Experimental approach
This investigation was completed as a randomized, double-blind, single-dose, comparator-controlled crossover trial. Ten apparently healthy men (n = 6) and women (n = 4) between the ages of 22–34 years were pre-screened using health history questionnaires, vital signs, and blood work prior to being enrolled in the study. All subjects were required to report to the laboratory after observing an eight hour fast (including caffeine) with all testing sessions taking place at near identical times in the morning. Additionally, subjects were asked to avoid exercising for 72 h prior to each research visit. Research procedures included venous blood draws and vastus lateralis muscle biopsies during a primed, constant infusion of L-[ring-d5]-phenylalanine (Cambridge Isotope Laboratories, Andover, MA). The fractional rate of muscle protein synthesis (FSR) was measured using the stable isotope tracer incorporation technique from vastus lateralis muscle biopsies performed two, four, and eight hours after initiating stable isotope tracer infusion. Blood samples were collected at baseline (time 0) and over an eight-hour time period (240, 270, 300, 330, 360, 390, 420 and 480 min) to assess changes in amino acid concentrations. Similarly, glucose and insulin concentrations were analyzed in venous blood samples collected 240, 270, 300, 330, 360, 390 and 480 min after tracer infusion. A skeletal muscle biopsy was performed two and four hours after tracer initiation followed by a single dose of the assigned test product administered orally. Study participants then completed eight sets of bilateral isotonic leg extension resistance exercise at a load equivalent to approximately 80% of their estimated one-repetition maximum (1-RM). A third biopsy was obtained four hours after test product ingestion. A washout period of 5 to 7 days was utilized before each subject was crossed over to the opposite condition and scheduled to complete an identical testing session. The order in which test products were provided was counterbalanced to prevent any order effect.
Study participants
Ten healthy male (n = 6) and female (n = 4) participants (mean ± SD: 26.6 ± 3.7 years, 175.5 ± 10.9 cm, 78.56 ± 17.4 kg) were recruited to participate in this study. All participants read and signed an IRB-approved informed consent to participate document prior to their participation in the study (Integreview, Austin, TX; approval date: January 13, 2015). All participants completed a medical history and were screened by a study physician and determined to be normotensive and euglycemic with normal fasting insulin and HOMA-IR values. Potential participants were excluded if they had a history of diabetes, smoking, malignancy in the previous 6 months or any other clinical condition that the researchers felt would compromise their safe participation. Individuals who recently lost more than ten pounds, had prior bariatric procedures or were diagnosed or being treated for any chronic inflammatory condition or disease (Lupus, HIV/AIDS, etc.) were also excluded. Participants were not allowed to be taking any form of chromium supplements or any other dietary ingredient deemed by the research team to affect insulin sensitivity or glucose tolerance. Participants must have been regularly consuming animal proteins and agreed to continue following their normal resistance training and protein/amino acid supplementation patterns. Finally, participants were also excluded if they had a known allergy to wheat proteins, amylopectin or chromium, were regularly using any form of corticosteroids, anabolic-androgenic steroids or were already participating in another research study.
Adverse event monitoring
All study participants were required to record any adverse events throughout the entire study protocol. Participants were queried for symptoms during and after their completion of the study protocol to assess both the incidence and severity of adverse events according to CTCAE grading and MedDRA guidelines.
Dietary and physical activity controls
All study participants were asked to maintain their current dietary and exercise/physical activity habits. Care was taken to control diet and physical activity levels 24 h prior to each experimental trial as all participants were required to complete a 24-h dietary recall prior to their initial experimental trial. A copy of this recall was made and all study participants were instructed to duplicate their dietary intake 24-h prior to their subsequent trial. As mentioned previously, all study participants were asked to refrain from exercise for 72 h prior to each visit and to fast for eight hours prior to testing. All dietary records were analyzed by the same research team member using the clinical edition of NutriBase IX (Phoenix, AZ).
Subject preparation
Participants reported to the laboratory after an overnight fast, were asked to void prior, and then height (in bare feet) and body mass were determined using a SECA Medical Scale (model 767, Hanover, Maryland USA). An 18–22-gauge polyethylene catheter was inserted into each arm by a research nurse; one was placed in a distal vein for heated blood sampling, and another was placed in the forearm for infusion of the stable isotope tracers.
Blood sampling
All blood samples were collected into lithium heparin tubes and centrifuged. Plasma samples were then aliquoted to minimize future freeze/thaw cycles and stored at −80° C until analyses. Plasma blood samples (5 ml) were collected at baseline (0 min) and after the beginning of isotope infusion (240, 270, 300, 330, 360, 390, 420 and 480 min) for analysis of amino acid concentrations and isotopic enrichment. Insulin and glucose concentrations in plasma were measured at 240, 270, 300, 330, 360, 390 and 480 min after baseline sampling.
