Subjects
Forty-two resistance-trained male subjects participated in this study. Subjects had to have at least of one year prior resistance training at a frequency of three times per week to be considered for this study. Subjects were excluded from participation if they were not between 18 – 50 years of age; had reoccurring or chronic joint problems; were diagnosed with cardiovascular or pulmonary disorders and/or disease; had abnormal heart rate or blood pressure; had less than one year of training experience; and/or had been recently instructed by a physician not to participate in high intensity exercise. A physical readiness questionnaire was completed to assess general health of the participant. Subjects were not currently (or in the past six months) taking dietary supplements containing creatine, glutamine, arginine, HMB, androstendione, thermogenics, or any other ergogenic supplement. Subjects meeting eligibility criteria were informed of the requirements of the study and signed an informed consent statement in compliance with the Human Subjects Guidelines of Baylor University and the American College of Sports Medicine. Subjects were descriptively 27 ± 9 years; 178 ± 8 cm, 85.15 kg, and 18.6 ± 6% body fat.
Study Design
This study was conducted as a randomized, double blind, placebo controlled clinical trial. Subjects completed two familiarization sessions one week prior to baseline testing. During the familiarization sessions, subjects completed a health history questionnaire, personal information sheet, and signed an informed consent statement. In addition, subjects completed one set of leg press and bench press as well as a Wingate anaerobic power test. Each participant was given personal instruction on how to fill out their training and diet logs. Diet logs were kept in order to ensure caloric intake did not differ significantly between groups. Training logs were kept to document number of sets, repetitions and weight used during each training session. This was done to calculate training volume for each participant.
Subjects were then scheduled to report to the lab for baseline testing (T1). Participants were instructed to refrain from exercise for 48 hours before each testing session and fast for 10 hours prior to donating blood. Additionally, subjects were instructed to keep a four day diet record (including one weekend day) prior to each testing session. Testing sessions included assessment of body weight and body composition, procurement of fasting blood samples, and performing 1-RM and 80% of 1-RM tests on the bench press and leg press and a Wingate anaerobic power test. Subjects were then randomized to ingest placebo or ZMA supplements and to begin training as described below. Participants returned to the lab after four (T2) and eight weeks (T3) of training.
Testing Procedures
Nutritional records were evaluated and analyzed by a registered dietitian using the Food Processor nutritional analysis software (ESHA Research Inc., Salem, OR). Height was measured using standard anthropometry and total body weight was measured using a calibrated electronic scale with a precision of ± 0.02 kg (
Bridgeview, Illinois). Total body water was estimated using a Xitron 4200 Bioelectrical Impedance Analyzer (
Xitron Technologies Inc., San Diego, CA). The BIA method has been determined to be a valid measure for total body water [
17]. Whole-body (excluding cranium) composition was estimated by certified personnel using a Hologic QDR-4500W dual-energy x-ray absorptiometry (DEXA) using Hologic software version 9.80C (
Waltham, MA). This test evaluates body composition and body density by scanning the entire body with a low dose of radiation taking approximately 6 min. An analysis of the subject's fat mass, soft tissue (muscle) mass, and bone mass was provided and used to determine body composition changes throughout the duration of the study. The DEXA scans regions of the body (right arm, left arm, trunk, right leg, left leg) to determine bone mass, fat mass and lean mass within each region. The scanned bone, fat, and lean mass for each region is then subtotaled to determine whole-body (excluding cranium) values. Percent body fat was determined by dividing the amount of fat mass by the total scanned mass (bone mass, fat mass and lean mass). Test-retest reliability studies performed on male athletes with this DEXA machine yielded a mean deviation for total BMC and total fat free/soft tissue mass of 0.31% with a mean intra-class correlation of 0.985. This method of determining body composition has been shown to be valid [
18].
Subjects then donated approximately 25 ml of fasting blood using venipuncture techniques of an antecubital vein in the forearm according to standard procedures. Two 10 mL serum separation (SST) vacutainers and one 5 mL anticoagulant tube containing K
3 (EDTA) vacutainer was inserted for blood collection using multiple sample phlebotomy techniques. Serum from the SST were centrifuged at 5,000 rev × min
-1 for 10 min using a Biofuge 17R centrifuge (
Heraeus Inc., Germany). Serum from both SSTs was transferred into three microcentrifuge tubes and frozen at -80°C for subsequent analysis. Remaining serum was transferred from the SST and placed into a sterile collection tube. Serum and whole blood samples (EDTA) were refrigerated and sent to Quest Diagnostics Labs (
Dallas, TX). A complete 31 panel clinical chemistry profile and various markers of muscle/protein breakdown, shown in Table
1, was run on serum samples using the Technicon DAX model 96-0147 automated chemistry analyzer (
Technicon Inc. Terry Town, NY) following standard clinical procedures. Whole blood cell counts with percent differentials were run on whole blood samples using a Coulter STKS automated analyzer using standard procedures (
Coulter Inc., Hialeah, FL). These analyzers were calibrated daily to controls according to manufacturer's recommendations and federal guidelines for clinical diagnostic laboratories. Test to test reliability of performing these assays ranged from 2 to 6% for individual assays with an average variation of ± 3%. Samples were run in duplicate to verify results if the observed values were outside control values and/or clinical norms according to standard procedures. Total and free testosterone, IGF-1, growth hormone, and cortisol levels were assayed in duplicate in the Exercise Biochemical Nutrition Laboratory (EBNL) using standard ELISA techniques. Test to test reliability of performing these assays were ± 3–5%. Serum zinc and magnesium levels were assayed using standard procedures by the Department of Human Nutrition at The Ohio State University under the supervision of Robert DiSilvestro, PhD. Test to test reliability of performing these assays were ± 2–4%.
