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Leukocyte ROS Measured with Flow Cytometry before and after a Strength Training Session
Hilde G. Nielsen1,2* and Torstein Lyberg2
1The Research Council of Norway, Lysaker, Norway
2Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Norway
Submission: July 23, 2018; Published: August 02, 2018
*Corresponding author: Hilde G. Nielsen, The Research Council of Norway, 1327 Lysaker, Norway, Tel: +47 40 92 22 60;
How to cite this article: Hilde G Nielsen, Torstein Lyberg. Leukocyte ROS Measured with Flow Cytometry before and after a Strength Training Session. J Phy Fit Treatment & Sports. 2018; 5(1): 555653. DOI:10.19080/JPFMTS.2018.05.555653
According to the literature there is a link between the intensity of exercise and the occurrence of infections and disease. Strength training is physical exercise with the use of resistance to induce muscular contraction. Reactive oxygen species (ROS) are unstable molecules that contains oxygen and are formed in the body. Antioxidants are important to prevent chemical damage to the cell components by oxidation. Oxidative stress is defined as a disturbance in the balance between the production of ROS and antioxidant defenses. The apprehension of increased rates of ROS production during exercise is part of a rationale why many athletes ingest antioxidant supplements beyond recommended dose. The aim of this present study was to investigate changes in leukocyte levels of ROS measured with flow cytometry, total antioxidant status (TAS) and catalase before and after a strength training session. Ten men and ten women were recruited to this study. All participants went through a strength training session consisting of nine exercises executed at 80% of 1RM, 6-8 repetitions/3 sets. The labelled samples were analysed within 24h in a flow cytometer. Examining the post-training blood samples, they were all non-significantly changed compared to the pre-training samples. The TAS and the levels of plasma catalase did not change as a result of the training session. The strength training session were not enough to influence the leukocyte production of ROS and the total antioxidant system in trained students. Based on these findings, there is no rationale to advice athletes to ingest antioxidant supplementation in addition to their regular diet.
Keywords: Leukocytes; Reactive oxygen species; Total antioxidant status; Strength training
Abbreviations: ROS: Reactive Oxygen Species; TAS: Total Antioxidant Status; 1RM: One Repetition Maximum; PMA: Phorbol Myristate Acetate; WBC: White Blood Cell; PBS: Phosphate- Buffered Saline; MFI: Mean Fluorescence Intensity; TAS: Total Antioxidant Status
Physical exercise can enhance or maintains physical fitness and overall health and wellness [1-4]. According to the literature, there is a link between the intensity of exercise and the occurrence of infections and disease [1,5-8]. Regular physical exercise and exercise at moderate levels are important factors for disease prevention [1,9].
Strength training is physical exercise with the use of resistance to induce muscular contraction. The effects of the training are increased muscle strength, anaerobic endurance and increased size of skeletal muscles. Strength training is dependent of the amount of work required to achieve the activity of the training (intensity), number of muscles worked, exercise, sets and reps (volume) and how many training sessions performed per week (frequency) [3,4].
Reactive oxygen species (ROS) are formed in the body. ROS include oxygen ions, free radicals and peroxides, both inorganic and organic. Both granulocytes and monocytes produce ROS, e.g superoxide anion (O2-), hydrogen peroxide (H2O2), peroxinitrite (ONOO-) and hydroxyl radical (•OH). Superoxide anions are unstable free radicals that kills bacteria directly and is generated both in intra- and in extracellular compartments. Nitric oxide and O2- react with each other and form rapidly ONOO-. Peroxinitrite is a strong radical, which can damage DNA, proteins and other cellular elements. Many sources of heat, stress, irradiation, inflammation and any increase in metabolism, including exercise lead to increased production of ROS [10-12]. The uncertainty of increased rates of ROS production during exercise is part of a rationale why many athletes ingest antioxidant supplements beyond suggested doses [9,11,13,14].
We have used the highly sensitive flow cytometric method for measuring ROS generation in leukocytes. Using fluorescence probes and flow cytometry we have earlier shown that the phorbol ester phorbol myristate acetate (PMA) may induce an increase in the levels of ROS in granulocytes by at least a factor
of 150 . In endurance running, e.g. marathon race, we could
show that the capacity of leukocytes (both granulocytes and
monocytes) to respond with ROS after in vitro PMA stimulus
was heavily suppressed . A tendency in this direction also
applied for a half-marathon race  whereas a short- term
maximal physical exercise (test of maximal oxygen consumption
(VO2max)) did not induce such a suppression of ROS generation,
but instead a significant increase in intracellular granulocytes
ROS following PMA stimulation .
Antioxidants are important to prevent chemical damage to the
cell components by oxidation. An antioxidant is a chemical compound
and is thought to defend the body cells from the destructive
effects of oxidation (ROS). Catalase is an enzyme found in
nearly all living organisms exposed to oxygen (such as bacteria,
plants, and animals). Catalase decompose hydrogen peroxide to
water and oxygen. Catalase is a very important enzyme in protecting
the cell from oxidative damage by reactive oxygen species
(ROS). Antioxidants and training prevent against ROS production
in exhaustive exercise [5,9].
