Anda di halaman 1dari 9

Original Research

Changes in Blood Concentration of Adenosine

Triphosphate Metabolism Biomarkers During
Incremental Exercise in Highly Trained Athletes of
Different Sport Specializations
Michał Włodarczyk,1 Krzysztof Kusy,1 Ewa Słomińska,2 Zbigniew Krasiński,3 and Jacek Zieliński1
Human Movement Laboratory “LABTHLETICS”, Department of Athletics, Strength and Conditioning, Poznan University of Physical
Education, Poznań, Poland; 2Department of Biochemistry, Medical University of Gdansk, Gdańsk, Poland; and 3Department of
Vascular and Endovascular Surgery, Angiology and Phlebology, Poznan University of Medical Sciences, Poznań, Poland

Włodarczyk, M, Kusy, K, Słomińska, E, Krasiński, Z, and Zieliński, J. Changes in blood concentration of adenosine triphosphate
metabolism biomarkers during incremental exercise in highly trained athletes of different sport specializations. J Strength Cond Res
XX(X): 000–000, 2019—We hypothesized that (a) high-level specialized sport training causes different adaptations that induce
specific biomarker release dynamics during exercise and recovery and (b) skeletal muscle mass affects biomarker release. Eleven
sprinters (21–30 years), 16 endurance runners (18–31 years), 12 futsal players (18–29 years), and 12 amateur runners as controls
(22–33 years) were examined. Hypoxanthine (Hx), xanthine (X), uric acid (UA), ammonia (NH3), and lactate (LA) concentrations were
determined at rest, during an incremental treadmill exercise test (every 3 minutes), and during recovery (5, 10, 15, 20, and 30
minutes after exercise). Hx, X, and UA concentration was determined from plasma, while LA and NH3 from whole blood, and muscle
mass was assessed using dual X-ray absorptiometry method. At rest, during incremental exercise, and up to 30 minutes into the
postexercise recovery period, sprinters had lowest Hx, X, and UA concentrations, and endurance athletes had lowest NH3
concentrations. For LA during exercise, the lowest concentrations were noted in endurance athletes, except when reaching
maximum intensity, where the differences between groups were not significant. There were no significant correlations observed
between skeletal muscle mass and biomarker concentration at maximal intensity and recovery in any group. In conclusion, the
magnitude of exercise-induced biomarker concentration is only related to training adaptations through specific training profile but
not to muscle mass. In addition, the results suggest that combined measuring of LA, NH3, and Hx concentration in blood is useful in
indirectly reflecting key changes in exercise- and training-induced energy status. Further research should focus on studying how
specific training sessions affect individual biomarker response in highly trained athletes.
Key Words: lactate, ammonia, hypoxanthine, xanthine, uric acid, training

Introduction exercise (10). As exercise duration and intensity increase, the

adenine nucleotide (AdN) pool and the ATP/adenosine di-
Skeletal muscles are the main organs used during exercise. Un-
phosphate ratio decreases (7), which causes specific patterns and
fortunately, performing muscle biopsies on highly trained athletes
is unpractical, and other methods such as blood measurement magnitude of accumulation of these biomarkers in blood.
must be used to reflect muscle biomarker concentration levels. In Biomarkers such as LA (3,5,8,9) and NH3 (1,10,11,18,19,31–34)
have been extensively researched in difference stages of exercise
addition, highly trained athletes also possess different levels of
muscle mass which can affect magnitude of biomarker release. (1,11,15,18,19,32–34). New purine metabolite biomarkers have
Because muscles use adenosine triphosphate (ATP) for energy been studied during and after exercise (13,22,30), after short-term
production, measuring ATP metabolism biomarkers can help to training lasting 1–7 weeks (27–29), during a 1-year training cycle in
better understand energy system utilization during different types highly trained athletes (36,38–40), and even considered a universal
of training and exercise (1,3,10,11,20,33). Mechanisms related to indicator of training status (37) as well as a predictor of performance
depletion and replenishing of ATP stores due to increased con- in highly trained athletes (35). To our knowledge, there is an in-
sumption or decreased provision are reflected by blood concen- adequate amount of research, studying ATP metabolism biomarker
tration of 3 basic biomarkers, i.e., lactate (LA), ammonia (NH3), efflux before, during exercise, and during the postexercise recovery
and oxypurines (7), as well as decreased power output during period in highly trained athletes. Sjodin and Hellsten-Westing (24)
studied only hypoxanthine (Hx) and uric acid (UA) before, during,
Address correspondence to Jacek Zieliński, and after exercise only in 4 well-trained runners. Ogino et al. (19)
Supplemental digital content is available for this article. Direct URL citations appear studied LA, NH3, and Hx before, during, and after exercise but only
in the printed text and are provided in the HTML and PDF versions of this article on in healthy male volunteers. Finally, Stathis et al. (28) studied LA and
the journal’s Web site ( purine derivatives in 7 active but nonspecifically trained males with
Journal of Strength and Conditioning Research 00(00)/1–9 the main focus on biomarker release during recovery as opposed to
ª 2019 National Strength and Conditioning Association release during exercise. As shown, there is a lack of research

Copyright © 2019 National Strength and Conditioning Association. Unauthorized reproduction of this article is prohibited.
ATP Metabolism Biomarkers During Exercise (2019) 00:00