Table 1
Metabolic and clinical chemistry panels and various hormones analyzed
Sodium | White blood cell (WBC) count |
Potassium | Red blood cell (RBC) count |
Calcium | Hemoglobin. |
Chloride | Hematocrit |
Carbon | Mean corpuscular volume (MCV) |
Glucose | Mean corpuscular hemoglobin (MCH) blood cell. |
Blood urea nitrogen (BUN) | Mean corpuscular hemoglobin concentration (MCHC) |
Creatinine | Red cell distribution width (RDW) |
BUN/creatinine ratio | Platelet count |
Total protein | Neutrophils |
Albumin | Lymphocytes |
Globulin | Monocytes |
Albumin/globulin ratio | Eosinophils |
Bilirubin | Basophils |
Alkaline phosphatase |
Lipid panel
|
Aspartate aminotransferase (AST) | Triglycerides |
Alanine aminotransferase (ALT) | High-density lipoprotein (HDL) |
Gamma glutamyl transpeptidase (GGT) | Low-density lipoprotein (LDL) |
Lactate dehydrogenase | Cholesterol to HDL ratio (Total/HDL) |
Uric acid |
Hormones
|
Creatine kinase | Total testosterone |
| Free testosterone |
| IGF-1 |
| Growth hormone |
| Cortisol |
Subjects warmed-up (2 sets of 8 – 10 repetitions at approximately 50% of anticipated maximum) on the bench press. Subject's then performed successive 1 RM lifts starting at about 70% of anticipated 1-RM and increasing by 5 – 10 lbs until the subject reached their 1-RM. After the acquisition of max the participant rested five minutes and completed as many repetitions as possible at 80% 1-RM to asses' muscular endurance. Subjects were instructed on proper technique and mechanics of the movement. Hand position was also recorded to ensure test to test reliability. Subjects then rested for 10 minutes and warmed-up on the Nebula 45° Leg press (2 sets of 8 – 10 repetitions at approximately 50% of anticipated maximum). Subjects then performed successive 1-RM lifts on the leg press starting at about 70% of anticipated 1-RM and increasing by 10 – 25 lbs until reaching the subject's 1-RM. Subjects then perform an 80% of 1-RM endurance repetition tests on the hip/leg sled. Foot placement and sled height were recorded to ensure test to test reliability. All strength testing was supervised by a Certified Strength and Conditioning Specialist (CSCS). Test to test reliability of performing these strength tests on resistance-trained subjects in our laboratory have yielded low mean coefficients of variation and high reliability for the bench press (1.9%, intraclass r = 0.94) and leg press/hip sled (0.7%, intraclass r = 0.91).
Subjects then performed a single standardized Wingate cycle ergometry test to assess anaerobic capacity and power. This test consisted of having each participant sprint in an all out fashion on the bicycle ergometer for 30-sec against a standard workload of 0.075 kg/kg of body weight. Correlation coefficients of test-retest reliability for absolute peak power and mean power has been found to be r = 0.692 and r = 0.950, respectively.
Supplementation Protocol
Subjects were matched into two groups according to fat free mass, age, and resistance training experience. Subjects were then randomly assigned to receive in a double blind manner a dextrose placebo or Z-Mass PM™ (Cytodyne Technologies, Lakewood, NJ). Subjects ingested 4 capsules a day on an empty stomach 30–60 minutes prior to going to bed for 8-weeks according to label instructions. Each daily dose of Z-Mass PM™ contains 11 mg of Vitamin B-6 (pyridoxine hcl), 450 mg of magnesium (as magnesium aspartate), 30 mg of zinc (as monomethionine and aspartate), 10 mg of potassium (as potassium aspartate), and 706 mg of a proprietary blend of mucuna pruriens (seeds) standardized for 50 mg of L-Dopa (purported as a muscle relaxant) and Polypodium Vulare/Suma root (herb) standardized for 30 mg of 20-hydroxyecdysone (a plant sterol purported to reduce catabolism). Supplementation compliance was monitored on a weekly basis by a research nurse as well as by having the subjects return the bottles and left over supplement at the end of 4 and 8 weeks of supplementation. The pill count method was used to determine supplement usage and subject compliance. On a weekly basis subjects were asked to complete a "weekly follow-up assessment" chart that addressed possible symptoms or side effects related to the supplementation and training.
Training Protocol
Subjects participated in a periodized 4-day per week resistance-training program split into two upper and two lower extremity workouts per week for a total of 8-weeks. The subjects performed an upper body 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 seven exercise lower extremity program (leg press or squat, back extension, step ups, leg curls, leg extension, heel raises, and abdominal crunches) performed twice per week. Subjects performed 3 sets of 8 repetitions during weeks 0 – 4 with as much weight as they can lift and 3 sets of 10 repetitions during weeks 4 – 8 with as much weight as they could handle (typically 60 – 80% of 1-RM). Rest periods between exercises were no longer than 3 minutes and rest between sets were no longer than 2 minutes monitored with a standardized timer. Each participant was given instruction on technique by a Certified Strength and Conditioning Specialist (CSCS).
Statistical Analysis
Dependent variables were analyzed by univariate analysis of variance (ANOVA) with repeated measures using SPSS version 11.5 software (Chicago, IL). Due to loss of some frozen samples, serum zinc, magnesium, and hormone levels were determined on a subset of 26 subjects (n = 13 P, n = 13 ZMA). Data are presented as means and ± standard deviation changes form baseline for the P and ZMA groups, respectively. Data were considered significantly different when the probability of error was < 0.05. Power analysis in a 2 × 3 design indicates that an n-size of 20 per group yields high power (>0.9) for delta values of 0.75 to 1.25.