- ICH GCP
- US Clinical Trials Registry
- Clinical Trial NCT05672758
Effect of Long-lasting Adaptation to Endurance and Speed-power Training on Plasma Free Amino Acids Concentration (AAdaptation)
The Effect of Long-lasting Adaptation to Intensive Speed-power and Endurance Training on Plasma Free Amino Acids Concentration at Rest, During Graded Exercise and Post-exercise Recovery
The goal of this observational study was to detect the long-term effect of two different training modalities - speed-power and endurance training - on changes in plasma free amino acid (PFAA) concentration at rest, during graded exercise and post-exercise recovery period. It was assumed that these training modalities cause different amino acids concentration in human blood depending on long-term sport specialization and predominant exercise type (the contribution of high-intensity exercise related to anaerobic metabolism). The hypotheses were:
- highly-trained speed-power have higher concentrations of PFAA than endurance athletes;
- PFAA concentration varies with the change in training loads in a one-year training cycle. Higher PFAA concentrations is expected in training phases with larger contribution of high-intensity exercise;
- PFAA concentration per 1 kg muscle mass differ between speed-power and endurance athletes.
Forty-eght highly-trained athletes aged 18-32 years with longer competitive sport experience - sprinters vs triathletes/distance runners - and 10 recreationally trained controls were examined. Laboratory tests were conducted in consecutive training subphases.
(i) Body composition and muscle mass was assessed using densitometry. (ii) Participants underwent a graded exercise treadmill test until exhaustion. (iii) Blood samples were drawn at rest, during exercise (every 3 min, at each speed change), and after exercise (immediately and 5, 10, 15, 20 and 30 min post exercise).
(iv)The analysis of PFAA profiles was based on the Liquid Chromatography Electrospray Ionization tandem Mass Spectrometry (LC-ESI-MS/MS) technique and the aTRAQ reagent. This allowed to quantify 42 PFAAs.
The results improve the understanding of metabolic adaptation to long-term exercise programmes. Possible practical application encompasses the domains of exercise medicine, sport and public health.
The novelty of the project: (1) comparing the effect of two different training models on PFAA concentration, (2) tracking the changes in PFAAs across a one-year training cycle, (3) repeated multiple sampling in one exercise session including resting conditions, (4) introducing skeletal muscle mass as a factor potentially affecting PFAA profiles, (5) a large number (42) of proteinogenic- and non-proteinogenic PFAAs, (6) homogenous highly-trained athletic groups, and (7) a proven state-of-the-art method to determine PFAAs.
Study Overview
Status
Conditions
Intervention / Treatment
Detailed Description
I. Study goal
Long-term effect of two different training modalities - speed-power and endurance training - on changes in plasma free amino acid (PFAA) concentration at rest, during graded exercise until exhaustion and recovery period was compared. The model cohorts were highly-trained athletes. The assumption was that structured long-lasting speed-power and endurance training cause adaptations resulting in different PFAA concentration and that the changes depend on long-term sport specialization (predominant exercise type) and training phase of the one-year cycle (contribution of high-intensity exercise based on anaerobic metabolism characterized by rapid adenosine-5'-triphosphate degradation). A larger contribution of high-intensity exercise is used by speed-power athletes and in phases of specific preparation/competition. There is a lack of research on long-term effects of physical training on changes in PFAAs. This picture is supllemented by monitoring changes in a set of 42 proteinogenic and non-proteinogenic PFAAs. Short- and long-lasting effects of training on levels and time course of PFAA concentration during exercise and recovery will be shown. The main goals were:
- To compare the effect of the two entirely different training modalities (speed-power and endurance) on PFAA concentration.
- To observe the effect of training load character on changes in resting, exercise and post-exercise PFAA concentration in consecutive training phases of a one-year training cycle.
- To determine the link between PFAA concentration and exercise modality, taking into account skeletal muscle mass.
II. Background
Only 3-6% of the total energy utilized during prolonged endurance exercise is derived from oxidation of AAs. However, the proportion of energy from AA catabolism considerably increases during exercise. Although protein turnover does not contribute substantially to the energy expenditure, it may fill other important functions during exercise. AAs may delay muscle glycogen depletion.
The free AA pool amounts only 2% of the total AA amount and is stored within the plasma and intra- and extracellular spaces. This small pool accounts for a continuous exchange of AAs. Blood plasma serves as a temporary reservoir of AAs, the size of which changes in response to exercise. Differences in regular training, exercise, and daily activities result in differences in PFAA levels.
