Eighteen male and ten female participants (n = 28; age 25 ± 5 years, height 175 ± 8 cm, body weight 74 ± 13 kg) completed the study. All participants were physical active, accustomed to strength training, conducting regularly strength training 1–4 times per week before the start of the study. Physical activity prior to the study was reported with a questionnaire. All participants gave written informed consent before entering the study, and were informed about potential risks related to the experiment. The study was approved by the Regional Ethics Committee of Southern Norway (2010/1352) and was performed in accordance with the Helsinki Declaration.
The detailed experimental design and limitations of the present study has already been described . Results on muscle mass and more strength measurements have been published previously . In a randomized double blinded manner, participants were allocated to receive either a vitamin C and E or a placebo supplement based on baseline maximal strength tests (1RM) and sex.
The vitamin C and E and placebo pills were produced by Petefa AB (Västra Frölunda, Sweden) under Good Manufacturing Practice (GMP) requirements. Each vitamin pill contained 250 mg of ascorbic acid and 58.5 mg DL-alpha-tocopherol acetate. The placebo pills had the same shape and appearance as the vitamins pills. All supplements were stored in similar unlabeled boxes, and were consumed orally with an artificially favored sucrose (30 g) drink to mask any potential taste from the pills.
The participants ingested 2 pills (total 500 mg of vitamin C and 117 mg vitamin E) 1–3 h before every training session and 2 pills in the first hour after training. On non-training days, the participants ingested 2 pills in the morning and 2 pills in the evening. The intake of pills was confirmed with an online training diary. Thus, daily dosage was 1000 mg of vitamin C and 235 mg vitamin E. The total supplemental dosage of vitamin C was ~13 times higher and ~23 times higher for vitamin E than the recommended daily dietary allowance in the Nordic countries.
Beside the supplementation given in the study, the participants were asked to not take any form of nutritional supplement or medication that could affect the strength training adaptations, such as NSAIDs. They were also asked not to drink more than 2 glasses of juice and 4 cups of coffee or tea per day. Juices rich in antioxidants, such as grape juice, were completely avoided.
The exercise consisted of strength training with heavy loads (6-11RM) for 10 weeks. The first six weeks the loads were 3 × 9-11RM, and 3-4 × 6-8RM the last four weeks. Sets were separated by a 1–1.5 min break. The exercise program included exercises for all major muscle groups in a 4-split exercise program (two upper- and two lower body sessions per week), with a seven upper and six lower body exercises. The exercise program was designed with the main goal to stimulate both maximal strength and muscle growth. The adherence and control of exercise and supplementation was monitored and logged using an online training diary. For variation and motivation participants were allowed to do alternative exercise forms (e.g. cycling or cross country skiing) once per week in addition to the planned training sessions.
Acute exercise session
After 4–6 weeks, ten male and five female participants (n = 15; age 26 ± 7 years, height 177 ± 7 cm, body weight 73 ± 13 kg) volunteered to an acute exercise experiment. The exercise session consisted of 4x10RM of leg press and knee-extension, with 1 min rest between sets and 3 min rest between exercises. Muscle biopsies were collected from m. vastus lateralis before and 100 and 150 min after the standardized exercise session using a modified Bergström technique (described in the Muscle tissue sampling and pre-analytic handling). Participants ingested the supplements, vitamin C and E or placebo, together with a standardized breakfast (3 g oat per kg body weight boiled in water with 5 g sucrose) two hours before the exercise session. A second dose of supplements was taken immediately after the exercise session.
Muscle tissue sampling and pre-analytic handling
Muscle biopsies from the mid-portion of the right m. vastus lateralis were collected before and after the training intervention. The post training insertion was proximally located to the pre-training site (approximately 3 cm). For the participants that also took part in the mid-way, acute session experiments, the biopsies were collected from the left m. vastus lateralis. One insertion was made for the pre sample, and a new insertion was made proximally from this for the post exercise samples. The two post samples were collected from the same insertion site, at two different directions. The first was sampled proximally and the second distally from the insertion site. The procedure was conducted under local anesthesia (Xylocain adrenalin, 10 mg/ml + 5 μg/ml, AstraZeneca PLC, London, UK). Approximately 200 mg (2-3 × 50–150 mg) of muscle tissue was obtained with a modified Bergström-technique. Tissue intended for homogenization and protein measurements was quickly washed in physiological saline, and fat, connective tissue, and blood were removed and discarded before the sample was weighed and quickly frozen in isopentane cooled on dry ice. Tissue intended for mRNA analyses were placed in RNAlater (AM7020, Ambion, Life technologies, Carlsbad, CA, USA). All muscle samples were stored at −80 °C for later analyses.
