Participants
Eighteen apparently healthy, recreationally active, non-resistance trained [no consistent (at least thrice weekly) resistance training for one year prior to the study] males with an average age of 22.8 ± 4.67 yr, height of 179.5 ± 6.38 cm, and total body mass of 79.1 ± 16.13 kg completed the study. All participants passed a mandatory medical screening. Participants with contraindications to exercise as outlined by the American College of Sports Medicine and/or who had consumed any nutritional supplements (excluding multi-vitamins) such creatine monohydrate, nitric oxide, hydroxy-beta-methylbutyrate (HMB), various androstenedione derivatives, or pharmacologic agents such as anabolic steroids three months prior to the study were not allowed to participate. All eligible subjects signed a university-approved informed consent document. Additionally, all experimental procedures involved in this study conformed to the ethical considerations of the Helsinki Code.
Testing sessions
The study included baseline testing at day 0 followed by a follow-up testing session at day 29 in which blood and muscle samples were obtained and where body composition and muscle performance tests were performed.
Strength assessment
Upper- and lower-body one repetition maximum (1-RM) strength tests were performed using the free weight bench press and angled leg press exercises (Nebula, Versailles, OH), respectively. Initially, an estimated 50% (1-RM) measured from the previous testing 1-RM test, was utilized to complete 5 to 10 repetitions. After a two min rest period, a load of 70% of estimated (1-RM) was utilized to perform 3 to 5 repetitions. Weight was gradually increased until a 1-RM was reached with each following lift, with a two min rest period in between each successful lift. Test-retest reliability of performing these strength assessments on subjects within our laboratory has demonstrated low mean coefficients of variation and high reliability for the bench press (1.9%, intraclass r = 0.94) and leg press (0.7%, intraclass r = 0.91), respectively.
Body composition assessment
Total body mass (kg) was determined on a standard dual beam balance scale (Detecto Bridgeview, IL). Percent body fat, fat mass, and fat-free mass were determined using DEXA (Hologic Discovery Series W, Waltham, MA). Quality control calibration procedures were performed on a spine phantom (Hologic X-CALIBER Model DPA/QDR-1 anthropometric spine phantom) and a density step calibration phantom prior to each testing session. The DEXA scans were segmented into regions (right & left arm, right & left leg, and trunk). Each of these segments was analyzed for fat mass, lean mass, and bone mass.
Total body water volume was determined by bioelectric impedance analysis (Xitron Technologies Inc., San Diego, CA) using a low energy, high frequency current (500 micro-amps at a frequency of 50 kHz). Based on previous studies in our laboratory, the accuracy of the DEXA for body composition assessment is ± 2% as assessed by direct comparison with hydrodensitometry and scale weight. Test-retest reliability of performing assessments of total body water on subjects within our laboratory has demonstrated low mean coefficients of variation and high reliability (2.4%, intraclass r = 0.91).
Venous blood sampling and percutaneous muscle biopsies
Venous blood samples were obtained from the antecubital vein into a 10 ml collection tube using a standard vacutainer apparatus. Blood samples were allowed to stand at room temperature for 10 min and then centrifuged. The serum was removed and frozen at -80°C for later analysis.
Percutaneous muscle biopsies (50–70 mg) were obtained from the middle portion of the vastus lateralis muscle of the dominant leg at the midpoint between the patella and the greater trochanter of the femur at a depth between 1 and 2 cm. After sample removal, adipose tissue was trimmed from the muscle specimens, immediately frozen in liquid nitrogen, and stored at -80°C for later analysis.
Supplementation protocol and dietary monitoring
Participants were assigned to a 28-day supplementation protocol, in double-blind placebo controlled manner. Participants ingested either 27 g/day of placebo (maltodextrose) or 27 g/day of NO-Shotgun® (Vital Pharmaceuticals, Inc., Davie, FL). NO-Shotgun contains a proprietary blend of a number of compounds, but those assumed to target muscle strength and mass are creatine monohydrate, beta-alanine, arginine, KIC, and leucine. For each supplement, the dosage was ingested 30 min prior to each exercise session. For days where no exercise occurs, the full dosage of each supplement was ingested in the morning upon waking. Participants completed supplementation compliance questionnaires and returned empty bottles during the post-study testing session.
