Nineteen 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. Enrollment was open to men of all ethnicities. 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-stimulating, 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 participants signed a university-approved informed consent document based on the guidelines set forth by the Institutional Review Board for the Protection of Human Subjects of Baylor University. Additionally, all experimental procedures involved in this study conformed to the ethical considerations of the Helsinki Code.
The study included baseline testing at day 0 and 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.
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 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 during the previous year has demonstrated low mean coefficients of variation and high reliability for the bench press (1.7%, intra-class r = 0.92) and leg press (0.72%, intra-class r = 0.93), 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. Based on previous quality control testing in our laboratory from the previous year, the accuracy of the DEXA for body composition assessment is ± 2.3% as assessed by direct comparison with hydrodensitometry and scale weight. 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).
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.
Participants were assigned to a 28-day supplementation protocol, in a double-blind, placebo-controlled manner. Participants ingested either 54 g/day of maltodextrose (CARB) or 27 g/day of NO-Shotgun® and 27 g/day of NO-Synthesize® (NOSS). For CARB, 27 g were ingested 30 min prior to exercise and 27 g within 30 min following exercise. NOSS ingested 27 g of NO-Shotgun® 30 min prior to exercise and 27 g/day NO-Synthesize® within 30 min following exercise. Immediately upon waking on non-training days, CARB ingested 27 g of the supplement, whereas NOSS ingested 27 g of NO-Synthesize®. For supplementation compliance, participants completed questionnaires and returned empty containers during the post-study testing session on day 29.
In order to monitor dietary intake, participants were required to record their food and drink intake for four days prior to each of the two testing sessions at day 0 and day 29. For standardization purposes, 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 were evaluated with the Food Processor IV Nutrition Software (ESHA, Salem OR) to determine the average daily macronutrient intake of fat, carbohydrate, and protein for the duration of the study.
Based on our previous study , participants completed a periodized 28-day resistance-training program split into two upper-extremity and two lower-extremity exercise sessions each week. 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 or squat, 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 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 an automated hematology analyzer (Sysmex XS-1000i, Mundelein, IL). The instrument's flow system was primed and the background counts checked daily to ensure appropriate RBC and WBC linearity. Based on the quality control standards from the manufacturer, the coefficients of variation for the Sysmex XS-1000i were 0.82%, 0.84%, 0.026%, 0.75%, and 0.82% for neutrophils, lymphocytes, monocytes, eosinophils, and basophils, respectively, and fell within the recommended ranges.
Serum samples were out-sourced (Quest Diagnostics, Dallas, TX) and 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). Based on the methodology employed for analysis, the coefficients of variation for all analyses reported by Quest Diagnostics (Dallas, TX) were no greater than 6%.
Serum IGF-1 analysis
Serum samples were analyzed in duplicate for IGF-1 (Enzo Life Sciences, Plymouth Meeting, PA) and HGF (R&D Systems, Minneapolis, MN) using an ELISA. For IGF-1, this assay has a sensitivity of 34.20 pg/ml and does not cross-react with IGFBPs 2, 3, and 4, HGF, or insulin. For IGF-1, 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 (iMark, Bio-Rad, Hercules, CA). A set of standards of known concentrations for IGF-1 was utilized to construct a standard curve by plotting the net absorbance values of the standards against the respective peptide concentrations. By applying a four-part parameter curve using data reduction software (Microplate Manager, Bio-Rad, Hercules, CA), the serum IGF-1 concentrations were calculated. The overall intra-assay percent coefficient of variation was 5.3% for IGF-1.
Skeletal muscle cellular extraction
Each muscle sample was weighed and approximately 20 mg were 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.
Myogenic regulatory factor quantitation
The muscle protein expression of the MRFs was assessed through the use of ELISA . Polyclonal antibodies specific for Myo-D, myogenin, and MRF-4 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 (1X phosphate buffered saline, Tween-20), blocked (10X 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 (10X 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 were 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. The overall intra-assay percent coefficients of variation were 7.12%, 6.47%, and 8.03% for Myo-D, myogenin, and MRF-4, respectively.
Myofibrillar protein content
Myofibrillar protein was isolated from the skeletal muscle cellular extracts with repeated incubations in 0.1% SDS at 50°C and separated by centrifugation. Myofibrillar protein content was determined spectrophotometrically based on the Bradford method at a wavelength of 595 nm [13, 15]. A standard curve was generated (R = 0.99, p = 0.001) using bovine serum albumin (Bio-Rad, Hercules, CA), and myofibrillar protein content was expressed relative to muscle wet-weight.
Myosin heavy chain isoform protein quantitation
The MHC protein isoform composition within 20 μg muscle homogenates was determined under denaturing conditions using an Experion Pro260 automated electrophoresis system (Bio-Rad, Hercules, CA) using the principles of SDS-PAGE and LabChip (Caliper Life Sciences, Hopkinton, MA) technology . The Experion Pro260 analysis kit has a resolution and quantitation of 10-260 kDa proteins while also separating and detecting 2.5-2,000 ng/μl protein. The Experion Pro260 system combines electrophoresis, staining, destaining, imaging, band detection, and basic data analysis into a single, automated step. Gel images were then processed and displayed on a computer monitor and MHC bands identified by migration relative to the molecular weight marker (data not shown). The density of the MHC bands was determined using Experion Imaging software (Bio-Rad, Hercules, CA), expressed in arbitrary density units.
Reported side effects from supplements
Participants reported by questionnaire at the testing session on day 29 how well they tolerated the supplementation protocol, in addition to reporting any medical problems and/or symptoms they may have encountered during the study.
Data were analyzed with separate 2 (group) × 2 (time) ANOVA with repeated measures on the second factor with SPSS 16.0 software (SPSS inc., Chicago, IL). Significant differences among groups were identified by a Tukey HSD post-hoc test. A probability level of < 0.05 was adopted throughout.