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School of Sport and Exercise Sciences, College of Life and Environmental Sciences, and 2School of Immunity and Infection,
University of Birmingham, Birmingham, United Kingdom
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Age, yr
BMI, kg/m
O2max, ml kg1 min1
Measured V
O2max, beats/min
Maximum heart rate at V
LTPA, median METmin/wk*
32.6 13.2
23.8 2.5
59.7 5.2
187 9
3,497 (7207,290)
Data are means SD. LTPA, leisure time physical activity, as assessed by
the international physical activity questionnaire. *Data are medians (min-max).
J Appl Physiol VOL
Protein carbonylation. Total lymphocyte protein carbonyl concentration was measured by enzyme-linked-immunosorbent-assay
(ELISA) described by Buss et al. (10). Lymphocytes were lysed with
RIPA buffer containing a protease inhibitor cocktail, and the supernatant was used for analysis. Samples and standards (0.5 mg/ml in
sodium carbonate buffer; 50 mM, pH 7.4, 50 l) were allowed to bind
to 96-well multi-sorb plates for 60 min at 37C in triplicate (Nunc,
Fisher Thermo Scientific). 2,4-Dintrophenylhydrazine (DNP; 1 mM,
50 l) in 2 M hydrochloric acid was added, and plates were incubated
at room temperature for 60 min. Plates were blocked overnight at 4C
with 0.1% TBS-TWEEN-20. Mouse anti-DNP antibody (1:1,000, 50
l) was incubated with samples for 120 min at 37C, followed by a
peroxidise-labeled rat anti-mouse antibody (1:5,000, 50 l), incubated
for 60 min at 37C. A citrate phosphate-based substrate (0.15 M, pH
5) was added, and plates were left to develop in the dark for 30 min
at 37C. The reaction was stopped with 2 M sulphuric acid, and plates
were read at 490 nm. Values were expressed as nmol/mg of protein.
Lipid peroxidation. Lipid peroxides were assayed in plasma using
a modification of the method by el-Saadani et al. (19). Samples and
positive and negative controls (1:1,000 hydrogen peroxide and distilled water, respectively) were added to 96-well plates in triplicate.
Working reagent (0.2 M potassium phosphate, 0.12 M potassium
iodide, 0.15 mM sodium azide, 2 g/l Triton-X, 0.1 g/l alkylbenzyldimethylammonium, 10 M ammonium molybdate; 100 l) was added,
and plates were incubated at room temperature for 30 min. Plates were
read at 340 nm. Lipid peroxide concentration was calculated using the
Beer-Lambert-Law with an extinction coefficient of 24,600. Values
were expressed as nmol/ml plasma.
Total antioxidant capacity. Total antioxidant capacity was assessed
in plasma using the ferric reducing ability of plasma (FRAP) assay
(8). Briefly, standards (0 1,000 M ascorbic acid) and samples (10
l/well) were added to 96-well flat-bottomed cell culture plates in
triplicate. Working reagent (20 mM ferric chloride, 160 mM 2,4,6tripyridyltriazine, 300 mM acetate buffer; 300 l) was added to each
well, and plates were read at room temperature after 8 min. Values
were determined by linear regression from a seven-point standard
curve (36) and expressed as M of antioxidant power relative to
ascorbic acid.
Flow cytometry. To examine the mobilization of T lymphocyte
subpopulations with exercise, fresh lysed whole blood was prepared
as described previously (54). Fixed cell preparations were read within
36 h of exercise on a multi-parameter flow cytometer (BD FACS
CANTO II, BD Biosciences). Lymphocytes were enumerated using a
Coulter ACTdiff hematology analyzer (Beckman-Coulter).
In a separate investigation, blood was collected from an antecubital
vein at rest from eight healthy men (means SD: age, 31.0 10.4
O2max 56.2 5.0
yr; body mass index 23.5 2.1 kg/m2, V
mlkg1m1). Two of the men took part in the main investigation,
and the additional six subjects did not differ from those included in the
main study. Samples were collected at least 2 h after consuming food
or fluids. Intracellular GSH was measured in lymphocyte subpopulations using a modification of the procedure described by Cossarizza et
al. (13). Monobromobimane (MBB) is a nonfluorescent probe that
binds to intracellular thiols emitting light at 490 nm when excited with
a 405-nm violet laser. GSH is the most abundant intracellular thiol;
therefore, median fluorescence intensity (MFI) of MBB can be considered a measure of intracellular GSH concentration (13, 43).
Approximately 2 106 peripheral blood lymphocytes were incubated with monoclonal antibody panels for 20 min at room temperature. The first antibody panel included anti CD19 FITC, anti CD56 PE
(Pharmingen, San Diego, CA), and anti CD3 PerCp (BD Biosciences,
San Jose, CA). The second antibody panel included anti CD45RA
FITC, CD27 PE (Pharmingen), CD3 PerCP, CD28 PE-cy7, CD4
APC, and CD8 APC-cy7 (BD Biosciences). Samples were washed in
PBS by centrifugation at 300 g. Cells were re-suspended in 1 ml of
37C PBS, and a sample from each individual was incubated for 20
min at 37C with 100 M N-ethylmaleimide. Intracellular thiol
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Fig. 1. Immunological and oxidative responses to exercise. Data are means SE.
A: revertant effector memory CD8 T cells, which have re-expressed CD45RA
(EMRA; CD27CD45RA) and naive CD8 T cells (CD27CD45RA). Cells
expressed as a proportion (%) of total lymphocytes. B: lymphocyte protein
carbonyl concentration. C: plasma lipid peroxide concentration. D: plasma antioxidant capacity. Signficant difference compared with baseline (paired samples
t-tests): *P 0.05; **P 0.01; ***P 0.001.
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GRANTS
This study was funded by the University of Birmingham (Birmingham, UK).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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