Amino acid (isotopic) tracer
After insertion of peripheral catheters, a primed (5.04 μmol/kg), continuous (0.084 μmol/kg/min) infusion of the stable isotope ring-d5-phenylalanine was initiated. Stable isotopes were obtained from Cambridge Isotope Laboratories (Andover, MA), compounded by a licensed pharmacy (Cantrell Pharmacy, Little Rock, AR) and tested for sterility and pyrogenicity prior to administration. Prior to infusion into the subject, the isotope solution was passed through a sterile 0.22 μm (Millipore) filter.
Muscle biopsy procedure
Muscle biopsies from the vastus lateralis were performed two, four and eight hours after initiation of tracer infusion. After the biopsy at four hours, a single dose of WPACr or WP was administered orally under supervision. Muscle biopsies were performed under local anesthesia (using sterile 1% lidocaine, without epinephrine) for pain management. A 5 mm Bergström needle was advanced into the muscle through a small (~1 cm) incision. Immediately after applying suction, a sample of the muscle (approximately 100–120 mg) was removed with the needle. The sample was cleaned with sterile saline, trimmed of any visible connective tissue, blotted, and then cut into three equal portions (~30 – 40 mg). All three samples were immediately frozen in liquid nitrogen and stored at −80° C. One portion was utilized for determination of muscle protein synthesis, and the others were retained for backup analyses.
Supplementation protocol
Upon consent, study participants were randomly assigned in a double-blind fashion to one of two trials: 6 g of whey protein isolate (BiPro USA, Eden Prairie, MN) + 2 g of the test product (Velositol™) or 6 g of whey protein isolate. All provided supplements were prepared in powdered form and packaged in coded generic containers for double-blind administration and dissolved in 8 oz of water immediately prior to oral dosing. All samples were blinded and matched for appearance, color, aroma and flavor by the study sponsor. Batch analysis of provided product at a third-party facility (Eurofins Scientific, Inc, Des Moines, IA USA, Certificate of Analysis # AR-15-QD-031109-01) was completed of both WPACr and WP and results indicated that levels of all bioactive ingredients were consistent with those reported on the Supplement Facts Label (see Fig. 1). After a 5–7 day washout, subjects crossed over and completed the opposite trial. The order in which test products were provided was counterbalanced to prevent any order effect.
Resistance exercise protocol
As previously reported [25], all study participants then completed a single bout of bilateral leg extension exercise after supplement ingestion. Prior to beginning the study protocol all study participants determined their ten-repetition maximum and this load was used throughout the study. Each exercise trial consisted of eight sets of ten repetitions at their respective 10-repetition maximum load. A traditional plate-loaded leg extension machine was used and 90 s of rest was provided between each set. All repetitions were performed to near full-extension of the knee before returning to approximately 90–100° of knee flexion. Participants were instructed to extend the knee through the concentric phase for two seconds, briefly pause and return the knee eccentrically for a two second period. Each repetition was supervised by research personnel to ensure the proper load was used, each repetition was completed, and an appropriate range of motion and lifting cadence was followed. If a participant became too fatigued during the initial session to complete any repetition, the weight was lowered and this adjustment was matched during the subsequent visit. Thus, since all participants were required to complete the same number of repetitions at the same weight load, volume was equal between trials (within subjects).
Calculation of fractional synthesis rates of muscle protein synthesis
Upon thawing, muscle tissues were weighed, and tissue proteins were precipitated with 0.5 ml of 4% SSA. The tissues were then homogenized and then centrifuged for collection of supernatant. The procedure was repeated two more times, and tissue intracellular free AAs were extracted from the pooled supernatant via the same cation exchange chromatography stated in plasma analyses and then dried under the Speed Vac. The remaining muscle pellet was washed, dried, and hydrolyzed in 0.5 ml of 6 N HCl at 105 °C for 24 h. Enrichments from muscle free and bound tracers were determined as in plasma analyses. Calculation of the fractional rate of muscle protein synthesis (FSR) was accomplished by the following equation:
FSR (%/hr) = [(Ep2 – Ep1)/(Em X t)] X 60 X 100;
where EP1 and EP2 are the enrichments of bound l-[ring-2H5] phenylalanine in the first and second biopsies, respectively, and Em is the calculated mean value of the enrichments of [ring-2H5] phenylalanine in the plasma pool. t is the time in minutes elapsed between the first and second muscle biopsy. Factors 60 and 100 were used to express FSR in percent per hour (Kim et al. 2014).