Exercise duration affects PFAA levels, however, the direction of the changes may be different for specific AAs and exercise type, intensity, and duration. In contrast, muscle AAs concentration remains relatively stable. AA metabolism during prolonged exercise has been described. Reports based on a short training periods (few weeks) in power-type athletes suggest that sprint and endurance training sessions have a distinct effect on serum AAs. However, there is a lack of studies on the differences in long-term adaptation changes in PFAAs between endurance- and sprint-trained individuals, taking into account multiple blood sampling (rest, exercise, recovery) and muscle mass.
III. Methods
Over 70 athletes aged 18-32 years - speed-power (sprinters), endurance (triathletes, distance runners), and recreationally trained - were recruited. Eventually, the data of 58 of them were considered for analysis. Athletes were examined four times during one-year training cycle: (1) general preparation phase, (2) specific preparation phase, (3) competition phase, and (4) transition phase.
The main statistical tool was one-way and two-way ANOVA with repeated measures. The required sample size was computed based on given alpha (significance) level, statistical power, and effect size. The significance level α < 0.05 and statistical power 0.8 were assumed. Based on earlier studies that compared sprint- and endurance-trained athletes in terms of other metabolic phenomena related to training and exercise, minimum partial eta square (η2) of 0.2 was adopted, i.e. a large effect size for differences between examined groups and consecutive examination across was expected. Other assumptions were nonsphericity correction = 0.75, number of measurements = 4, and correlation between repeated measurements = 0.5. The minimum total sample size of 22 athletes was obtained.
Body composition was assessed using dual X-ray absorptiometry (Lunar Prodigy, GE Healthcare, USA). Skeletal muscle mass was estimated using regression equations.
Graded exercise tests on a treadmill (h/p/cosmos, Germany) were conducted before 12 a.m., two hours after a standard meal. The initial speed was 8 km/h and increased every 3 min by 2 km/h until exhaustion. Respiratory parameters and heart rate were measured (ergospirometer MetaLyzer 3B, Cortex, Germany; Polar Elektro RS 400, Finland). Maximum oxygen consumption was measured.
Blood sampling. Peripheral venous catheter was placed into dorsal metacarpal vein. Blood samples were drawn at rest, during exercise (every 3 min, at each speed change), and immediately, 5, 10, 15, 20, and 30 min after exercise completion. The volume of venous blood obtained during one examination was 25 ml, i.e. 2.5 ml for each sample, up to 10 samples. Each sample was collected into plasma-separation tube containing EDTA for further plasma analysis. Samples were centrifuged at 13,000 revolution/min for 3 min at 4°C. Obtained plasma was then pipetted into 0.5 ml vials and immediately frozen in liquid nitrogen. Samples were stored in -80°C until analysis.
Forty two PFAAs were assayed: O-Phospho-L-serine, O-Phosphoethanolamine, Taurine, L-Asparagine, L-Serine, Hydroxy-L-proline, Glycine, L-Glutamine, Ethanolamine, L-Aspartic acid, L-Citrulline, Sarcosine, β-Alanine, L-Alanine, L-Threonine, L-Glutamic acid, L-Histidine, 1-Methyl-L-histidine, 3-Methyl-L-histidine, L-Homocitrulline, Argininosuccinic acid, γ-Amino-n-butyric acid, D, L-β-Aminoisobutyric acid, L-α-Amino-n-butyric acid, L- α- Aminoadipic acid, L-Anserine, L-Carnosine, L-Proline, L-Arginine, δ-Hydroxylysine, L-Ornithine, Cystathionine, L-Cystine, L-Lysine, L-Valine, L-Methionine, L-Tyrosine, L-Homocystine, L-Isoleucine, L-Leucine, L-Phenylanine, L-Tryptophan. The analysis of PFAAs was based on the LC-ESI-MS/MS technique and the aTRAQ (Sciex) reagent, characterized by high sensitivity and specificity, short analytical run time, low sample volume required to perform the analysis, and high amount of analytes being quantified in one run.