Muscle tissue was homogenized using a commercial homogenization buffer (78510, T-PER/Tissue Protein Extraction Reagent, Thermo Scientific, Rockford, IL, USA) with a cocktail of protease and phosphatase inhibitors (1861281, Halt protein and phosphatase inhibitor cocktail, Thermo Scientific) and EDTA (1861274, Thermo Scientific). Quantification of protein extracts was assessed with the BioRad DC protein micro plate assay (0113, 0114, 0115, Bio-Rad, CA, USA). A filter photometer (Expert 96, ASYS Hitech, Cambridge, UK) was used to measure the colorimetric reaction and sample protein concentration was calculated by the provided software (Kim, ver. 22.214.171.124, Daniel Kittrich, Prague, Czech Republic).
Extracted proteins were analyzed by western blotting. Equal amounts of protein were loaded per well (15 μg) and separated by 4–12% SDS gradient gels under denaturized conditions. Proteins were transferred onto PVDF membranes (162–0177, Immuno-blot, Bio-Rad or iBlot Gel transfer stacks, IB4010, Invitrogen, Carlsbad, CA, USA) before blocked in a 5% fat free skimmed milk and 0.1% TBS-t solution (TBS, 170–6435, Bio-Rad; Tween 20, 437082Q, VWR International, Radnor, PA, USA; Skim milk, 1.15363, Merck, Darmstadt, Germany). Blocked membranes were incubated in antibodies against GPx1 (ab22604, Abcam, Cambridge, UK), mnSOD (ab16956, Abcam), IκBα (ab32518, Abcam), HSP70 (ADI-SPA-810, Enzo Life Sciences, Farmingdale, NY, USA) or αB-crystallin (ADI-SPA-222, Enzo Life Sciences), followed by incubation in an appropriate secondary antibody (31430, Thermo Scientific; 7074, Cell Signaling Technology, Danvers, MA, USA). Between stages, membranes were washed in 0.1% TBS-t solution. Bands were visualized using a HRP-detection system (34076, Super Signal West Dura Extended Duration Substrate, Thermo Scientific). Chemiluminescence was measured using a CCD image sensor (Image Station 2000R or Image Station 4000R, Eastman Kodak Inc., Rochester, NY, USA) and band intensities were calculated with the Carestream molecular imaging software (Carestream Health Inc., Rochester, NY, USA). All samples were run as duplicates and mean values were used for statistical analyses.
HSP27 in the cytosolic and cytoskeletal fractions was measured as previously described in detail . Briefly, HSP27 was detected using a in house-made double antibody sandwich ELISA. By using capture antibodies (25 ng/well; ADI-SPA-800, Enzo Life Sciences) and detection antibodies against HSP27 (ADI-SPA-803, Enzo Life Sciences), HSP27 was determined by using a filter photometer (Expert 96, ASYS Hitech) measuring optical density at 450 nm.
Total RNA was extracted from muscle biopsies (n = 14) from the acute study by homogenization in TRIzol reagent (15596, Invitrogen, Life Technologies). DNase I digestion was performed using RNase free-DNase from Qiagen (79254, Qiagen Inc., Germantown, MD, USA) in order to prevent genomic DNA contamination. Quantitative Reverse Transcription PCR (RT-qPCR) analysis was performed in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) by using Power SYBR Green RNA-to-Ct™ 1-step Kit (4389986, Applied Biosystems) supplemented with forward and reverse primer in a total volume of 20 μl. The Reverse Transcription step was performed at 48 °C for 30 min in the presence of RNase inhibitor by using 6 ng of total RNA Thermocycling conditions were according to the recommendations of the manufacturer. Ct values for gene expression were calculated according to the comparative Ct method . Relative quantification was performed by simultaneous quantification of GAPDH and 18S gene expression. The primers used for RT–qPCR analyses can be found as Additional file 1.
All values are presented as means ± standard deviations (SD). A two-way ANOVA was used to evaluate the effects of training (time) and supplementation (interaction), and a Holm-Sidak multiple comparisons test was chosen for post hoc analyses. In general, figures display individual data points, mean and standard deviations. The level of significance was set to P < 0.05. Graphpad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA) was used for the statistical analyses.