering the balance between free and bound vitamin D fractions (Bouillon et al., 2019).In such circumstances, 25-(OH)D T concentration estimations may be misleading.Some studies suggest (Cashman et al., 2015;Ginsberg et al., 2020) that evaluating the molar ratio of 24,25-(OH) 2 :25-(OH)D may be a better index of vitamin D insufficiency in healthy men (Alonso et al., 2022) as it is not affected by race (Berg et al., 2015).Recent evidence demonstrates that VMR strongly associates with higher bone mineral density (Ginsberg et al., 2018) and parathyroid hormone (PTH) (Bosworth et al., 2012) than 25-(OH)D T .Therefore, VMR may provide additional functional information on vitamin D metabolism in athletes.
The vitamin D receptor (VDR) is expressed in skeletal muscle and has essential roles in maintaining mitochondrial function and recovery (Latham et al., 2021).Latest evidence indicates that vitamin D signaling contributes to muscle regeneration.In animal models, VDR protein expression associates closely with 25-(OH)D serum concentration (Srikuea et al., 2020).The VDR and vitamin D-activating enzyme CYP27B1 are expressed at a low level in homeostatic skeletal muscle in vitro and in vivo, evidenced by immunocytochemical and immunohistochemical visualization and immunoblotting in C2C12 myoblasts and whole mouse muscle (Srikuea et al., 2012;Srikuea et al., 2020;Latham et al., 2021).Vitamin D play role in muscle regeneration supported by rapidly raised Pax7 and VDR protein expression in skeletal muscle to take action on the repair response after an acute bout of damaging high-intensity physical effort (Puangthong et al., 2021), demonstrating that the myogenic repair and vitamin D systems are both rapidly and contemporaneously initiated after skeletal muscle damage (Latham et al., 2021).

Study design, participants, and blood draws
Forty-two football players were recruited from one club competing in the highest male football Polish league, the "Ekstraklasa," providing a total of 180 records.After initial screening, the study included 20 participants with a mean age of 26.9 ± 4.7 years.Athletes with injuries, those not present at more than two blood draws, and participants who used calcium (Ca) or vitamin D supplementation were excluded.However, sporadic vitamin D intake was permitted.The competitors who participated in the study were active in all training periods and had similar athletic performance levels, career duration, and training loads.
Blood draws followed the first round of the Polish "PKO BP Ekstraklasa" from June 2021 to January 2022.Sample collections were performed in June -V1 (before the pre-season), July-V2 (after the pre-season), August -V3 (during the competitive season), October -V4 (during the competitive season), December-V5 (after the competitive season), and January -V6 (after off-season).The team trained regularly and played at latitudes between 50 °and 54 °N.Table 1 details the content of each of the training periods, considering different training sessions and durations.

Biochemical analyses
Blood samples were collected into plain tubes containing a clot activator (Vacutest, Kima, Italy), stored at room temperature for As previously described (Książek et al., 2022), total serum Ca was determined by colorimetric assay using the Konelab 60 system (bioMérieux, Marcy-l'Etoile, France).Albumin was assayed on a Siemens Dimension Xpand Plus clinical chemistry system (Siemens, Munich, Germany).
Plasma CK activity was evaluated using diagnostic kits for the Konelap 60 kinetic enzyme analyzer (bioMérieux, Marcy-l'Etoile, France).The CK detection limit for the kits was 6 U/l, with an intraassay CV of 1.85%.
Serum ferritin and cortisol levels were measured by ECLIA on the Cobas e601 analyzer (Roche, Mannheim, Germany).Intra-assay and inter-assay CVs for ferritin and cortisol were 2.5% and 8.1%, and 5.4% and 10.1%, respectively.Serum total testosterone was measured by ECLIA on the Cobas e411 analyzer (Roche, Mannheim, Germany) and had intra-assay and inter-assay CVs of 4.7% and 8.4%, respectively.
The testosterone-to-cortisol ratio (T/C) was calculated as a surrogate marker of overtraining and psychophysical stress.
Aspartate transaminase (AST) and alanine transaminase (ALT) levels were measured by enzymatic assay on the Alinity m analyzer (Abbott Laboratories, IL, USA).The AST intra-and inter-assay CVs were 0.7% and 1.0%, respectively, and the limit of detection was 3 U/ L. The ALT intra-and inter-assay CVs were 0.9% and 1.5%, respectively, and the limit of detection was 2 U/L.