reflecting a greater range of ATP biomarker release around exercise of the transition period of their annual training cycle (beginning
(rest, exercise, and recovery) in highly trained athletes in the same of preparatory phase). Controls, however, were not specifically
training phase. Moreover, the contribution of specific muscle fibers, trained, recreationally performed common forms of physical ac-
genetically predetermined or resulting from training adaptation, is of tivity (e.g., jogging, swimming, and team games) in their spare
importance. Fast-twitch muscle fibers contain more ATP and phos- time, and did not exceed the 150 minutes of moderate-intensity
phocreatine (16), are more glycolytically active (5) suited for LA physical activity per week recommended by the World Health
production (8), and contain more AMP deaminase leading to greater Organization. The project was approved by the Ethics Committee
AMP deamination producing more NH3 (13,33) and ultimately at the Poznan University of Medical Sciences and was performed
more Hx in muscle compared with slow-twitch muscle fibers. Fur- according to the ethical standards laid down in the Declaration of
thermore, in the context of exercise, there were no attempts to elu- Helsinki. Each subject was informed of the testing procedure,
cidate the relationships between skeletal muscle mass (SMM) and purpose, and risks of the study and submitted their written con-
ATP metabolism biomarker concentration. It is unknown whether sent to participate.
changes in ATP metabolism biomarker concentrations are related to
the changes in muscle mass. Greater muscle mass is related to the
total amount of ATP-PCr in muscle that can be used during exercise
and increases the amount of ATP in muscle that can be produced
through anaerobic glycolysis (21). Because muscle mass levels and Subjects were instructed not to participate in any high-intensity or
muscle properties are different in athletes of diverse physiological long-duration training sessions at least 24–48 hours before test-
and training profiles, this could cause different release dynamics ing. Testing was performed in the morning hours, 3 hours after
throughout exercise. a light breakfast (no coffee or tea). Before the exercise test, sub-
The aim of this study was to determine changes in blood con- jects underwent body composition analysis. Afterward, an in-
centration of ATP metabolism biomarkers (LA, NH3, and purine cremental treadmill exercise test until volitional exhaustion was
metabolites) during graded exercise and in the recovery period, in performed. During all examinations, room temperature remained
athletes of different training profiles and differences in SMM. We at 20–21° C.
hypothesize that (a) high-level specialized sport training causes
different adaptations resulting in specific exercise-related release Anthropometric and Body Composition Analysis. The anthro-
dynamics of ATP metabolism biomarkers during standard exer- pometric measurements taken were body mass (kg) and height
cise and in the postexercise recovery period, and that (b) differ- (cm) using a digital stadiometer (SECA 285; SECA, Hamburg,
ences in muscle mass of athletes will affect biomarker release. Germany). Body mass index was calculated by dividing body
mass by height squared. The dual X-ray absorptiometry method,
using Lunar Prodigy Pro (GE Healthcare, Madison, WI, USA)
Methods and enCORE v. 16 SP1 software, was used for body composition
Experimental Approach to the Problem analysis. During examination, subjects only wore their under-
garments, without jewelry and metal objects to minimize mea-
To obtain the full-blood ATP biomarker spectrum of release surement error. Skeletal muscle mass was calculated based on
around exercise, the testing procedures included an incremental regression models appropriate for age and sex (17).
exercise test, where blood samples were obtained at rest, whereas
subjects ran on a mechanical treadmill with increasing speed, and Respiratory Parameters. An incremental exercise test until voli-
up to 30 minutes after exercise. Elite athletes of different sport tional exhaustion on a mechanical treadmill (H/P Cosmos Pulsar,
specializations were chosen to represent diverse metabolic and Sports & Medical, Nussdorf-Traunstein, Germany) was per-
physiological adaptations (speed-power, endurance, and mixed) formed to determine the maximal oxygen uptake (V̇ O2max).
to see whether long-term specialized training would produce Initial speed was set at 4 km·h21 and increased after 3 minutes to
different biomarker responses. Subjects underwent body com- 8 km·h21. After that point, treadmill speed increased pro-
position analysis to obtain SMM levels. A control group of rec- gressively by 2 km·h21 every 3 minutes until volitional exhaus-
reationally trained athletes was used to represent a group of tion. Once a 10-km·h21 speed was reached, blood samples were
general training adaptations to exercise contrary to the highly drawn from the participant at the end of each 3-minute stage.
specialized athletic groups. Respiratory parameters were measured (breath by breath) by
an ergospirometer (Cortex Metamax 3B R2, Leipzig, Germany)
and analyzed using MetasoftStudio v. 5.1.0 Software (Cortex-
Metamax 3B R2; Cortex Biophysik, Leipzig, Germany). Heart
Four groups of athletes participated in the study: sprinters (SP, rate (HR, b·min21) was monitored with a Polar Bluetooth Smart
n 5 11) specialized in the 100 and 200 m events, aged 24.2 6 3.2 H6 (Polar Electro Oy, Kempele, Finland) HR monitor. All sub-
(range 21–30) years, and practicing sport for 8.5 6 2.5 years, jects were familiar with the exercise protocol since they already
endurance athletes (EN, n 5 16) consisting of triathletes and long- performed this test previously.
distance runners aged 23.4 6 3.6 (range 18–31) years, and
practicing sport for 8.7 6 1.9 years, futsal players (FU, n 5 12) Blood Sampling. Subjects wore a catheter (BD Venflon Pro 1.3
aged 24.5 6 3.8 (range 18–29) years, and practicing sport for 3 32 mm; Becton Dickinson, Helsingborg, Sweden) patent
10.0 6 3.4 years, and amateur runners (AM, n 5 12) representing with isotonic saline (0.9% NaCl), from which blood was drawn
the control group aged 27.7 6 4.1 (range 22–33) years with no from one of the antecubital veins. Blood samples were collected
past or current competitive sport history (recreational sports ac- at rest, at the end of each 3-minute stage above 10 km·h21
tivities 3–5 times per week). A more detailed description of the treadmill speed, immediately after exercise, and 5, 10, 15,
subjects is presented in Table 1. Sprinters, EN, and FU were 20, and 30 minutes into the postexercise recovery period.
members of the Polish National Team and were tested at the end Later, a 2.7-ml blood sample was taken into 2 monovettes

Copyright © 2019 National Strength and Conditioning Association. Unauthorized reproduction of this article is prohibited.
ATP Metabolism Biomarkers During Exercise (2019) 00:00 |