To compare with our different historical groups on running
exercises, the aim of the present study was to investigate in
athletes the changes in leukocyte levels of ROS measured with
flow cytometry, total plasma antioxidant capacity and catalase
before and after a strength training session.
Ten men and ten women were recruited to this study. The
average age of the men were 25 years old (range 22-28), and the
average age of the women were 26 years old (range 23-30). The
participants were all students and were recruited in an exercise
room at the Oslo university campus. The men trained in average
3,6hours/week, while the women trained in average 3,9hours/
week. The average time of each training sessions were for the
men 1,8hours/time (range 1,5 - 2,5hours/time) and 1,6hours/
time (range 1,0 - 2,5hours/time) for the women. The main
exercise was in addition to strength training, endurance training
as running, bicycling, spinning and football. All participants
were asked to avoid training the day before the test. None of the
participants ingested vitamins regularly, but two participants
eat cod liver oil daily. The subjects were informed about the
study and gave their written informed consent for participation.
The study was approved by the Regional Committee for Medical
and Health Research Ethics (REC) before the start of the project.
Venous blood samples were taken before (before the warm
up) and immediately after the training session (within 10
minutes). Venous blood was sampled into anticoagulated (EDTA
or heparin) vacuum tubes (Becton Dickinson, Plymouth, UK).
The blood samples were kept on ice and transported within
30 minutes to the appropriate laboratory at Oslo university
After 20 minutes warm up running at a treadmill or biking, all
participants went through a strength training session consisting
of nine exercises; squats, bench press, shoulder press, biceps,
curl, leg curve, leg extension, back and belly. Every exercise was
done at about 80% of One Repetition Maximum (1RM) with six
to eight repetitions and three sets.
White blood cell (WBC) and platelet counts, hemoglobin and
hematocrit were determined in EDTA blood using the Technicon
H2•TM System (Bayer, Tarrytown, NY, USA) at the Department of
Medical Biochemistry, Oslo University Hospital, Ullevål.
Aliquots of 50μl EDTA whole blood were incubated for
15min at 37ºC in a 5% CO2 /humidified air atmosphere in 5ml
polystyrene round-bottom tubes (2052 Falcon, Oxnard, CA,
USA) with dihydroethidium (DHE) (Sigma, St. Louis, MO, USA)
(final concentration 5μmol/l), recognizing mainly the oxygen
species superoxide anion (O2-) (18) or with dihydrorhodamine
123 (DHR) (Molecular Probes, Leiden, Netherlands) (final
concentration 5μmol/l), recognizing the oxygen species
peroxynitrite (ONOO-), hypochlorous acid and hydrogen peroxide
(9) or with phosphate- buffered saline (PBS) (Sigma) (10mmol/l
phosphate buffer, 2.7mmol/l KCl, 137mmol/l NaCl, pH 7.4) as
autofluorescence control. After ended incubation, 1.5ml red
cell lysing solution containing 156mmol/l NH4Cl, 10mmol/l
NaHCO3 and 0,12mmol/l EDTA was added to the tubes. The
tubes were incubated in the dark at room temperature for
15min and centrifuged at 300g for 5min at 4°C. The leukocytes
were washed once in 2ml cold PBS and finally resuspended in
0.5ml 1% (w/v) paraformaldehyde in PBS, pH 7.4. The samples
were stored in the dark at 4°C until flow cytometry could be
performed (within 24h).
Aliquots of 1.5ml EDTA whole blood were incubated in
polystyrene tubes with ventilation caps with 15μl phorbol
myristate acetate (PMA) (final concentration 100ng/ml) (Sigma)
or PBS (control) at 37ºC (DHE) and 30ºC (DHR) for 60min in
a CO2 incubator. Aliquots of 50μl blood were then labeled with
DHE and DHR as described for basal ROS levels.
The labeled samples were analysed within 24h in a
FACSort™ flow cytometer (Becton Dickinson, San Jose, CA, USA).
The flow cytometer was equipped with an argon laser and CellQuest™
software (Becton Dickinson). Ten thousand events
were collected from each sample. The leukocyte subpopulations
monocytes and granulocytes, were identified by their light
scatter characteristics, enclosed in electronic gates and
separately analysed for fluorescence intensity from the separate
(DHE/DHR) fluorescent probes. The results are expressed as
mean fluorescence intensity (MFI). The intra assay coefficient
of variation (CV) was < 5% in unstimulated and < 10% in PMAstimulated
Blood samples were collected in heparin tubes and
centrifuged at 2300g, 4°C for 12min and plasma stored at - 70°C
until analysis. The Randox Total Antioxidant Status (TAS) kit
(Crumlin, UK) and The Catalase Assay kit (Cayman Chemical,
UK) were used according to the manufacturer’s instructions.
Results are given as means and standard errors of the mean
(mean, SEM). Difference within groups was analyzed using twotailed
paired sample t-test (from pre to post). Independent
sample t-test was used in order to compare men and women.
Since we did not observe any statistically significant differences
between the women and men, we have chosen to present the
results as one group (except the hematology values which are
presented separately for each group).
All the twenty subjects completed the training session.