Free 25-(OH)D levels from vitamin D binding protein analysis
VDBP concentration was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) (R&D Systems, MN, United States).The intra-assay CV ranges between 5% and 7%, and the inter-assay CV ranges from 5% to 8%.

VDBP, 25-(OH)D F , vitamin D metabolites, VMRs and psychophysical stress markers
We found significant seasonal rhythms for VDBP, 25-(OH)D T , and 25-(OH)D B .Similarly, Vitale el al. (Vitale et al., 2018).observed a significant circannual rhythm in 25-(OH)D T concentrations in male and female professional skiers.The higher vitamin D concentrations appeared in July, with the rhythm-adjusted mean and amplitude comparable between the two groups.Lombardi et al. (Lombardi et al., 2017)    The primary aim of athletes training is to supply stimulation that disrupts homeostasis to bring about adaptive responses that enhance physical performance.Therefore, maximizing the training stimulus is a key rule of athletic training.On the other hand, the ability to recover fast is crucial so that competitors can perform at high intensities more frequently.Human skeletal muscle counters to training stimuli and/or tissue damage through remodeling (Dahlquist et al., 2015).Some recent studies suggest that vitamin D might play an important role in skeletal muscle repair and Frontiers in Physiology frontiersin.org08 remodeling (Owens et al., 2018), and others have reported on the relationship between 25-(OH)D T muscle damage biomarkers, and overtraining symptoms.The results of these studies found no relationship between 25-(OH)D T and ferritin (Jastrzebska et al., 2017) or CK (Lombardi et al., 2017;Ferrari et al., 2020) in athletes from different sports disciplines.Based on the free hormone hypothesis, some vitamin D functions may be closely linked to its free fraction than total serum 25-(OH)D concentrations (Powe et al., 2011;Shieh et al., 2016).Hence, we examined whether 25-(OH)D F was associated better with psychophysical stress markers than 25-(OH)D T .However, we documented no significant association of 25-(OH)D fractions (total and free) with psychophysical stress markers in football players in each training period.
Testosterone is the principal male sex hormone and stimulates anabolic metabolism, causing an increase in muscle and skeletal system volume, strength, and endurance.Moreover, testosterone facilitates muscular adaptations to exercise and improves their recovery ability.Male reproductive physiology is influenced by 25-(OH)D (Crewther et al., 2011), with VDRs and vitamin D metabolizing enzymes expressed in Leydig cells (Jensen, 2012;Boisen et al., 2017), suggesting a direct role for vitamin D in steroidogenesis regulation.Based on this evidence, vitamin D may have a role in regulating testosterone levels.However, we found no correlation between 25-(OH)D T , 25-(OH)D F , and testosterone concentration in football players, which is in line with our previous study (Książek et al., 2021) in young, healthy men.Krzywański et al. (Krzywański et al., 2020) also evaluated the relationship between plasma 25-(OH)D T and testosterone concentrations in professional track and field athletes.Similar to the current study, they found no significant correlation between 25-(OH) D T and testosterone concentrations in male and female athletes, from strength or endurance disciplines, in any season.Crewther et al. (Crewther et al., 2020) assessed the interplay between 25-(OH)D T , cortisol, and testosterone and their effects on exercise performance in 88 male ice hockey players (<20 years), with no correlation evident between vitamin D and both hormones.Other studies also demonstrated no relationship between 25-(OH)D T and cortisol and testosterone concentration in elite soccer players (Lombardi et al., 2017;Ferrari et al., 2020) and ice hockey players (Fitzgerald et al., 2018).
This study had strengths and limitations.One of the study's strengths is the homogeneity of the cohort and the athletes having similar training loads/volumes.In addition, vitamin D metabolites were measured using gold-standard methods.The main limitation of this study was that the data collection period covered only half of the year instead of the whole year.Also, a greater number of participants would have increased the power of statistical   Frontiers in Physiology frontiersin.org09 analysis.Nevertheless, football teams usually have no more than 25 players.Moreover, we assessed 25-(OH)D F concentrations using a calculated method rather than directly measuring free vitamin D metabolite levels.Nonetheless, findings emerging from various studies indicate that VMRs, particularly the 24,25-(OH) 2 D-to-25-(OH)D ratio (i.e., the ratio indicating how much precursor could be converted into the bioactive form), may better represent the vitamin D status than 25-(OH)D alone since it considers the different metabolic fates.Specifically, the ratio of 24,25-(OH) 2 D:25-(OH)D should range between 4% and 12%, which reflects correct vitamin D status regardless of the absolute 25-(OH)D level (Alonso et al., 2022).