Protocol for determination of PFAAs. Plasma samples of 40 µl were transferred to Eppendorf tubes. In order to precipitate proteins present in plasma, 10 µl of 10% sulfosalicylic acid was added and the content was mixed and centrifuged (10,000 g for 2 minutes). After that, the supernatant was transferred to a new tube and mixed with 40 µl of borate buffer. An aliquot of 10 µl of the obtained solution was subsequently labeled with the aTRAQ reagent Δ8 solution (5 µl), mixed, centrifuged, and incubated at room temperature for 30 minutes. The labeling reaction was then stopped by addition of 5 µl of 1.2% hydroxylamine, mixed, and incubated at room temperature for 15 minutes. After that, 32 µl of the internal standard solution was added and the content was mixed. In the next step, the sample was evaporated in a vacuum concentrator for 15 minutes to reduce the volume of the sample to about 20 µl. The residue was then diluted with 20 µl of water, mixed, and transferred to an autosampler vial with an insert. This procedure was modified in order to measure the concentrations of PFAAs that could not be detected using the original method. For this purpose, plasma samples of higher volume were used for the sample preparation procedure. Instead of 40 μl, plasma samples of 80 μl were transferred to Eppendorf tubes and mixed with 20 μl of 10% sulfosalicylic acid. After that, 20 μl of the supernatant was transferred to a new tube and mixed with 30 μl of borate buffer. The subsequent steps of the procedure remained unchanged. The internal standard solution contained the same AAs labeled with the aTRAQ reagent Δ0. Thus, each determined PFAA had its corresponding internal standard. Norleucine and norvaline, two non-proteinogenic AAs, were used to evaluate the labeling efficiency and recovery. Their corresponding internal standards were also present in the internal standard solution.
The analyses were performed using the liquid chromatography instrument 1260 Infinity (Agilent Technologies) coupled to the 4000 QTRAP (quadrupole ion trap) mass spectrometer (Sciex). This mass spectrometer is equipped with an electrospray ionization source and three quadrupoles and allows to conduct the quantitative PFAA analysis planned within the presented project. The chromatographic separation was performed with the Sciex C18 (5 μm, 4.6 mm x 150 mm) chromatography column. The flow rate of mobile phases was maintained at 800 μl/min. The method uses the following mobile phases: water (phase A) and methanol (phase B), both with addition of 0,1 % formic acid and 0.01 % heptafluorobutyric acid. The time of analysis was 18 minutes and during that time the chromatographic separation was carried out with the following gradient elution: from 0 till 6 min - from 2% to 40% of phase B, then maintained at 40% of phase B for 4 minutes, increased to 90% of phase B till 11 min and held at that ratio phases for 1 minute, then decreased to 2% of phase B and finally maintained at 2% of phase B from 13 to 18 min. The injection volume was set at 2 μl and the separation temperature at 50 °C. The ion source settings were the following: curtain gas 20 psig; ion spray voltage 4500 V; ion source temperature 600 °C; ion source gas 1 = 60 psig, ion source gas 2 = 50 psig. The mass spectrometer operated in positive ionization mode and the following parameters were applied: entrance potential = 10 V; declustering potential = 30 V; collision cell exit potential = 5 V; collision energy = 30 eV (50 eV in case of 7 compounds); collision gas: nitrogen. The PFAAs were measured in scheduled multiple reaction monitoring (sMRM) mode. This mode ensures high specificity and sensitivity in quantitative analyses. A system suitability test (analysis of a standard mixture of AAs) was conducted before each batch of samples in order to warm up and check the inter-day performance of the whole system. Data acquisition and processing were performed using the Analyst 1.5 software (Sciex). The described LC-ESI-MS/MS method for determination of AAs is well established in the Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, the co-investigator of the project.
Total blood count was performed using the device Mythic 18 (Orphée, Swiss). Capillary blood samples from fingertip (20 μl per sample) were drawn simultaneously with venous blood pre-, during and postexercise. Blood lactate concentration was measured using C-line analyzer (EKF-Diagnostic, Germany).
Study Type
Enrollment (Actual)
Contacts and Locations
Study Locations
-
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Wielkoolska
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Poznań, Wielkoolska, Poland, 60-687
- Poznan University of Physical Education
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Participation Criteria
Eligibility Criteria
Ages Eligible for Study
Accepts Healthy Volunteers
Genders Eligible for Study
Sampling Method
Study Population
Description
Inclusion Criteria for professional athletes:
- highly trained sprint- and endurance-trained athletes
- national or international performance level
- current participation in training programs organized by professional sports clubs or national team (licence) for at least 5 years
- current medical eligibility for competitive sport
Inclusion Criteria for amateur/recreational athletes:
- regular recreational activity for at least 5 years (preferred endurance disciplines)
- participation in amateur competition
- good health status
- non-smoking
Exclusion Criteria for professional athletes:
- untrained individuals
- athletes that do not meet the above criteria for participation in professional sport
- injured athletes or those who are not able or willing to participate for other reasons
Exclusion Criteria for amateur/recreational athletes:
- inactive/sedentary individuals
- medical contraindications to high-intensity exercise and testing
- injured individuals or those who are not able or willing to participate for other reasons
- current smokers or heavy past smokers
Study Plan
How is the study designed?