Table 1
Basic characteristics of the athletic groups and controls.*†
Futsal players Endurance athletes Sprinters Control group One-way Effect
(n 5 12) (n 5 16) (n 5 11) (n 5 12) ANOVA size
Age (y) 24.50 6 3.8 (22.1–26.9) 23.38 6 3.6 (21.5–25.3) 24.18 6 3.2 (22.0–26.3) 27.70 6 4.1‡ (25.1–30.3) 0.025 0.18
Training experience (y) 10.00 6 3.4 (7.8–12.2) 8.69 6 1.9 (7.7–9.7) 8.45 6 2.5 (6.8–10.1) — 0.299 0.06
Height at examination (cm) 181.38 6 5.5 (177.9–184.9) 181.80 6 6.1 (178.6–185.0) 186.19 6 4.6 (183.1–189.3) 180.44 6 5.6 (176.9–184.0) 0.080 0.13
Mass at examination (kg) 77.12 6 7.2 (72.6–81.7) 73.41 6 7.1 (69.6–77.2) 81.61 6 5.5‡ (77.9–85.3) 76.72 6 7.7 (71.8–81.6) 0.039 0.16
BMI (kg·m22) 23.47 6 2.2 (22.1–24.8) 22.23 6 2.1 (21.1–23.4) 23.53 6 1.0 (22.9–24.2) 23.68 6 2.6 (22.0–25.3) 0.221 0.09
SMM (kg) 33.65 6 3.2{ (31.6–35.7) 32.40 6 3.2# (30.7–34.1) 39.08 6 3.7 (36.6–41.6) 33.12 6 3.1{ (31.2–35.1) ,0.001 0.39
HRVT (b·min21) 146.33 6 14.7 (137.0–155.7) 156.31 6 13.8 (149.0–163.7) 147.09 6 15.4 (136.8–157.4) 151.50 6 13.6 (142.8–160.2) 0.244 0.08
VO2VT (L·kg21) 2.97 6 0.4 (2.7–3.2) 3.26 6 0.5 (3.0–3.5) 2.70 6 0.5‡ (2.3–3.1) 3.02 6 0.4 (2.8–3.3) 0.034 0.17
VO2VT (ml·kg21·min21) 38.45 6 3.9‡ (35.9–40.9) 44.52 6 6.1 (41.3–47.8) 33.25 6 6.9‖ (28.6–37.9) 39.44 6 3.9 (37.0–41.9) ,0.001 0.38
VO2VT (ml·kg smm · 88.16 6 9.4{ (82.2–94.1) 100.88 6 14.1# (93.3–108.4) 69.61 6 14.6 (59.8–79.5) 91.14 6 6.8{ (86.8–95.5) ,0.001 0.49
HRRCP (b·min21) 174.83 6 11.3 (167.6–182.0) 181.61 6 9.5 (176.6–186.7) 179.09 6 8.4 (173.5–184.7) 179.17 6 10.2 (172.7–185.6) 0.366 0.06
VO2RCP (L·kg21) 3.73 6 0.5‡ (3.4–4.0) 4.29 6 0.5 (4.0–4.6) 3.84 6 0.4 (3.6–4.1) 4.05 6 0.6 (3.7–4.4) 0.025 0.18
21 21
VO2RCP (ml·kg ·min ) 48.36 6 4.3‖ (45.6–51.1) 58.55 6 5.6 (55.6–61.5) 47.08 6 4.4‖ (44.1–50.1) 52.82 6 4.6‡ (49.9–55.8) ,0.001 0.50
VO2RCP (ml·kg 110.93 6 10.9‖ (104.0–117.9) 132.82 6 14.6 (125.0–140.6) 98.60 6 10.2‖ (91.8–105.4) 122.41 6 12.8# ,0.001 0.54
smm21·min21) (114.3–130.5)
HRmax (b·min21) 187.50 6 10.0 (181.1–193.9) 191.94 6 8.5 (187.4–196.5) 188.27 6 9.9 (181.6–195.0) 187.33 6 7.7 (182.4–192.2) 0.482 0.05
VȮ 2max (L·kg )
4.44 6 0.5 (4.2–4.7) 4.91 6 0.6 (4.6–5.2) 4.34 6 0.4‡ (4.1–4.6) 4.31 6 0.4‡ (4.1–4.6) 0.004 0.24
VȮ 2max (ml·kg21· 57.53 6 1.1‖ (56.8–58.2) 66.86 6 5.0 (64.2–69.5) 53.27 6 3.6‖ (50.8–55.7) 56.34 6 2.7‖ (54.6–58.1) ,0.001 0.71
VȮ 2max (ml·kg 132.01 6 6.4§ (128.0–136.1) 151.65 6 13.5 (144.5–158.8) 111.54 6 8.8‖ (105.6–117.5) 130.46 6 7.0‖# ,0.001 0.70
smm21·min21) (126.0–134.9)
Time to complete test 17.14 6 1.4§ (16.3–18.0) 19.11 6 1.2 (18.5–19.8) 16.24 6 1.2‖ (15.4–17.0) 16.51 6 1.2‖ (15.7–17.3) ,0.001 0.50

*ANOVA 5 analysis of variance; BMI 5 body mass index; SMM 5 skeletal muscle mass; VO2VT 5 oxygen consumption at ventilatory threshold; VO2RCP 5 oxygen consumption at respiratory compensation
point; VȮ 2max 5 maximal oxygen uptake.
†Values are mean values 6 SDs (confidence intervals 95%).
‡Post hoc tests: p , 0.05, significantly different from endurance athletes.
§p , 0.01, significantly different from endurance athletes.
‖p , 0.001, significantly different from endurance athletes.
{p , 0.01, significantly different from sprinters.
#p , 0.001, significantly different from sprinters.

