Males and females aged 18-45 years who participated in at least five hours of cardiovascular activity (running, biking, swimming, etc.) per week were recruited for this study. Exclusion criteria included the presence of any underlying injury as well as the prescription of selective serotonin reuptake inhibitor (SSRI) medications. SSRIs inhibit the reuptake of serotonin within the synaptic cleft, resulting in increased levels of serotonin in the brain (Sangkuhl 2009). Safety and Ethics approval was granted by the Montana State University Review Board (Project No. DF081319). All participants gave written informed consent prior to initiation of study activities.
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 and serotonin to verify supplementation induced increases in BCAA serum concentration, as well as for serotonin to assess the impacts of BCAAs. An 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 (VO2 max) using a motorized treadmill. Each participant was fitted with an electronic heart rate (HR) monitor, a noseclip and mouthpiece connected to a one-way Rudolph valve and breathing tube to deliver expired air to a metabolic cart (TrueOne 2400, ParvoMedics, Sandy, Utah). Participants self-selected a speed between 5 and 7.5 miles per hour (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 VO2 max was defined as the highest 30 second average value collected during the test. Heart rate at 65% of VO2 max was then determined and used for the experimental trials.
Participants were asked to return to the lab no earlier than 72 hours after the completion of the VO2 max 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, and they were asked to match pre-testing conditions from trial 1 to trial 2. 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 BCAA or a placebo solution. Participants consumed the experimental beverage three 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 VO2 max. 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.
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.
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 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.
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 to generate a standard concentration curve and to determine 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 (Chambers 2012, Katajamaa 2006). 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 (Chong 2019, Dührkop 2015, Dührkop 2019). The BCAA supplement was also examined using LCMS and the results were analyzed to confirm the presence of BCAA and additives including flavoring and sweetening agents.
Secondary study to assess immediate impact of BCAA consumption on serotonin
The timing of blood samples immediately before and after exercise was intended to isolate the impact of BCAA on serotonin and metabolism during exercise. Supplements were consumed three minutes prior to the pre-exercise blood sample. It was anticipated that three minutes was too short for BCAA absorption to impact tryptophan uptake and serotonin release into the blood. However, lower serotonin concentrations were measured in the pre-exercise blood samples in the BCAA compared to placebo condition. To determine if there was an impact of the experimental drink on blood serotonin concentration three minutes after ingestion, ten healthy males and females aged 23 to 28 years were selected for participation in a validation study to determine the impact of the BCAA supplement drink on serotonin. Potential participants were screened for the absence of underlying pathologies and the presence of an active lifestyle. Several of the participants were endurance athletes fitting the original criteria although some were not. Blood was drawn from each participant after which they ingested an experimental beverage containing either BCAA or the placebo solution. Three minutes after consuming the beverage, a second blood draw was performed. Serum was collected from the blood samples as previously described and stored at -80°C until LCMS analysis. All samples were extracted and analyzed concurrently within 24 hours of metabolite extraction. LCMS and data analysis methods were identical to those from the original cohort.
Serotonin concentrations were normalized and center scaled using the caret R package. After normalization, a nested analysis of variance (ANOVA) was completed with serotonin concentration as the dependent variable and treatment and time as independent variables. The significant results of the nested ANOVA analysis comparing treatment by time led to a post-hoc analysis of the groups and t-tests were performed between treatment and temporal groupings. A second statistical analysis, including normalization, a nested ANOVA and paired t-tests, was performed with the second cohort as with the first cohort.