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Serum levels of bone formation and resorption markers in relation to vitamin D status in professional gymnastics and physically active men during upper and lower body high-intensity exercise

Ethics statement

Experimental procedures and study design were approved by the Bioethics Committee for Clinical Research at the Regional Medical Chamber in Gdansk, Poland. The study was conducted according to the 1964 Declaration of Helsinki and its later amendments. Each participant gave a written consent to participate in the study, and was informed about the purpose and test procedures, and the possibility of withdrawal of consent at any time and for any reason.


A group of 16 elite male artistic gymnasts (EG) aged 20.6 ± 3.3 years-old and 16 physically active men (PAM) aged 19.9 ± 1.0 years-old participated in the study. Descriptive physical characteristics are shown in Table 1.

Table 1 Physical characteristics of the study participants (n = 32)

All gymnasts have been training regularly since 6–8 years old. (five times per week, 2-3 h per unit). At the time of the research, all gymnasts were in the highest form of preparation for participation in national and international competitions (senior level). During 1 week of training, the athletes completed from 9 to 10 training units lasting no more than 200 min (per unit). The four training session included mainly high intensity anaerobic performance. The effective duration of each session ranged from 12 to 18 min during which it was performed; jumping; squat jump; drop jump; sprint with the load; push-ups; pull up exercises including abdominal muscles, core, and arm strength training; back; and other specialized exercises using various training simulators. The remaining five training sessions included technical skills (floor exercises, pommel horse, rings, vault, parallel bars, and horizontal bar). The effective duration of a single session ranged from 40 to 50 min during which 210 to 270 gymnastic exercises were performed on selected aparatus, including 10 to 20 vault jumps. Elements from the higher difficulty groups (C, D and E) according to the Code of Points 2017–2020 International Gymnastics Federation classification constituted from 30 to 40% of all performed exercises.. PAM population declared regular participation in recreational sports, such as running, swimming, and team sports (on average, 2–3 times per week, 45–60 minunit). PAM were untrained in professional gymnastics and served as a control group for long-term gymnastic training.

All of the participants had a normal health status 3 months prior to the study, specifically, no injuries to the bone or the muscle tissue; no intake of drugs during the study; negative medical history regarding disorders of the cardiovascular system, autonomic nervous system, mental disorders, craniocerebral trauma, and other diseases that might directly affect the obtained results. The participants were informed of the nature and possible inconveniences associated with the experiment and the fact that they can opt out at any stage of the study.

Experimental overview

The study consisted of two parts:

  1. (1)

    measurement of the anaerobic components of fitness using WAnT, i.e. upper and lower body high-intensity exercises;

  2. (2)

    assessment of the serum levels of PINP (bone formation marker) and CTX (bone resorption marker), and vitamin D metabolites in serum samples collected in part (1).

The participants were instructed to avoid caffeine, alcohol, and any substances that could have influenced their physical performance 1 month before the experiment. All participants attended a 1-h familiarization session 1 week prior to the experiment, to ensure that they were familiar with the testing equipment and procedures. Further, 48 h prior to testing, the participants were asked to refrain from exhaustive exercise, to maintain their normal dietary habits, and to come to the laboratory in a euhydrated state.

All participants performed the lower and upper body WAnT (LBWT and UBWT, respectively). The actual measurements began with LBWT. Before the test, venous blood for serum isolation was taken at rest, and 5 and 30 min after test completion. One week later, the participants completed the UBWT. Blood samples were collected at rest before and after the test, as for the LBWT.

Measurements of the anaerobic components of fitness: LBWT and UBWT

LBWT was conducted using a cycle ergometer (Monark 894E, Peak Bike from Sweden) with MCE 5.1 software package („JBA” Zb. Staniak, Poland). For each participant, the saddle height was adjusted so that the knee remained slightly flexed after the completion of the downward stroke (with the final knee angle of approximately 170–175°). Toe clips were used to ensure that the participant’s feet were held firmly in place and in contact with the pedals. Before any experimental testing, each individual completed a standardised warm-up on the cycle ergometer (5 min at 60 rpm, 1 W/kg). Each participant was required to pedal with maximum effort for a period of 30 s against a fixed resistive load of 75 g/kg of total body mass as recommended by Bar-Or O [17].