(S-Monovette 2.7 ml KE; Sarstedt, Nümbrecht, Germany) one Purines in plasma were determined by high liquid performance
with a lithium anticoagulant (heparin) and another containing chromatography (HPLC) with UV detection as described earlier
an anticoagulant (EDTA). (26). The analyses were performed using 1100 HPLC system
(HPLC System; Agilent, Santa Clara, California). Separation was
Lactate and Ammonia. Biosen C-line (EKF diagnostic GmbH, achieved with analytical column BDS Hypersil C18 (150 3 4.6 mm
Barleben, Germany) was used to measure lactate level using 3 3 mm; Thermo ScientificTM, Waltham, Massachusetts) main-
whole blood. To determine lactate level, 20 ml of whole blood tained at 18° C, protected by SecurityGuard precolumn (Secur-
was placed into a capillary. The apparatus measurement accu- ityGuard precolumn; Phenomenex, Torrance, California). The
racy (CV) was 1.5% for 12 mmol·L21 concentration. To de- mobile phase consisted of A: 122 mM KH2PO4, 150 mM KCL,
termine ammonia level, PocketChem BA PA-4140 (Arkay, and 28 mM K2HPO4 and B: 15% (vol/vol) acetonitrile in A. The
Kyoto, Japan) was used. To perform a measurement on a testing percentage of B changed from 0 to 100% in 8 minutes, was
strip (Ammonia Test Kit II; Arkay), 20 ml of blood was placed maintained at 100% for 1 minute, and then returned to 0% for re-
using a pipette. The apparatus measuring range is 8–285 equilibration. The separation time with re-equilibration was 13.5
mmol·L21. The apparatus measurement accuracy (CV) minutes and was conducted at a flow rate of 0.9 ml·min21. The
was 2.3%. sample injection volume was 20 ml. The quantitative analyses were
performed based on external calibration of the signal at 254 or 280
Purine Metabolites (Hx, X, UA). The vial (Eppendorf, nm. Data acquisition and processing was managed by the Chem-
Wesseling-Berzdorf, Germany) containing the blood mixture station software (Chemstation Software; Agilent, Santa Clara,
with EDTA was centrifuged (Universal 320R; Hettich Lab California). The within-run/between-run %CVs were 3.1/4.1, 3.3/
Technology, Tuttlingen, Germany) at 4° C for 30 seconds at 4.4, and 2.7/3.2% for Hx, X, and UA, respectively.
14,000 rpm (using short spin function). After centrifugation, 0.2
ml of plasma was pipetted into 1.5 ml of vials (Eppendorf,
Statistical Analyses
Wesseling-Berzdorf), in duplicate and immediately frozen in
liquid nitrogen. All samples were then stored in a freezer (280° The sample size was a priori estimated based on the assumption
C). At the time of extraction, 0.2 ml of 1.2 mol·L21 HClO4 was that effect size will be at least medium. Using an a-level of 0.05,
added to frozen plasma for deproteinization. After centrifuga- a power (1 2 b) of 0.80, it was calculated that at least 8 par-
tion, to remove the protein pellet an acid supernatant was ticipants would be needed to detect a significant change or dif-
neutralized with 3 mol·L21 K3PO4, centrifuged to remove pre- ferences in lactate and plasma purine metabolite concentration
cipitated KClO4, and stored in 280° C. (G*Power; Heinrich-Heine-Universitat Dusseldorf, Dusseldorf,

Copyright © 2019 National Strength and Conditioning Association. Unauthorized reproduction of this article is prohibited.
ATP Metabolism Biomarkers During Exercise (2019) 00:00

Germany). A one-way repeated-measures analysis of variance and X concentration was shifted by 5–10 minutes (even 15
(ANOVA) was performed to test the changes in LA, NH3, and minutes in controls) into recovery period (after exhaustion). In
purine metabolite (Hx, X, and UA) concentration over time case of UA, its concentration in all groups still increased after 30
(exercise and recovery measurement points) within each group minutes of recovery, and thus actual maximal values could not
of participants. A one-way repeated-measures ANOVA was also be measured.
used to test differences between groups at the same measurement
times. A post hoc Scheffe test was conducted if a significant
difference was found (p , 0.05). All effect sizes for ANOVA Correlations Between Biomarker Concentration and Skeletal
were calculated using h (2) and defined as small (0.01), medium Muscle Mass
(0.06), and large (0.14) while confidence intervals (CI 95%) There were no significant correlations observed between SMM
were also calculated. Pearson correlation coefficients (r) and and biomarker concentration at maximal intensity and recovery
effects sizes were used to describe the relationship between within groups. Pearson’s correlation coefficients (r) during exer-
biomarker concentrations within each group at maximal exer- cise and recovery for LA were 20.02 to 0.28, 20.13 to 0.15,
cise and recovery compared with total-body SMM and defined 0.10–0.22, and 20.13 to 0.17 (FU, EN, SP, and AM, re-
as small (0.1), medium (0.3), or large (0.5). All statistical anal- spectively), for NH3 were 20.2 to 0.27, 0.15–0.46, 0.16–0.30,
yses were performed using STATISTICA 13.0 software (Stat- and 20.05 to 0.44 (FU, EN, SP, and AM, respectively), for Hx
Soft, Tulsa, OK, USA). Significance level for all statistical were 20.13 to 0.18, 20.47 to 20.44, 20.45 to 20.03, and 0.25
analysis was set at p # 0.05. All values were presented as mean to 0.46 (FU, EN, SP, and AM, respectively), for X were 20.12 to
6 SD. 0.18, 20.47 to 20.42, 20.45 to 20.03, and 0.21 to 0.37 (FU,
EN, SP, and AM, respectively), and for UA were 20.43 to 20.27,
20.11 to 0.19, 0.16–0.40, and 0.06–0.36 (FU, EN, SP, and AM,
Descriptive Characteristics
Sprinters had a significantly greater amount of SMM compared
with the remaining groups and significantly greater weight at ex- Discussion
amination than EN. EN differed significantly from the SP and AM This is the first study to measure ATP metabolism biomarker (LA,
groups in maximal absolute (L·min21) and relative (ml·kg21·min21 NH3, and purine) concentration in blood at rest, during in-
and ml·kg smm21·min21) V̇ O2max. The AM group was signifi- cremental exercise, and in the postexercise recovery period with
cantly older than the other groups. Other baseline anthropometric many sampling points in highly trained athletes. Each group of
and physical characteristics were similar in all groups and are our elite athletes represented a distinct physiological and meta-
presented in Table 1. bolic training profile dictated by sport discipline. Sprinters rep-
resented speed/power athletes engaged in very high-intensity
exercise requiring mostly anaerobically dominant energy contri-
Biomarker Concentration Changes
bution (6). Triathletes and distance runners represented endur-
All detailed mean values with SD, effects sizes, and confidence ance athletes performing predominantly aerobic exercise in their
intervals are presented as supplementary digital content (see training (4). Futsal players represented the team sport group
Tables 1 and 2, Supplemental Digital Content 1 and 2, http:// characterized by mixed energy system contribution from both and aerobic and anaerobic pathways (2).
A129). Absolute LA concentration at rest, at the point of ex- Our main finding is that the pattern and magnitude of bio-
haustion, and during recovery did not differ between groups. marker concentration depends on the specific training profile of
During consecutive stages of exercise, sprinters showed highly trained athletes in distinct sport disciplines. At rest, during
a rapid increase in LA from the initial stage of the test, whereas incremental exercise, and up to 30 minutes into the postexercise
in endurance runners, the increase was noticeably delayed recovery period sprinters had lowest purine metabolism bio-
(Figure 1A). After converting LA per 1 kg muscle mass, the marker concentrations, and endurance athletes had lowest am-
groups also significantly differed at exhaustion and during monia concentrations. For LA during exercise, the lowest
recovery. Highest LASMM values were obtained by endurance concentrations were noted in endurance athletes, except when
athletes and lowest by sprinters (Figure 1B). reaching maximum intensity, where the differences between
For absolute NH3 values, significant between-group differ- groups were not significant. In this study, sprinters had signifi-
ences were observed at rest, during exercise, and recovery but not cantly greater absolute SMM than all remaining groups. Finally,
at maximum exertion. During exercise, sprinters had a higher there were no correlations between SMM and biomarker con-
concentration and most rapid increase in NH3 and endurance centrations at maximal intensity and during recovery within each
athletes the lowest values compared with other groups, whereas group.
during recovery, controls showed highest and endurance athletes We observed that groups differed in regard to specific bio-
the lowest NH3 levels (Figure 1C). Converting the values relative marker concentration. Sprinters had overall lower purine bio-
to SMM resulted in lowest values in sprinters during recovery marker concentration than all groups. Sprint training is mainly
(Figure 1D). composed of short, high-intensity exercise bouts with complete or
Across the entire measurement spectrum (rest, exercise, ex- incomplete rest depending on training goal (6,36). This type of
haustion, and recovery), the groups significantly differed in both exercise causes a decrease in the muscle adenine nucleotide pool
absolute and relative (per 1 kg muscle mass) values of Hx, X, (including ATP), through degradation of inosine monophosphate
and UA. Regardless of metabolite and measure unit, highest (IMP) into Hx and X, which further effluxes into the bloodstream
values were consistently obtained by controls and lowest values and forms UA, later being excreted with urine (13,14). Zieliński
by sprinters (Figure 2A‒F). Unlike LA and NH3, maximum Hx et al. (36–40) have demonstrated that short-term and long-term

