Metabolomic Impacts of Branched-chain Amino Acid Supplementation During Endurance Exercise
Condition: Healthy
Study type: Interventional

Summary: 
      The overall goal of this investigation was to determine the impact of branch-chained amino
      acids (BCAAs) supplementation on serotonin and associated metabolites during endurance
      exercise on healthy individuals. Specifically, despite the strong theoretical basis for
      supplementation of BCAAs to attenuate serotonin production and fatigue during exercise, most
      human clinical trials have failed to demonstrate these benefits. To shed light on this
      discrepancy, the purpose of this study was to measure the impact of BCAA supplementation on
      serotonin and associated metabolites during exercise.

      To achieve this purpose, investigators determined the effects of the consumption of a BCAA
      beverage on endurance exercise performance and serum metabolites. The supplementation
      protocol consisted of two trials in which each participant consumed 8 grams of BCAA
      supplement in a beverage or a placebo in randomized order. It was hypothesized that
      post-exercise metabolites would differ between BCAA and placebo conditions.
    

Detailed description: 
      Research Design This study utilized a randomized, placebo-controlled, double-blind crossover
      design comparing supplementation of BCAA versus placebo beverages consumed immediately before
      and halfway through a 60-minute run at 65% of maximal aerobic capacity. To isolate the impact
      of BCAA on serotonin and metabolism during exercise, blood samples were collected immediately
      before and after exercise. A targeted metabolomic analysis was designed for BCAA to verify
      supplementation induced increases in serum concentration, as well as for serotonin to assess
      impacts of BCAAs. Untargeted metabolomic analysis was performed to identify global metabolic
      impacts of BCAA supplementation on metabolism during endurance exercise.

      Maximal Aerobic Capacity A graded exercise test was used to measure maximal oxygen
      consumption (VO2max) using a motorized treadmill. Each participant was fitted with an
      electronic heart rate (HR) monitor, a mouthpiece and a two-way non-rebreathing tube and a
      nose clip, all of which were attached to a standard laboratory metabolic cart (TrueOne 2400,
      ParvoMedics, Sandy, Utah). Participants self-selected a speed between 5 and 7.5 mph and began
      running at a 0% incline. Each minute, the treadmill incline was increased by 1.5% while speed
      remained constant until the participant voluntarily terminated the test due to exhaustion.
      Each participant's VO2max was defined as the highest 30 second average value collected during
      the test. Heart rate at 65% of VO2max was then determined and used for the experimental
      trials.

      Experimental procedure Participants were asked to return to the lab no earlier than 72 hours
      after the completion of the VO2max test to complete the first of two trials in the
      experimental procedure. In the 48 hours prior to each trial, participants were asked to
      record what they were eating, drinking and if they took any medications. All participants
      were asked to refrain from consuming alcohol within the 48 hours prior to the trials. With
      the exception of the experimental beverage, each of the two trials were identical in
      procedure and were separated by at least 72 hours. Testing was performed at the same time of
      day for each participant and all testing was completed between 4:00 and 8:00 p.m.

      Participants were provided with a randomly assigned beverage containing either BCAAs or a
      placebo solution. Participants consumed the experimental beverage five minutes prior to their
      running trial. Each participant was then fitted with a HR monitor and blood was drawn three
      minutes after ingestion. The blood draw was immediately followed by a warm-up on the
      treadmill at a self-selected speed for two to five minutes. Each participant then started
      their 60-minute running trial at 0% incline at 65% of their established VO2max. Halfway into
      the 60-minute trial, each participant ingested another serving of the assigned placebo or
      BCAA beverage. Heart rate and the rate of perceived exertion (RPE) were collected every 10
      minutes during the 60-minute trial. At the conclusion of the 60 minutes, participants cooled
      down for two minutes before exiting the treadmill and completing the post-exercise blood
      draw. Blood samples were allowed to clot for 15 minutes followed by centrifugation at 1200
      RPM for 15 minutes at 4°C. The serum supernatant was then collected in clean vials and
      immediately stored at -80°C until liquid chromatography mass spectrometry (LCMS) analysis.
      Treadmill speed during the first trial were recorded and replicated during the second trial.