UBWT was conducted using a hand cycle ergometer (Monark 891E). Participants sat in a chair affixed to the ground, and were advised to keep their feet flat on the ground and remain seated throughout the WAnT. The seat height and backrest were adjusted so that, with the crank position on the opposite side to the body and the hand grasping the handles, the elbow joint was almost fully extended (140–155°) and the shoulders were in line with the centre of the ergometer’s shaft. A standard resistive load equivalent to 50 g/kg of total body mass was used for each participant. Before the test, the participants completed a warm-up that involved 5 min of arm cranking at a power output of 1 W/kg and a crank rate of 60 rev/min.

For both WAnTs, each participant was instructed to cycle as fast as possible and was given a 3-s countdown before the set resistance was applied. Verbal encouragement was given to all participants to maintain their highest possible activity throughout the tests. Both cycle ergometers were connected to a PC to allow data capture via the MCE 5.1 software. The following WAnT variables were measured: peak power (W) and relative peak power (W/kg), calculated as the single highest point of power output (recorded at 0.2-s intervals); mean power (W) and relative mean power (W/kg), calculated as the average power output during the 30-s test.

Sample collection and methodology

The blood for biochemical analyses was collected three times on the day of each test (immediately before, and 5 and 30 min after WAnT completion) into serum separation tubes (Becton Dickinson, Oxford, UK). The blood was collected at rest, fasting, and in the morning (07:00–08:00). The tubes were centrifuged at 2000×g for 10 min at 4 °C and stored at − 80 °C until analysis.

Serum CTX assay

Serum CTX levels were determined by using IDS-iSYS CTX (CrossLaps®) (Immunodiagnostic Systems, Tyne and Wear, UK), a chemiluminescence immunoassay, and iSYS analyser, according to the manufacturer’s protocol. Before the analysis, the serum aliquots were kept frozen at − 80 °C, and had only be thawed once, i.e. prior to the analysis. All analyses were performed immediately after sample thawing. Assay performance was verified by using the manufacturer-supplied controls.

Serum PINP assay

Serum PINP levels were determined by using IDS-[kit name] (Immunodiagnostic Systems) and iSYS analyser, according to the manufacturer’s protocol. The two-site chemiluminometric assay involved a two-point calibration in triplicate (the top calibrant value was approximately 135 μg/l), quality control material (in duplicate), and 20 μl of sample analysed in single. Specimens with PINP values above the assay range (2–230 μg/L) were diluted in specimens with low PINP levels. At each study centre, several analytical batches of specimens were analysed to establish reference intervals, by using the method during routine analyses of clinical samples. The stored specimens were analysed within 2 months of collection. Total PINP was measured electrochemiluminometrically using a Roche E170 automated analyser (Roche Diagnostics, Burgess Hill, UK, and Vivoord, Belgium). As described by the manufacturer and corroborated by published studies, the within- and between-calibration CVs of the assay were < 3.7% and < 2.9%, respectively [7].

Serum vitamin D assays

Vitamin D active metabolites, 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3], as a proportion of the total serum concentration of 25-hydroxyvitamin D [25(OH)D], were analysed using the commercially available Total 25OH Vitamin D ELISA kits (BIOHIT OYJ, Helsinki, Finland), according to the manufacturer’s protocol. All assays were performed in duplicate. In the current study, the intra-assay CV for 25(OH) D was below 4%. Only samples that were not haemolysed were analysed.

Statistical analysis

Descriptive statistics were used to analyse mean ± SD for all measured variables. The normality of distribution was checked using the Shapiro–Wilk’s test. One-way analysis of variance (ANOVA) was used to determine the difference in WAnT performance characteristics (all variables) between the EG and PAM groups. In addition, the effect size of the analysed correlations was determined (Cohen’s d-value). All calculations and graphics were generated using GraphPad Prism 6.0 (ftx.pl/program/graphpad-prism). Differences were considered statistically significant at p ≤ 0.05.

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