Copyright © 2019 National Strength and Conditioning Association. Unauthorized reproduction of this article is prohibited.
ATP Metabolism Biomarkers During Exercise (2019) 00:00 |

Figure 1. Venous plasma LA (A), LASMM (B), NH3 (C), and NH3SMM (D), concentrations at rest, during incremental
treadmill exercise test until exhaustion, and after exercise in futsal players (FU), endurance athletes (EN), sprinters
(SP), and control group (AM). Data are mean values 6 SDs. Vertical arrows indicate point of volitional exhaustion
(V̇O2max). Horizontal arrows indicate first significant difference from blood sampling points at rest and maximal
exercise within groups. Significant differences between blood sampling points: ***p , 0.001, **p , 0.01. Significant
differences between groups at same sampling point: #p , 0.001, ‡p , 0.01, §p , 0.05.

training decreases postexercise Hx levels, which coincides with change differences between groups compared with absolute val-
increased activity of the enzyme hypoxanthine-guanine phos- ues. This demonstrates that blood purine metabolite concentra-
phoribosyltransferase (HGPRT) that catalyzes the conversion of tion is not sensitive to differences in SMM but only to specific
Hx to IMP. This Hx decrease occurs during periods of training metabolic adaptations.
with high-intensity intermittent-type sessions, especially during In terms of NH3, endurance athletes overall had lower blood
the special preparatory and competitive period. Also, this is more concentrations than all groups. Endurance training is mostly
evident in highly trained athletes, since they engage in more composed of high-volume, low- to moderate-intensity-type ex-
metabolically challenging training sessions producing greater ercise bouts with some higher-intensity training sessions in-
ATP precursor loss (36,37,39). Thus, high-intensity intermittent corporated into certain phases of the annual training plan
training results in higher erythrocyte HGPRT activity (36–40) (4,36,38). Endurance training has been shown to reduce ammo-
causing more efficient utilization of the salvage pathway in re- nia production during submaximal exercise suggesting that en-
covering derivatives of adenine nucleotide breakdown, limiting durance training does not decrease ammonia production capacity
purine efflux from skeletal muscle, and, consequently, decreasing but instead delays it (33). Endurance training also increases the
postexercise plasma Hx concentrations (12,14,28,36–40). Futsal mitochondrial content and blood flow capacity of muscles
players also had relatively lower purine concentrations at rest, resulting in less adenosine monophosphate (AMP) deaminase
during exercise, and after exercise compared with endurance activity and decreased ammonia production. This most likely
athletes and controls. Since gameplay during matches requires the causes greater utilization of the aerobic system during this type of
ability to repeat sprints with incomplete rest while continuously incremental exercise, thus decreasing overall ATP breakdown
running, team sport training also encompasses higher-intensity through AMP degradation that is indicative of cellular energetic
intermittent training interspersed with aerobic training sessions stress and cellular energy status (7). Endurance athletes also have
(25,27). Plasma purine concentration relative to SMM did not a smaller amount of fast-twitch (FT) muscle fibers, which contain

Copyright © 2019 National Strength and Conditioning Association. Unauthorized reproduction of this article is prohibited.
ATP Metabolism Biomarkers During Exercise (2019) 00:00

Figure 2. Venous plasma Hx (A), HxSMM (B), X (C), XSMM (D), UA (E), and UASMM (F) concentrations at rest, during
incremental treadmill exercise test until exhaustion and after exercise in futsal players (FU), endurance athletes
(EN), sprinters (SP), and control group (AM). Data are mean values 6 SDs. Vertical arrows indicate point of
volitional exhaustion (V̇ O2max). Horizontal arrows indicate first significant difference from blood sampling points at
rest and maximal exercise within groups. Significant differences between blood sampling points: ***p , 0.001, **p
, 0.01, *p , 0.05. Significant differences between groups at same sampling point: #p , 0.001, ‡p , 0.01. UA 5
uric acid.

more AMP deaminase associated with greater NH3 accumulation containing more AMP deaminase, leading to greater NH3 accu-
in blood (33). Oxidative capacity of muscle has shown to be mulation in blood (33). Blood NH3 concentration relative to
sensitive to training, and at the same exercise intensities, aerobi- SMM (NH3SMM) at maximum intensity and recovery was lowest
cally trained muscle will have a smaller increase in glycolytic rate, in sprinters compared with absolute values. This is most likely due
suggesting that AMP deaminase is less active in FT fibers of aer- to greater absolute SMM in sprinters.
obically trained muscles (33). Sprint-trained athletes, on the other For LA, during exercise, the lowest concentrations were noted
hand, possess higher amounts of fast-twitch (FT) muscle fibers, in endurance athletes, except when reaching maximum intensity,

