Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT04969536 |
| Other study ID # |
DF081319 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
October 1, 2019 |
| Est. completion date |
February 23, 2021 |
Study information
| Verified date |
July 2021 |
| Source |
Montana State University |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial 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.
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.