Design Details
- Observational Models: Cohort
- Time Perspectives: Prospective
Cohorts and Interventions
Group / Cohort |
Intervention / Treatment |
---|---|
Sprint-Trained Athletes
Highly trained track sprinters at the national or international level aged 18-35 years with sport experience 5-10 years; structured periodized training
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One-year cycle including specific sprint-oriented athletic training
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Endurance-Trained Athletes
Highly trained long-distance runners and triathletes at the national or international level aged 18-35 years with sport experience 5-10 years; structured periodized training
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One-year cycle including specific endurance-oriented athletic training
|
Amateur/Recreational Athletes (Controls)
Individuals participating in amateur non-professional sport aged 18-35 years with activity experience 5-10 years; unstructured non-periodized training
|
One year of nonspecific recreational training
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What is the study measuring?
Primary Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
---|---|---|
absolute Plasma Free Amino Acids Concentration (42 amino acids)
Time Frame: 4 times in a 1-year training cycle: at rest, during graded exercise (every 3 min), and during recovery (5, 10, 15, 20, and 30 min)
|
µmol/L
|
4 times in a 1-year training cycle: at rest, during graded exercise (every 3 min), and during recovery (5, 10, 15, 20, and 30 min)
|
relative Plasma Free Amino Acids Concentration (42 amino acids)
Time Frame: 4 times in a 1-year training cycle: at rest, during graded exercise (every 3 min), and during recovery (5, 10, 15, 20, and 30 min)
|
µmol/L/kg muscle mass
|
4 times in a 1-year training cycle: at rest, during graded exercise (every 3 min), and during recovery (5, 10, 15, 20, and 30 min)
|
Secondary Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
---|---|---|
absolute Skeletal Muscle Mass (aSMM)
Time Frame: 4 times in a 1-year training cycle
|
kg
|
4 times in a 1-year training cycle
|
relative Skeletal Muscle Mass (rSMM)
Time Frame: 4 times in a 1-year training cycle
|
percent of total body mass
|
4 times in a 1-year training cycle
|
absolute Lean Body Mass (aLBM)
Time Frame: 4 times in a 1-year training cycle
|
kg
|
4 times in a 1-year training cycle
|
relative Lean Body Mass (rLBM)
Time Frame: 4 times in a 1-year training cycle
|
percent of total body mass
|
4 times in a 1-year training cycle
|
absolute Fat Mass (aFM)
Time Frame: 4 times in a 1-year training cycle
|
kg
|
4 times in a 1-year training cycle
|
relative Fat Mass (rFM)
Time Frame: 4 times in a 1-year training cycle
|
percent of total body mass
|
4 times in a 1-year training cycle
|
absolute Maximum Oxygen Uptake (aVO2max)
Time Frame: 4 times in a 1-year training cycle
|
L/min
|
4 times in a 1-year training cycle
|
relative Maximum Oxygen Uptake 1 (r1VO2max)
Time Frame: 4 times in a 1-year training cycle
|
L/min/kg total body mass
|
4 times in a 1-year training cycle
|
relative Maximum Oxygen Uptake 2 (r2VO2max)
Time Frame: 4 times in a 1-year training cycle
|
L/min/kg skeletal muscle mass
|
4 times in a 1-year training cycle
|
Other Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
---|---|---|
White Blood Cells
Time Frame: 4 times in a 1-year training cycle
|
10^9/L
|
4 times in a 1-year training cycle
|
Red Blood Cells
Time Frame: 4 times in a 1-year training cycle
|
10^12/L
|
4 times in a 1-year training cycle
|
Hematocrit
Time Frame: 4 times in a 1-year training cycle
|
percent
|
4 times in a 1-year training cycle
|
Mean Corpuscular Volume (MCV)
Time Frame: 4 times in a 1-year training cycle
|
fL
|
4 times in a 1-year training cycle