Copyright © 2019 National Strength and Conditioning Association. Unauthorized reproduction of this article is prohibited.
ATP Metabolism Biomarkers During Exercise (2019) 00:00 |

where endurance athletes had the highest concentrations. Blood mass will seem to have lower biomarker levels at the same exercise
lactate response to incremental and constant intensity tests dif- intensity, which could falsely imply better training level.
ferentiates 3 intensity zones, which reflect 3 distinct metabolic Determining LA and NH3 plasma concentration is more fa-
responses (3): (a) exercise intensity that does not require an in- vorable during exercise because both increase proportionately to
crease in glycolytic ATP resynthesis (intensity below “lactate exercise intensity and achieve peak concentration at maximum
threshold”), (b) exercise intensity that causes an increase in gly- intensity. Determining purine metabolite (Hx, X, and UA) con-
colytic rate leading to blood lactate concentration steady state, centration is more useful after exercise because purine efflux is
matched by pyruvate utilization and therefore leading to intensity indicative of adenine nucleotide loss and consequently reflects en-
between lactate threshold and maximal lactate steady state ergy loss. Determining LA threshold is often used in endurance
(MLSS), and (c) exercise intensity leading to a glycolytic rate that athletes to prescribe appropriate training loads below, at, or above
cannot be matched by aerobic pyruvate utilization indicating this threshold (23). However, because endurance athletes have LA
need for ATP generation through anaerobic glycolysis (intensity thresholds at relatively high intensities (percentage of their
above MLSS). Training of highly trained endurance athletes is V̇ O2max), and because most engage in low- to moderate-intensity
mostly dedicated to the first intensity domain (70–90% of exercise below this threshold, it leaves a large unquantifiable area
training volume) and less to the second and third intensity to prescribe training intensity. Therefore, when quantifying train-
domains (1). This most likely leads to LA concentrations being ing load at intensities below LA threshold, NH3 is more intensity-
very low during initial stages of an incremental exercise test. Also, sensitive. LA is a more adequate marker to assess exercise intensity
endurance athletes’ LA thresholds are relatively high, which in sprint-trained and game sport athletes, and mainly during
allows them to exercise at a higher relative intensity (%V̇ O2max) higher-intensity interval–type sessions (HIIT), since it is an in-
for longer duration. In endurance athletes, glycolytic rate is dicator of anaerobic glycolysis utilization during exercise (7).
matched by pyruvate utilization at much higher intensities than in NH3 may be used as an indirect indicator of muscle ATP loss
untrained and recreationally trained subjects (3). This may in- during exercise (11) because it increases proportionately to ex-
dicate that lactate shuttling to other tissues that use lactate as fuel ercise intensity and is more intensity-sensitive at lower intensities
in the body may be higher in these athletes, which is reflected by below lactate threshold. This verifies the study by Banister et al.
decreased LA blood concentration. Endurance athletes also have (1), where NH3 blood concentration was elevated even at work
more slow-twitch (ST) muscle fibers, which produce less LA rates at 40–50% V̇ O2max, whereas LA blood concentration in-
through anaerobic glycolysis. Sprinters have lower LA thresholds, creased much more slowly during exercise. Itoh (15) observed
a greater percentage of fast-twitch (FT) muscle fibers therefore use that peak ammonia concentration did not differ between 15-, 30-,
anaerobic glycolysis to a higher degree leading to greater LA and 45-second sprint bouts, but LA concentration did. This
production and release. In addition, sprinters have greater levels demonstrates that ammonia is more intensity-sensitive because
of PCr that can be used during exercise. Utilization of PCr in turn each sprint was performed at maximal volitional exertion leading
is coupled closely with glycolysis; depending on intensity and to similar ammonia concentrations (depletion of TAN pool after
duration of exercise, greater depletion of PCr will lead to in- 15 seconds in all cases). LA concentrations increased because of
creased glycolysis (21). Finally, futsal athletes had the second increases in utilization of anaerobic glycolytic pathways in longer
largest LA concentration during incremental exercise (sprinters sprint bouts. Determining plasma NH3 concentration can be used
the most). Since futsal athletes represent mixed energy system to evaluate, monitor, and prescribe exercise intensity during
contribution, their LA concentration increase could be caused by clinical exercise tests, conditioning programs, occupational tasks,
greater anaerobic glycolytic activity than endurance athletes and and athletic performance.
controls, which relied more on aerobic contribution. For LAmax, Purine metabolites, especially Hx can be used during exercise
sprinters had the lowest concentrations relative to SMM because Hx increases proportionately to exercise intensity, but it
(LASMM). In contrast to absolute LA concentration, LASMM is worthwhile to measure Hx concentration in the postexercise
concentration during exercise was lowest in sprinters compared recovery period to determine total purine derivative loss. This can
with other groups most likely due to greater absolute SMM. help assess recovery needs of athletes after high-intensity exercise
LA, NH3, and Hx are all valuable for monitoring training sessions. Hx level also indicates adaptation to high-intensity ex-
status in highly trained athletes, and each demonstrates different ercise because of reduced purine efflux from skeletal muscle and
exercise-induced changes in skeletal muscle. Gorostiaga et al. (11) can be viewed as a marker of anaerobic metabolism (14,36). Hx
found that LA concentration in muscle correlated with plasma LA can be used in highly trained athletes as an indicator of training
concentration in blood, and that ATP concentration in muscle status in different training phases of a 1-year training cycle re-
correlated with plasma NH3 concentration in blood. This indi- gardless of age and sport discipline (37). Using Hx is particularly
cates that by measuring plasma concentration of LA or NH3, valuable in highly trained athletes because commonly used
energy status of skeletal muscle can be indirectly determined. measures such as V̇ O2max, “anaerobic threshold” or LA con-
Purine concentration, on the other hand, mirrors adenine nucle- centration are not sensitive to changes in training status and
otide derivative loss in muscle. Stathis et al. (29) and Sahlin et al. performance of highly trained athletes. Training prescription
(22) demonstrated that subjects with a high blood and muscle should be revised based on recent knowledge regarding purine
purine concentration exhibited a decreased muscle total adenine metabolism adaptations to fit training programs of highly trained
nucleotide (TAN) pool or muscle ATP level. Overall, plasma LA athletes. Hx can also be used during tapering periods to determine
concentration reflects muscle LA concentration that further preparedness and readiness to competition (37). Notably, UA
reflects anaerobic glycolysis utilization, plasma NH3 concentra- plasma concentration did not reach peak values during exercise
tion reflects muscle ATP content, and plasma purine concentra- and even after 30 minutes after exercise. This questions its use-
tion reflects TAN pool depletion. However, calculating fulness as a biomarker of muscle status since it is difficult to
biomarker concentration relative to muscle mass (per 1 kg of achieve a peak value in the specified time frame.
SMM) does not present a clear and accurate picture and does not Finally, there were also no correlations observed between
have diagnostic value. Athletes having larger amounts of muscle SMM and biomarker concentrations at maximal intensity and