Free
The levels of bioavailable 25-(OH)D (25-(OH)D B ) were calculated using equations adapted from Powe et al. (Powe et al., 2011) Bioavailable 25 − OH ( )D 1 + 6 x 10 5 x albumin x Free 25 − OH ( )D The concentration of 25-(OH)D F and 25-(OH)D B were derived from the respective total (sum of D 3 + D 2 ) values.

except for 1 ,
25-(OH) 2 D, when fitted to cosinor curves.Our cohort of professional football players showed similar trends.Indeed, 3-epi-25-(OH)D 3 and 24,25-(OH) 2 D 3 had a propensity to fluctuate with 25-(OH)D T , with changes between summer and winter periods.Furthermore, 25-(OH)D 3 :24,25-(OH) 2 D 3 and 25-(OH)D 3 3 VMRs were less susceptible to seasonal fluctuation.It should be noted that sunlight exposure is one of the major factor, which influences on vitamin D metabolites levels, especially in countries situated at latitude above 40 °.Therefore, the results of this study show that higher concentration of vitamin D metabolites (25-(OH)D T , 25-(OH) D B, 25-(OH)D F , 24,25-(OH) 2 D 3 , 3-epi-25-(OH)D 3 ) occurred in summer time (July, August) compare to fall or winter months.Other factors, which may affecting on vitamin D metabolite levels is dietary intake of vitamin D or use of supplements.
have been documented.However, there are no such data on the effects of varied training loads over different training periods on the vitamin D metabolome.Therefore, we explored the relationship between 25-(OH)D 3 , 24,25-(OH) 2 D 3 , and 3-epi-25-(OH)D 3 in each training period, and found that 25-(OH)D 3 correlated strongly with 24,25-(OH) 2 D 3 in all periods.Although 24,25-(OH) 2 D and 3-epi-25-(OH)D are deemed to be biologically inactive metabolites, studies in animal models indicated that 24,25-(OH) 2 D exerts a pivotal role in maintaining bone integrity, function, and healing conclusion, a seasonal rhythm was present for VDBP, 25-(OH)D T , 25-(OH)D B , vitamin D metabolites (24,25-(OH) 2 D 3 , 3epi-25-(OH)D 3 ), and VMRs (25-(OH)D 3 :24,25-(OH) 2 D 3 , 25-(OH) D 3 :3-epi-25-(OH)D 3 ), though none was detected for 25-(OH)D F or psychophysical stress markers (ferritin, liver enzymes, CK, testosterone, cortisol, and T/C ratio).Furthermore, no correlation was observed between total or free 25-(OH)D, VMRs, or psychophysical stress markers.As such, the association between free and total 25-(OH)D and psychophysical stress markers do not demonstrate the superiority of free measurements over total measurements.The results of the present study did not provide evidence that 25-(OH)D T and 25-(OH)D F influence testosterone concentration in football players during different training periods.Moreover, training loads in different training periods did not affect resting vitamin D metabolite concentrations.

TABLE 1
Training content for each of the training periods.

TABLE 2
Changes in the levels of the biochemical parameters of football players (n = 20) during the training periods (V1-V6).

TABLE 3
Changes in the levels of the biochemical parameters of football players (n = 20) between summer and winter period.

TABLE 4
Rhythmometric analysis of VDBP, vitamin D metabolites and ratios of football players.PR: percentage of rhythm.MESOR: Midline Estimating Statistic of Rhythm.Amplitude: half the difference between the highest and the lowest points of the cosine function best fitting the data.Acrophase indicates the time in which the highest values occur.

TABLE 5
Correlations between 25-(OH)D 3 and vitamin D metabolites in each training period.