      Experimental beverages Each participant was provided a BCAA supplement solution or a placebo
      solution in a double-blind, randomized crossover design. The BCAA solution was 8 oz of water
      mixed with approximately 8 grams of a standard BCAA supplement in powder form. Each 8-gram
      dose contained 2.5 grams of leucine, 1.25 grams of isoleucine and 1.25 grams of valine. The
      presence of BCAA was confirmed by LCMS. The placebo solution was 8 oz of water mixed with
      approximately 2.0 mL of a sucralose-based drink mix. Both the BCAA solution and the placebo
      solution were similar in color and in taste. Participants were allowed to drink additional,
      plain water during the 60-minute running trial if desired.

      Metabolite extraction Serum samples were thawed and 20μL of serum was removed and placed in a
      clean vial. Protein precipitation was completed with the addition of 80μL of cold acetone
      followed by agitation on a vortex machine and two hours in a -80°C freezer. Serum was then
      centrifuged at 20,000g for 10 minutes at -4°C. The metabolite rich supernatant was collected
      and concentrated using negative pressure to dryness (ConcentratorPlus, Eppendorf, Hamburg,
      Germany). Samples were then stored at -80°C for no more than 24 hours until ready for LCMS
      analysis. Directly before LCMS analysis, metabolite samples were reconstituted with 40μL of
      methanol:water (50:50) and placed in a clean mass spectrometry vial.

      LCMS conditions LCMS analysis was performed on an Agilent 6538 Q-TOF MS (Agilent
      Technologies, Santa Clara, CA) coupled to an Agilent 1290 UHPLC (Agilent Technologies, Santa
      Clara, CA) using a 1.8μm, 2.1mm X 150mm Waters HSST-3 UPLC column (Waters Corp., Milford,
      MA). Electrospray ionization was in positive mode. LC mobile phases were water (A), and
      acetonitrile (B), both with 0.1% formic acid. Flow was kept constant at 300µL per minute. The
      mobile phase gradient began with 95% A and finished with 5% A after seven minutes before
      returning to 95% at eight minutes and continuing to the end of the ten-minute run time.
      Column compartment temperature was kept constant at 30°C. MSMS analysis was completed using
      identical conditions with pooled extracted serum samples.

      Data analysis Serotonin standards (Thermo Fisher Scientific, Waltham, MA) were analyzed under
      the described LCMS conditions and the retention time and m/z value were determined.
      Concentrations of 0.001µM, 0.01µM, 0.05µM, 0.1µM, 0.5µM, 1µM and 5µM were analyzed allowing
      for the creation of a standard concentration curve and the determination of the limit of
      detection. Peaks for serotonin from participant serum samples were then integrated and the
      concentration was calculated from the standard curve using MassHunter (Agilent Technologies,
      Santa Clara, CA). For the untargeted data analysis, the raw data files were converted to
      .mzML and .mgf files using MSConvert and then mined using mzMine. Blank samples were also
      created using the same metabolite procedure without serum and were analyzed under identical
      LCMS condition concurrently. The resulting sample blank data was also converted and mined
      with the sample data and were used to remove machine background and mobile phase
      contributions to the data. Cleaned datasets and MSMS data were then statistically analyzed
      using MetaboAnalyst and Sirius software, respectively. The BCAA supplement was also examined
      using LCMS and the results were analyzed to confirm the presence of BCAAs and additives
      including flavoring and sweetening agents.

      Statistical Analysis Serotonin concentrations were normalized and center scaled using the
      caret R package. After normalization, a nested ANOVA was completed with serotonin
      concentration as the dependent variable and treatment and time as independent variables. The
      results of the nested ANOVA analysis led to further exploration of the groups and t-tests
      were performed between treatment and temporal groupings.
    

Intervention name(s): Branched Chain Amino Acids; Placebo
Intervention type(s): Dietary Supplement; Other
Intervention description(s): Supplementation with 8 g Nutrex Research Amino Acid Drive BCAA in 8 ounces of water once each immediately pre-exercise and after 30 minutes of exercise; Supplementation with 2.0 milliliters of a sucralose-based drink mix with 8 ounces of water once each immediately pre-exercise and after 30 minutes of exercise
Allocation: Randomized
Intervention model: Crossover Assignment
Intervention model description: 60 minutes of moderate (65% of VO2max) endurance exercise with either a branched chain amino acid supplement or a placebo consumed immediately prior to and 30 minutes into the exercise bout.
Masking: Double (Participant, Investigator)
Primary purpose: Other
Keywords: Branch-chained amino acids; Endurance exercise; Metabolomics; Serotonin