For dietary analysis, participants were required to record their dietary intake for four days prior to each of the two testing sessions at day 0 and day 29 blood and muscle samples were obtained. The participants' diets were not standardized and subjects were asked not to change their dietary habits during the course of the study. The four-day dietary recalls will be evaluated with the Food Processor IV Nutrition Software (ESHA, Salem OR) to determine the average daily macronutrient consumption of fat, carbohydrate, and protein in the diet for the duration of the study.
Resistance-training protocol
Participants completed a periodized 28-day resistance-training program split into two upper-extremity and two lower-extremity exercise sessions each wk for 28 days. This constituted a total of 16 exercise sessions, with eight upper-body and eight lower-body exercise sessions. Prior to each exercise session, participants performed a standardized series of stretching exercises. The participants then performed an upper-extremity resistance-training program consisting of nine exercises (bench press, lat pull, shoulder press, seated rows, shoulder shrugs, chest flies, biceps curl, triceps press down, and abdominal curls) twice per week and a program consisting of seven lower-extremity exercises (leg press, back extension, step ups, leg curls, leg extension, heel raises, and abdominal crunches). Participants performed three sets of 10 repetitions at 70 – 80% 1-RM. Rest periods were two min between exercises and between sets. The resistance exercise sessions were not supervised; however, it was required that each participant completed detailed daily resistance-training logs.
Whole blood and serum clinical chemistry analyses
Whole blood was collected and immediately analyzed for standard cell blood counts with percentage differentials (hemoglobin, hematocrit, RBC, MCV, MCH, MCHC, RDW, WBC counts, neutrophils, lymphocytes, monocytes, eosinophils, basophils and leukocyte differentials) using a Cell-Dyne 3500 (Abbott Diagnostics, Dallas, TX) automated hematology analyzer. The instrument's flow system was primed and the background counts checked daily to ensure appropriate RBC and WBC linearity. The coefficients of variation for the Cell-Dyne 3500 are 0.8747%, 0.8830%, 0.0296%, 0.7903%, and 0.8534% for neutrophils, lymphocytes, monocytes, eosinophils, and basophils, respectively.
Using a Dade Dimension RXL Analyzer (Dade Behring, Newark, DE), serum samples were assayed for general clinical chemistry markers (total cholesterol, high-density lipoproteins, low-density lipoproteins, triglycerides, albumin, glucose, GGT, LDH, uric acid, BUN, creatinine, BUN/creatinine ratio, calcium, creatine kinase, total protein, total bilirubin, ALP, ALT, and AST). This clinical chemistry analyzer was calibrated daily using liquid assay multiqual (BIO-RAD, Hercules, CA). For all assays mentioned above, the coefficients of variation are less than 5%.
Serum IGF-1 and HGF analyses
Serum samples were analyzed in duplicate for free/bioactive IGF-1 (Diagnostic Systems Laboratories, Webster, TX) and HGF (Biosource, Camarillo, CA) using an ELISA. For IGF-1, this assay has a sensitivity of 0.06 ng/ml, and does not cross-react with albumins or GH binding proteins. For HGF, the sensitivity is 10 pg/ml. For both IGF-1 and HGF, the subsequent absorbances, which were directly proportional to the concentration of analyte in the sample, were measured at a wavelength of 450 nm using a microplate reader (Wallac Victor 1420, Perkin Elmer, Boston MA). A set of standards of known concentrations for IGF-1 and HGF were utilized to construct standard curves by plotting the net absorbance values of the standards against their respective protein concentrations. By applying a four part parameter curve using MikroWin microplate data reduction software (Microtek Lab Systems, Germany), the free IGF-1 and HGF concentrations in the serum samples were calculated. The overall intra-assay percent coefficient of variation was 4.9% and 3.3% for IGF-1 and HGF, respectively.
Skeletal muscle phosphorylated c-met content and MRF ELISAs
Approximately 20 mg of each muscle sample was weighed and subsequently homogenized using a commercial cell extraction buffer (Biosource, Camarillo, CA) and a tissue homogenizer. The cell extraction buffer was supplemented with 1 mM phenylmethanesulphonylfluoride (PMSF) and a protease inhibitor cocktail (Sigma Chemical Company, St. Louis, MO) with broad specificity for the inhibition of serine, cysteine, and metallo-proteases.