One subject did train before the test day, even that they were
informed of no training before the test. None of the participants
ingested any nutrition supplement the day before strength
The total number of leukocytes was within normal range
(3.0-11.0 x 109.L-1) before the strength training session. After the
session the numbers had increased but not significantly. Both
the haematocrit, the haemoglobin and thrombocytes remained
at the same level after the training session (Table 1).
Basal and stimulated leukocytes ROS levels before and after
the training session Table 2 summarise the separate figures
(MFI±SEM) for PBS (control) and PMA- stimulated leukocytes
and the stimulating index (SI = PMA-stimulated/PBS) for
the separate fluorechromes (DHR/DHE) in granulocytes and
monocytes, comparing the pre- versus post-training sessions.
Using the DHR probe, the stimulation index in granulocytes of
pre-training blood samples was 199±16 (monocytes 2.1±0.2)
whereas the corresponding figures using the DHE probe was
62±5 (monocytes 12.2±1.0).
Examining the post-training blood samples demonstrated
that both PBS (control samples) and PMA-stimulated samples,
as well as the calculated stimulation index (SI=PMA-stimulated/
control) were all non-significantly changed compared to the pretraining
Figure 1 illustrates the granulocyte basal ROS levels the ROS
increase induced by PMA and the stimulation index (SI) before
and after a strength training session measured with the DHR and
the DHE probes. Total antioxidant status and catalase before and
after the training session.
The total plasma antioxidant status and the levels of plasma
catalase did no change after the training session (Figure 2).
We have in the present study investigated the changes in
selected haematological parameters, the leukocyte expression
of ROS measured with flow cytometry using the DHE- and the
DHR probes basal and after in vitro stimulation with PMA, total
plasma antioxidant capacity and catalase in males and females’
athletes before and after a strength training session.
We observed after the strength training session unchanged
levels of total number of leukocytes. This is in contrast to what we
observed in our study with the marathon and the half-marathon
runners where we found a 3.2-fold and a 2.4- fold increase in
leukocytes after the races . Similarly, we observed a 1.6-fold
increase of circulating leukocytes after a short term physical
exercise until exhaustion (test of maximal oxygen consumption
(VO2max)) . Our former studies confirm earlier studies;
changes in white blood cells are dependent of type of exercise
and the intensity of the exercise [1,2,5,7,18-20].
As in our earlier studies [16,17,21] we have in this study
used the highly sensitive flow cytometric method for measuring
ROS generation in leukocytes. We observed after a VO2max test
a significant increase in ROS levels in leukocytes . Based
upon that observation, we infer that the leukocytes have been
primed for ROS production during the primary phase. Upon
prolonged running the ROS generation machinery in leukocytes
seems to be temporal exhausted, more exhausted after a full
marathon race than after the shorter half-marathon race .
In the present work on strength training, we could not observe
significantly changes in the leukocyte ROS levels comparing
pre-training values with post- training values. A marathonand
a half marathon race is executed at an intensity where the
production and utilisation of blood lactate ([La-]b) is equal. The
average running time in marathon race was 3h 40min and the
half- marathon race was finished in 1h 53min for the women
and 1h 41min for the men . The test of VO2maxv ends with
at deflating of heart frequencies about 8-10 beats below the
athletes’ maximum heart frequency (HFmax) and the test is over
within 10 min. The present strength training session consisted
of nine exercises which were executed at 80% of One Repetition
Maximum (1RM), 6-8 repetitions and 3 sets. The exercise
session lasts for about 60minutes in average. The reason for
non-responses in leukocytes ROS is probably due to the short
duration of the exercise session even though quite high heart
frequencies were applicable after every single exercise. The total
work load seems to be not high (intensity) or last long (duration)
enough to influence the leukocyte generation of ROS.
The different levels of fitness form of the subjects in our
studies could also be an explanation for the different responses
of leukocyte generation of ROS. This in combination with
the different types of exercise which were performed. The
optimal situation would be to test the same persons executing
all the different training regimes, but this is nearly impossible
with active athletes/students who have their own training
The total plasma antioxidant status level and catalase levels
were not affected by the strength training session. We observed
increased TAS levels after the marathon and the half-marathon
race. The increase was higher in the marathon runners compared
to the half- marathon runners, probably due to the duration of the
activity . The results corresponds also to what we observed
in 10 cadets after a ranger-training course . We observed
no changes in TAS levels comparing values before and after the
test of VO2max . A session of strength training was not enough
(total intensity of the work) or last long enough (duration of the
exercise) to influence any of the measured parameters. Based
on this, a normal diet including daily recommended doses of
vitamins and minerals seems to be suitable for performing this
type of exercise.
One-hour strength training consisting of nine exercises
performed at 80% of 1RM/6-8 repetitions/3 sets were not
enough to influence the leukocyte production of ROS and the
total plasma antioxidant system in trained students. Based on
these findings, there is no rationale to advice athletes to ingest
antioxidant supplementation in addition to their regular diet.
The authors would like to thank the participants who
voluntarily participated in this project. A special thanks to MLT
Lisbeth Saetre for excellent technical and practical assistance.
Thanks to Oslo University Hospital for funding of the project and
to Kristiania University College for economical support.