Copyright © 2019 National Strength and Conditioning Association. Unauthorized reproduction of this article is prohibited.
ATP Metabolism Biomarkers During Exercise (2019) 00:00

recovery in all groups. This indicates that muscle mass levels are Acknowledgments
not significantly related to magnitude of biomarker release in
The authors do not have professional relations with any
blood. Because we did not perform muscle biopsies, we were not
companies or manufacturers who will benefit from the results
able to determine biomarker concentrations in muscle and their
of this study. The results of this study do not constitute
correlations with blood biomarker levels. However, this may in-
endorsement of the product by the authors or the NSCA. The
dicate that training level and especially training profile as a result
authors thank the coaches, athletes, and volunteers for their full
of metabolic adaptations in muscle dictates pattern of biomarker
participation in the study. The authors declare that they have no
release in blood, not muscle mass itself. Although more muscle
conflicts of interest. This work was funded by the Polish Ministry
mass may indeed produce greater absolute amounts of these
of Science and Higher Education from financial resources of the
biomarkers in muscle, the blood concentration is the combined
National Science Center within the OPUS 5 program (application
result of many factors (production and utilization in various
and grant number: 2013/09/B/NZ7/02556).
metabolic processes).
A limitation in this study was that subjects could not perform an
additional familiarization session with V̇ O2max testing a few days
before the actual exercise (with blood sampling). Because most
athletes are part of the Polish National Team, it is difficult to ask References
them to perform additional tests since they have a very rigorous 1. Banister EW, Allen ME, Mekjavic IB, Singh AK, Legge B, and Mutch
training cycle, and additional loading in the form of this exercise BJC. The time course of ammonia and lactate accumulation in blood
test would not be accepted by their respective coaches. Nonetheless, during bicycle exercise. Eur J Appl Physiol Occup Physiol 51: 195–202,
all subjects performed these tests regularly throughout their annual 1983.
2. Beato M, Coratella G, Schena F, and Hulton AT. Evaluation of the ex-
training plan in our laboratory to monitor training, so they were ternal and internal workload in female futsal players. Biol Sport 34:
accustomed to such a test. Another limitation in this study is the 227–231, 2017.
differences in the average time it took participants to complete the 3. Beneke R, Leithäuser RM, and Ochentel O. Blood lactate diagnostics in
test. The EN group, being the most endurance-trained group, took exercise testing and training. Int J Sports Physiol Perform 6: 8–24, 2011.
4. Billat VL, Demarle A, Slawinski J, Paiva M, and Koralsztein JP. Physical
the longest to perform the exercise test. However, the aim of using and training characteristics of top-class marathon runners. Med Sci Sports
such a test was to reach V̇ O2max that would standardize intensity Exerc 33: 2089–2097, 2001.
for all groups although duration was greater in the EN group. 5. Brooks GA. Lactate: Link between glycolytic and oxidative metabolism.
Sports Med 37: 341–343, 2007.
6. Duffield R, Dawson B, and Goodman C. Energy system contribution to
Practical Applications 100-m and 200-m track running events. J Sci Med Sport 7: 302–313,
The magnitude and exact time course of exercise-induced 7. Finsterer J. Biomarkers of peripheral muscle fatigue during exercise. BMC
Musculoskelet Disord 13: 218, 2012.
biomarker concentration during a standard progressive ex-
8. Gladden LB. Lactate metabolism: A new paradigm for the third millen-
ercise until exhaustion is related to training adaptations nium. J Physiol 558: 5–30, 2004.
through specific training profile. Combined measuring of LA, 9. Goodwin ML, Harris JE, Hernández A, and Gladden LB. Blood lactate
NH3, and Hx concentration in blood is useful in indirectly measurements and analysis during exercise: A guide for clinicians.
reflecting key changes in exercise- and training-induced en- J Diabetes Sci Technol 1: 558–569, 2007.
10. Gorostiaga EM, Navarro-Amezqueta I, Calbet JAL, Hellsten Y, Cusso R,
ergy status without the need to use invasive methods such as Guerrero M, et al. Energy metabolism during repeated sets of leg press
muscle biopsies. It is essential to note that the 3 metabolites exercise leading to failure or not. Plos One 7: e40621, 2012.
play different roles. (a) Determining LA concentration in 11. Gorostiaga EM, Navarro-Amezqueta I, Calbet JAL, Sanchez-Medina L,
blood is useful for athletes when performing training sessions Cusso R, Guerrero M, et al. Blood ammonia and lactate as markers of
muscle metabolites during leg press exercise. J Strength Conditioning Res
using anaerobic glycolysis to a high degree and for athletes in
28: 2775–2785, 2014.
disciplines where LA threshold determination is needed to 12. Hellsten-Westing Y, Norman B, Balsom PD, and Sjodin B. Decreased
divide training-intensity zones (3). (b) NH3 concentration is resting levels of adenine-nucleotides in human skeletal-muscle after high-
helpful for monitoring exercise at lower exercise intensities intensity training. J Appl Physiol 74: 2523–2528, 1993.
and can be applied for monitoring ATP loss during exercise, 13. Hellsten Y, Richter EA, Kiens B, and Bangsbo J. AMP deamination and
purine exchange in human skeletal muscle during and after intense exer-
for training prescription and evaluation (33), and can be used cise. J Physiology-London 520: 909–920, 1999.
by endurance athletes to more precisely monitor exercise at 14. Ipata P and Pesi R. Metabolic interaction between purine nucleotide cycle
low intensities. (c) Purine, especially Hx concentration in and oxypurine cycle during skeletal muscle contraction of different in-
blood, is a valuable tool to determine postexercise purine tensities: A biochemical reappraisal. Metabolomics 14: 42, 2018.
15. Itoh H and Ohkuwa T. Ammonia and lactate in the blood after short-term
derivative loss. Hx is particularly important in highly trained
sprint exercise. Eur J Appl Physiol Occup Physiol 62: 22–25, 1991.
athletes (36–40). Hx can indicate adaptations to anaerobic 16. Karatzaferi C, de Haan A, van Mechelen W, and Sargeant AJ. Metabolic
exercise to check training status in different phases of a 1-year changes in single human muscle fibres during brief maximal exercise. Exp
training cycle, especially during tapering periods, regardless of Physiol 86: 411–415, 2001.
sport discipline. The differences in plasma purine concentra- 17. Kim J, Wang ZM, Heymsfield SB, Baumgartner RN, and Gallagher D.
Total-body skeletal muscle mass: Estimation by a new dual-energy X-ray
tion of metabolites and NH3 between specifically adapted absorptiometry method. Am J Clin Nutr 76: 378–383, 2002.
athletic groups are independent from SMM. Skeletal muscle 18. Mutch BJ and Banister EW. Ammonia metabolism in exercise and fatigue:
mass level does not significantly affect magnitude of bio- A review. Med Sci Sports Exerc 15: 41–50, 1983.
marker release, and thus sport practitioners do not have to 19. Ogino K, Kinugawa T, Osaki S, Kato M, Endoh A, Furuse Y, et al. Am-
monia response to constant exercise: Differences to the lactate response.
consider muscle mass levels when measuring biomarker con-
Clin Exp Pharmacol Physiol 2000;27: 612–617.
centration in blood. Further research should focus on studying 20. Pedersen BK. IL-6 signalling in exercise and disease. Biochem Soc Trans
how specific training sessions affect individual biomarker re- 35: 1295–1297, 2007.
sponse in highly trained athletes. 21. Sahlin K. Muscle energetics during explosive activities and potential effects
of nutrition and training. Sports Med 44(Suppl 2): S167–S173, 2014.

