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:

1. highly-trained speed-power have higher concentrations of PFAA than endurance athletes;
2. 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;
3. 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.

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:

1. To compare the effect of the two entirely different training modalities (speed-power and endurance) on PFAA concentration.
2. 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.
3. 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).

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

Highly trained competitive athletes aged 18-35 years with longer competitive sport experience, specialized in sports of different character - speed-power (sprinters) vs endurance (triathletes, distance runners). Amateur/recreational athletes participating in training on a regular basis.

All individual participant data that underlie results in a publication

Starting 1 year after publication of the data

Scientists specialized in amino acids and training research on request (kusy@awf.poznan.pl). Principal investigator will review the requests