|
Mean Corpuscular Hemoglobin (MCH)
Time Frame: 4 times in a 1-year training cycle
|
pg
|
4 times in a 1-year training cycle
|
Mean Corpuscular Hemoglobin Concentration (MCHC)
Time Frame: 4 times in a 1-year training cycle
|
g/dL
|
4 times in a 1-year training cycle
|
Red Blood Cell Volume Distribution Width (RDW-CV)
Time Frame: 4 times in a 1-year training cycle
|
percent
|
4 times in a 1-year training cycle
|
Blood Lactate Concentration
Time Frame: 4 times in a 1-year training cycler: at rest, during graded exercise (every 3 min), and during recovery (5, 10, 15, 20, and 30 min)
|
mmol/L
|
4 times in a 1-year training cycler: at rest, during graded exercise (every 3 min), and during recovery (5, 10, 15, 20, and 30 min)
|
Blood Ammonia
Time Frame: 4 times in a 1-year training cycle: at rest, during graded exercise (every 3 min), and during recovery (5, 10, 15, 20, and 30 min)
|
µmol/L
|
4 times in a 1-year training cycle: at rest, during graded exercise (every 3 min), and during recovery (5, 10, 15, 20, and 30 min)
|
Creatine Kinase at rest
Time Frame: 4 times in a 1-year training cycle
|
U/L
|
4 times in a 1-year training cycle
|
Maximum Heart Rate (HRmax)
Time Frame: 4 times in a 1-year training cycle
|
beats per min
|
4 times in a 1-year training cycle
|
Appendicular Lean Soft Tissue (ALST)
Time Frame: 4 times in a 1-year training cycle
|
kg
|
4 times in a 1-year training cycle
|
Relative Skeletal Muscle Mass Index (RSMI)
Time Frame: 4 times in a 1-year training cycle
|
kg/m^2
|
4 times in a 1-year training cycle
|
Weight
Time Frame: 4 times in a 1-year training cycle
|
kg
|
4 times in a 1-year training cycle
|
Height
Time Frame: 4 times in a 1-year training cycle
|
kg
|
4 times in a 1-year training cycle
|
Body Mass Index (BMI)
Time Frame: 4 times in a 1-year training cycle
|
kg/m^2
|
4 times in a 1-year training cycle
|
Maximum Minute Ventilation (VEmax)
Time Frame: 4 times in a 1-year training cycle
|
L/min
|
4 times in a 1-year training cycle
|
Maximum Tidal Volume (VT)
Time Frame: 4 times in a 1-year training cycle
|
L
|
4 times in a 1-year training cycle
|
Maximum Breathing Frequency (BF)
Time Frame: 4 times in a 1-year training cycle
|
/min
|
4 times in a 1-year training cycle
|
Age
Time Frame: baseline
|
years
|
baseline
|
Sports History (Experience)
Time Frame: baseline
|
years
|
baseline
|
Speed at VO2max (vVO2max)
Time Frame: baseline
|
km/h
|
baseline
|
Collaborators and Investigators
Investigators
- Principal Investigator: Krzysztof Kusy, PhD, Poznan University of Physical Education
Publications and helpful links
General Publications
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- Zielinski J, Kusy K. Training-induced adaptation in purine metabolism in high-level sprinters vs. triathletes. J Appl Physiol (1985). 2012 Feb;112(4):542-51. doi: 10.1152/japplphysiol.01292.2011. Epub 2011 Dec 8.
Study record dates
Study Major Dates
Study Start (Actual)
Primary Completion (Anticipated)
Study Completion (Anticipated)
Study Registration Dates
First Submitted
First Submitted That Met QC Criteria
First Posted (Actual)
Study Record Updates
Last Update Posted (Estimate)
Last Update Submitted That Met QC Criteria
Last Verified
More Information
Terms related to this study
Keywords
Other Study ID Numbers
- OPUS 14 2017/27/B/NZ7/02828
Plan for Individual participant data (IPD)
Plan to Share Individual Participant Data (IPD)?
IPD Plan Description
IPD Sharing Time Frame
IPD Sharing Access Criteria
IPD Sharing Supporting Information Type
- Study Protocol
- Statistical Analysis Plan (SAP)
- Informed Consent Form (ICF)
Drug and device information, study documents
Studies a U.S. FDA-regulated drug product
Studies a U.S. FDA-regulated device product
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