Copyright © 2019 National Strength and Conditioning Association. Unauthorized reproduction of this article is prohibited.
ATP Metabolism Biomarkers During Exercise (2019) 00:00 |

22. Sahlin K, Tonkonogi M, and Söderlund K. Plasma hypoxanthine and 32. Yuan Y and Chan KM. A longitudinal study on the ammonia threshold in
ammonia in humans during prolonged exercise. Eur J Appl Physiol Occup junior cyclists. Br J Sports Med 38: 115–119, 2004.
Physiol 80: 417–422, 1999. 33. Yuan Y and Chan KM. A review of the literature on the application of
23. Seiler KS and Kjerland GO. Quantifying training intensity distribution in blood ammonia measurement in sports science. Res Q Exerc Sport 71:
elite endurance athletes: Is there evidence for an “optimal” distribution? 145–151, 2000.
Scand J Med Sci Sports 2006;16: 49–56. 34. Yuan Y, So R, Wong S, and Chan KM. Ammonia threshold—
24. Sjodin B and Westing YH. Changes in plasma-concentration of hypo- Comparison to lactate threshold, correlation to other physiological
xanthine and uric-acid in man with short-distance running at various parameters and response to training. Scand J Med Sci Sports 2002;12:
intensities. Int J Sports Med 11: 493–495, 1990. 358–364.
25. Slattery KM, Wallace LK, Bentley DJ, and Coutts AJ. Effect of training 35. Zielinski J, Krasinska B, and Kusy K. Hypoxanthine as a predictor of
load on simulated team sport match performance. Appl Physiol Nutr performance in highly trained athletes. Int J Sports Med 34: 1079–1086,
Metab 37: 315–322, 2012. 2013.
26. Smolenski RT and Yacoub MH. Liquid-chromatographic evaluation of 36. Zielinski J and Kusy K. Training-induced adaptation in purine metabo-
purine production in the donor human heart during transplantation. lism in high-level sprinters vs. triathletes. J Appl Physiol 112: 542–551,
Biomed Chromatogr 7: 189–195, 1993. 2012.
27. Spencer M, Bishop D, and Lawrence S. Longitudinal assessment of the 37. Zielinski J and Kusy K. Hypoxanthine: A universal metabolic indicator of
effects of field-hockey training on repeated sprint ability. J Sci Med Sport training status in competitive sports. Exerc Sport Sci Rev 43: 214–221,
7: 323–334, 2004. 2015.
28. Stathis CG, Carey MF, Hayes A, Garnham AP, and Snow RJ. Sprint 38. Zielinski J, Kusy K, and Rychlewski T. Effect of training load structure on
training reduces urinary purine loss following intense exercise in humans. purine metabolism in middle-distance runners. Med Sci Sports Exerc 43:
Appl Physiol Nutr Metab 31: 702–708, 2006. 1798–1807, 2011.
29. Stathis CG, Febbraio MA, Carey MF, and Snow RJ. Influence of sprint 39. Zielinski J, Kusy K, and Slominska E. Alterations in purine metabolism in
training on human skeletal muscle purine nucleotide metabolism. J Appl middle-aged elite, amateur, and recreational runners across a 1-year
Physiol 76: 1802–1809, 1994. training cycle. Eur J Appl Physiol 113: 763–773, 2013.
30. Stathis CG, Zhao S, Carey MF, and Snow RJ. Purine loss after repeated 40. Zielinski J, Rychlewski T, Kusy K, Domaszewska K, and Laurentowska
sprint bouts in humans. J Appl Physiol 87: 2037–2042, 1999. M. The effect of endurance training on changes in purine metabolism: A
31. Wilkinson DJ, Smeeton NJ, and Watt PW. Ammonia metabolism, the brain longitudinal study of competitive long-distance runners. Eur J Appl
and fatigue; revisiting the link. Prog Neurobiol 91: 200–219, 2010. Physiol 106: 867–876, 2009.

Copyright © 2019 National Strength and Conditioning Association. Unauthorized reproduction of this article is prohibited.