Maximal exercise in normoxia results in oxidative stress due to an increase in free radical production. However, the effect of a single bout of moderate aerobic exercise performed in either relative or absolute normobaric hypoxia on free radical production and lipid peroxidation remains unknown. To examine this, we randomly matched {according to their normobaric normoxic V̇O2peak [peak V̇O2 (oxygen uptake)]} and assigned 30 male subjects to a normoxia (n=10), a hypoxia relative (n=10) or a hypoxia absolute (n=10) group. Each group was required to exercise on a cycle ergometer at 55% of V̇O2peak for 2 h double-blinded to either a normoxic or hypoxic condition [FiO2 (inspired fraction of O2)=0.21 and 0.16 respectively]. ESR (electron spin resonance) spectroscopy in conjunction with ex vivo spin trapping was utilized for the direct detection of free radical species. The main findings show that moderate intensity exercise increased plasma-volume-corrected free radical and lipid hydroperoxide concentration (pooled rest compared with exercise data, P<0.05); however, there were no selective differences between groups (state×group interaction, P>0.05). The delta change in free radical concentration was moderately correlated with systemic V̇O2 (r2=0.48, P<0.05). The hyperfine coupling constants recorded from the ESR spectra [aN=13.8 Gauss, and aHβ=1.9 Gauss; where 1 Gauss=10−4 T (telsa)] are suggestive of oxygen-centred free radical species formed via the decomposition of lipid hydroperoxides. Peripheral leucocyte and neutrophil cells and total CK (creatine kinase) activity all increased following sustained exercise (pooled rest compared with exercise data, P<0.05), but no selective differences were observed between groups (state×group interaction, P>0.05). We conclude that a single bout of moderate aerobic exercise increases secondary free radical species. There is also evidence of exercise-induced muscle damage, possibly caused by the increase in free radical generation.

INTRODUCTION

During aerobic exercise, oxygen utilization is increased which may lead to the incomplete reduction of oxygen molecules in one or more mitochondrial complexes, resulting in an extensive increase in oxidative stress [1,2]. Oxidative stress can occur during aerobic exercise not only via the mitochondria as a primary source, but from other mechanisms such as substrate auto-oxidation, xanthine oxidase activity, neutrophil activation, nitric oxide synthesis and metal-catalysed reactions [3].

Paradoxically, a growing body of evidence suggests that exercise performed in hypoxia may also stimulate oxidative stress due primarily to a decrease in mitochondrial respiration and a build-up of reducing equivalents that cannot be transferred to molecular oxygen at the level of cytochrome oxidase [4]. In addition to this concept, known as reductive stress, other potential sources of free radical generation in hypoxia include increased nitric oxide production, xanthine oxidase and phospholipase A2 activation, and increased availability of free Fe2+ and Cu2+ ions [2,5]. The influence of exercise and hypoxia on lipid peroxidation has been explored by a number of investigators [4,6,7], and work by Bailey et al. [8] has shown an increase in LH (lipid hydroperoxide) and MDA (malondialdehyde) production following maximal exhaustive exercise in normobaric hypoxia. Other research by Bailey et al. [9] has shown that 4 weeks of exercise training in intermittent hypoxia can attenuate the exercise-induced increase in these putative biomarkers of lipid peroxidation more effectively than normoxic training, suggesting that hypoxic exercise training may have influential molecular adaptive properties. These studies, however, used indirect indices of freeradical-induced molecular damage, thus the claim that hypoxia generates free radical species has not been confirmed in exercising humans. ESR (electron spin resonance) spectroscopy, which is the most direct method of measuring free radical molecules in conjunction with the spin-trapping technique, has largely been used to confirm the presence of free radical species in venous blood of humans exercising to exhaustion in normoxia [1012]. To our knowledge there are currently no studies that have used ESR spectroscopy to directly determine the pro-oxidant effects of moderate aerobic exercise performed in normobaric hypoxia. Therefore the purpose of the present study was to quantify the degree of freeradical-mediated oxidative stress in a single bout of moderate normobaric hypoxic exercise. Furthermore, we hypothesize that exercising at the same absolute workload in hypoxia would result in an increased free radical response compared with exercise in relative hypoxia.

METHODS

Subjects

Thirty apparently healthy male volunteers were recruited from a student population to participate in the present study. The subject characteristics are shown in Table 1. Subjects were free of any diseases or ailments as assessed by a medical history questionnaire prior to experimental exercise. All subjects were non-smokers, and any subjects taking antioxidant supplements were excluded. Volunteers provided written informed consent prior to participation, and the Local Medical Research Ethics Committee (Bro Taff, Cardiff, South Wales, U.K.) granted ethical approval.

Table 1
Subject characteristics

Values are means±S.D. All groups were equally matched for the above characteristics.

