All procedures were approved by the Bioethics Committee at the Regional Medical Chamber in Gdansk (KB-10/16). The study protocol was constructed according to the Declaration of Helsinki, and all participants gave their written consent, were fully informed prior to participation, informed about the possibility of withdrawal at any time for any reason and were given the opportunity to view their results.
From the group of 40 volunteers, 24 women meeting the inclusion criteria were selected. Only women at the age of 65 years and older, with BMI under 25, not taking any medications on a permanent basis, not smoking or having any other addictions. None of the women took any supplements for 3 months prior to the experiment. Only those qualified by a sports medicine doctor took part in the experiment. The women were subjected to tests typical for athletes, i.e. general medical interview, electrocardiogram, blood pressure and heart rate measurements, as well as basic laboratory determinations (morphology, urine, blood glucose, cholesterol). Participants were divided randomly into two groups: supplemented group (SUP) n = 12, mean age 72.8 ± 5.26 years, mean body mass 68.1 ± 8.3 kg, and control group (CON) n=12, mean age 72.4 ± 5.5 years, body mass 67.7 ± 7.5 kg. All participants have never been professional athletes. The participants did not know who received vitamin C supplementation or cellulose (products in caps, look similar, double-blinded).
Body mass analysis was performed using the InBody 720 composition analysis (In body, Department Poland). The basic characteristics of the SUP and CON are summarized in the Results section (Table 1). To eliminate women with poor diets we also performed diet analysis and participants were asked not to change their nutritional habits during the study.
The training program was similar to the one used and described earlier . Mainly the principles of health training were followed. Women participated in a 6-week multidisciplinary training program (three times per week for 60 minutes in duration) consisting of gyrokinesis, stabilization training, and Nordic walking at moderate intensity. Total duration of the training period overall was 1080 minutes. Heart rate was monitored during each training session (Polar H1). Participants were asked not to exceed a heart rate of 130/min.
During the 6 weeks of training, the SUP group received 1000mg of vitamin C (Max VitaC 1000, Colfarm, Poland) and CON group received cellulose in tablets (Colfarm, Poland). People conducting the experiment and participants did not know who was receiving supplementation and who was receiving placebo. The choice of vitamin C dose was chosen due to its easy availability and high frequency of consumption in the Polish population.
Determination of VO2 max.
The VO2 max was determined using the cycloergometer Ergoline Ergoselect 150p (Jaeger OxyconPro) and a gas analyzer (Jaeger OxyconPro) using the direct method. The measurement procedure was as follows: 2 minutes for registration of resting values followed by 5 minutes of a warm-up with 30W load and 60 rpm cadence. Participants performed the test with the load gradually increasing by 10W/min. The test was stopped if the participant was unable to continue with a given power and the required term of 60 rpm, or if symptoms occurred which indicated the need to end the trial. The results of VO2 max testing are presented in Table 1.
Blood collection, analysis of vitamin C concentration, total oxidative status (TOS)/total oxidative capacity (TOC), total antioxidative status (TAS)/total antioxidative capacity (TAC) analysis and analysis of gene expression.
Blood samples were collected twice: immediately before and 24 hours after the training period. Venous blood (5mL) was collected into BD Vacutainer tubes (Becton Dickinson, USA), in order to evaluate TOS/TOC and TAS/TAC. Plasma was separated by centrifugation (3000g at 4°C for 10 minutes). Collected plasma was stored immediately at -80°C for further analysis. We used photometric PerOx assay kit to evaluate TOS/TOC status (Immundiagnostik AG, Germany), and ImAnOx assay kit (Immundiagnostik AG, Germany) for TAS/TAC analysis. From the obtained results prooxidative/antioxidative balance was calculated.
Vitamin C plasma concentration measurement
Vitamin C concentration was determined in plasma using the method of Robitaille and Hoffer  described in our earlier paper .
The protocol used for gene expression evaluation has been previously described in detail by Grzybkowska , Żychowska . Briefly, 2ml of venous blood was collected and to remove red blood cells, the Red Blood Cell Lysis Buffer (RBCL) (A&A Biotechnology, Gdynia, Poland) was added and the samples were then centrifuged. Obtained leukocytes were lysed using Fenozol (A&A Biotechnology, Gdynia, Poland) and stored at - 20°C. The isolation of total RNA was performed based on Chomczynski and Sacchi method  using 200 µL of chloroform (POCH, Gliwice, Poland), centrifugation, and 500 µL of isopropanol (POCH, Gliwice, Poland). The obtained pellet was washed twice in 1ml of 75% ethanol and spun at 7500 g at 4°C. The dried RNA was then resuspended in the PCR-grade water, and after spectrophotometric evaluation of the purity and concentration of obtained material, the reverse transcription procedure was performed using 1000ng of pure RNA, 0.2 µM oligo(dT) and the Transcriptor First Strand cDNA Synthesis Kit as per the manufacturer’s instructions (Roche, Warszawa, Poland). Immediately after this step, the samples were frozen and stored at - 20°C without additional freeze-thaw cycles. For the quantitative real-time polymerase chain reaction (qRT-PCR) step, the 1:10 dilution of the cDNA has been used. This step was performed on AriaMx Real-Time PCR System (Agilent Technologies, Warszawa Poland) using FastStart Universal SYBR® Green Master (Rox) (Roche, Warszawa, Poland) according to the manufacturer’s protocol. Three replicates of 2 µL of diluted cDNA were used for qRT-PCR analysis. For each reaction, the melt curve analysis was performed to check for non-specific amplification.
The TUBB was used as a reference gene. All primers sequences were designed by authors in the Primer3 web tool and then in silico PCR tool in the USCS genome browser was used. All primers were delivered by Genomed, Warszawa, Poland. Primer sequences (5’-3’) used to perform this experiment were as follows:
For TUBB (tubulin beta class I, NM_001293213): Forward primer: CTAGAACCTGGGACCATGGA and Reverse primer: TGCAGGCAGTCACAGCTCT
For IL1 (NM_000575.5): Forward primer: AGT GCT GCT GAA GGA GAT GCC T and Reverse primer: CCT GCC AAG CAC ACC CAG TAG
For IL6 (NM_000600.5): Forward primer: AAT TCG GTA CAT CCT CGA CGG and Reverse primer: GAA TCC AGA TTG GAA GCA TCC
For IL10 (NM_000572.3); Forward primer: GAC ATC AAG GCG CAT GTG AAC Reverse primer: TCC ACG GCC TTG CTC TTG TTT
For CCL2 (NM_002982.4); Forward primer: CAG CCA GAT GCA ATC AAT GCC Reverse primer: CTTGGCCACAATGGTCTTGAA
For CRP (NM_000567.3): Forward primer: TCG TTA ACG GTG CTT TGA GG and Reverse primer: TCT TGG TCT TGA CCA GCC TCT
First, the presence of a normal distribution was checked with the Shapiro-Wilk’s test for all results. Data obtained before and after six weeks of training period were compared within each group using a paired test. The differences between groups were analyzed using parametric or non-parametric (Wilcoxon) tests, as appropriate, as well as two-way ANOVA. The pro and antioxidative balance were calculated as the pro/anti ratio. Relative expression was calculated in Microsoft Excel 2015, using Schmittgen and Livak’s method . Data were transformed into linear values, and subjected to the same methodology as the other parameters. To determine the significance of differences between the groups in gene expression, t-test and two-way ANOVA were used. Calculations and figures were generated using GraphPad Prism 6.0 software. P-values were considered statistically significant when ≤ 0.05.