Muscle homogenate samples were analyzed for phosphorylated c-met (Tyr1230/Tyr1234/Tyr1235) using a phosphoELISA kit (Millipore, Billerica, MA). This sensitivity of this particular assay is reported to be 0.78 U/ml. The absorbances, which are directly proportional to the concentration of c-met in the samples, were measured at 450 nm with a microplate reader (Wallac Victor 1420, Perkin Elmer, Boston MA). A set of standards of known concentrations for c-met were utilized to construct standard curves by plotting the net absorbance values of the standards against their respective protein concentrations. By applying a four part parameter curve using MikroWin microplate data reduction software (Microtek Lab Systems, Germany), the c-met concentrations in the muscle samples were appropriately calculated. The overall intra-assay percent coefficient of variation was 6.89%
The muscle protein expression of the MRFs was assessed through the use of ELISAs. Polyclonal antibodies specific for Myo-D, myogenin, MRF-4, and myf5 (where their target specificities had been verified by Western blotting) were purchased from Santa Cruz Biotech (Santa Cruz, CA). Initially, the antibodies were diluted to 1 μg/ml in coating buffer (Na2CO3, NaHCO3, and ddH2O, pH 9.6) and allowed to incubate at room temperature overnight. Following incubation, the plates were washed (1× phosphate buffered saline, Tween-20), blocked (10× phosphate buffered saline, bovine serum albumin, ddH2O), washed, and then incubated with a secondary antibody (IgG conjugated to HRP) diluted to 1 μg/ml in dilution buffer (10× phosphate buffered saline, Tween-20, bovine serum albumin, ddH2O). After washing, a stabilized TMB chromogen was added and the plates were covered and placed in the dark for the last 30-min prior to being stopped with 0.2 M sulphuric acid. The subsequent absorbances, which are directly proportional to the concentration of the MRFs in the samples, were measured at a wavelength of 450 nm. There were no standards used in these ELISAs, thus no standard curve was created. Therefore, the absorbances relative to muscle weight were assessed and compared as percent changes. The overall intra-assay percent coefficients of variation were 7.12%, 6.47%, 8.03%, and 6.57% for Myo-D, myogenin, MRF-4, and myf5, respectively.
Myofibrillar protein content
Total cellular RNA was extracted from biopsy samples with a monophasic solution of phenol and guanidine isothiocyanate contained within the TRI-reagent (Sigma Chemical Co., St. Louis, MO), and then isolated with 100% isopropanol. The interphase was removed and total (soluble + insoluble) muscle protein was then isolated from the organic phase with 100% isopropanol and washed with a 0.3 M guanidine HCl/95% ethanol solution. Myofibrillar (soluble) protein was further isolated with repeated incubations in 0.1% SDS at 50°C and separated by centrifugation. Total and myofibrillar protein content were determined spectrophotometrically based on the Bradford method at a wavelength of 595 nm [33]. A standard curve was generated (R = 0.98, p = 0.001) using bovine serum albumin (Bio-Rad, Hercules, CA), and total and myofibrillar protein content was expressed relative to muscle wet-weight [34].
Total DNA content
Total DNA was isolated from the remaining interphase from the total RNA isolation procedure using 100% ethanol, washed with a 0.1 M sodium citrate/10% ethanol solution, and resuspended in 75% ethanol. The DNA was then solubilized in 8 mM NaOH. The total DNA concentration was determined spectrophotometerically (Helio γ, Thermo Electron, Milford, MA) by optical density (OD) at 260 nm using an OD260 equivalent to 50 μg/μl [35]. At a wavelength of 260 nm, the average extinction coefficient for DNA is 0.024 μg/ml; therefore, an OD of 1.0 corresponds to a DNA concentration of 50 μg/ml. The final DNA concentration was expressed relative to muscle wet-weight.
Reported side effects from supplements
On day 29, participants reported by questionnaire whether they tolerated the supplement, supplementation protocol, as well as report any medical problems and/or symptoms they may have encountered throughout the study.
Statistical analysis
With the exception of the MRFs, all data were analyzed with separate 2 (group) × 2 (time) univariate ANOVA with repeated measures on the time factor with SPSS for Windows Version 16.0 software (SPSS inc., Chicago, IL). Significant differences among groups were identified by a Tukey HSD post-hoc test. For the MRFs, the percent changes from Day 0 to Day 29 were analyzed with separate independent group t-tests (p < 0.05). A probability level of ≤ 0.05 was adopted throughout.