Group
NormoxiaHypoxia (relative)Hypoxia (absolute)
n 10 10 10 
Age (years) 21±1 21±1 21±2 
Height (m) 1.76±0.5 1.73±0.4 1.78±0.4 
Body mass (kg) 75±13 72.4±8 73.1±9 
Body fat (%) 17.4±4 15±6 15.8±5 
V̇O2peak (ml·kg−1·min−146.7±5 48.1±6 48.6±9 
Group
NormoxiaHypoxia (relative)Hypoxia (absolute)
n 10 10 10 
Age (years) 21±1 21±1 21±2 
Height (m) 1.76±0.5 1.73±0.4 1.78±0.4 
Body mass (kg) 75±13 72.4±8 73.1±9 
Body fat (%) 17.4±4 15±6 15.8±5 
V̇O2peak (ml·kg−1·min−146.7±5 48.1±6 48.6±9 

Experimental design

Subjects were instructed to refrain from exercise and alcohol for 48 h before all tests, and to maintain their usual dietary pattern up until the last meal consumed 12 h before experimental exercise, where a standardized meal-replacement drink (Wake-up cereal; Retail Brand) was issued to all subjects. A quantity (0.32 g/kg of total body weight) of cereal powder was mixed with 2.7 ml of semi-skimmed milk per kg of total body weight by the same investigator. Dietary composition and caloric intake in the 72 h before the exercise test was recorded by means of a food diary and was assessed using a commercial nutritional assessment package (Nutri-check; Health Options Limited). All subjects attended a familiarization session 1 week prior to the commencement of the incremental test protocol.

Incremental test protocol

On arrival at the laboratory, body mass and height of the subjects were determined according to standard methods. Body fat was measured using Harpenden skin fold callipers (British Indicators) and the equations as described by Durnin and Womersley [13]. All incremental exercise tests were performed between 09.00 and 17:00 hours at the University of Glamorgan under the supervision of the same investigators to minimize inter-analytical subject variation. Each subject in a randomized double-blind placebo-controlled fashion performed two incremental cycling tests to volitional exhaustion, one in normobaric normoxia [FiO2 (inspired fraction of oxygen)=0.21] and the other in normobaric hypoxia (FiO2=0.16). Each test was separated by 7 days. The ergometer cycling test consisted of a 5 min warm up period at 0.5 kg at 80 rev./min. The subject was then required to maintain 80 rev./min while 0.4 kg was applied to the basket every 2 min (power output increase of 32 W/stage) until volitional exhaustion. This protocol was chosen as it had previously been validated to elicit V̇O2peak [peak V̇O2 (oxygen uptake)] in a hypoxic environment [14]. Pre- and post-exercise blood lactate was measured using an automated electrochemical analyser (Analox PGM7 Champion). V̇O2 was monitored during the last 60 s of each stage and at the point of volitional exhaustion using the off-line gas analysis Douglas bag system. HR (heart rate) was recorded continuously throughout exercise using a three-lead ECG system (Life pulse; HME). The relationship between V̇O2, power output (maximum workload) and HR was subsequently determined for each subject and was used to assess the individual exercise intensity level for the 2 h exercise protocol.

Experimental protocol

All exercise tests were performed between 08.00 and 13.00 hours at the University of Glamorgan. Subjects were randomly matched according to their normobaric normoxia V̇O2peak scores and randomly assigned to one of three groups prior to performing a single bout of moderate exercise: group 1 (normoxia), subjects (n=10) performed 2 h of cycling exercise in normobaric normoxia (FiO2=0.21) at a workload corresponding to 55% of the pre-determined V̇O2peak in normobaric normoxia; group 2 [hypoxia (relative)], subjects (n=10) performed 2 h of cycling exercise in normobaric hypoxia (FiO2=0.16) at a workload corresponding to 55% of the pre-determined V̇O2peak in normobaric hypoxia; and group 3 [hypoxia (absolute)], subjects (n=10) performed 2 h of cycling exercise in normobaric hypoxia (FiO2=0.16) at a workload corresponding to 55% of the pre-determined V̇O2peak in normobaric normoxia. V̇O2 was monitored during the last 60 s of each time period. HR and RPE (rate of perceived exertion) [15] were measured at selected intervals throughout. SaO2 (arterial oxygen saturation) was determined using a finger pulse oximeter (Nonin Model 8800) with a reported accuracy of ±1%.

Blood sampling

Venous blood was collected following a 12 h overnight fast from a prominent forearm vein after supine rest and during the last minute of experimental exercise using an intravenous cannula (Venflon IV cannula; Becton-Dickinson). Following blood collection, EDTA vacutainers were placed on ice, whilst blood in the serum separation tubes (SST) was allowed to clot at room temperature. After centrifugation at 996 g at 4°C for 10 min, the serum/plasma aliquots were stored at −80°C. ESR–PBN (α-phenyl-tert-butylnitrone) adduct analysis was completed on the experimental day, whereas the remaining aliquots were assayed within 6–8 weeks of collection. Post-exercise blood samples were corrected for the possibility of an exercise-induced haemoconcentration using the equations as described by Dill and Costill [16].

Blood biochemistry

Oxidative stress

Free radicals were measured using methodology published previously [11]. Briefly, toluene extracts of PBN-trapped species were analysed at room temperature on a Bruker EMX X-band ESR spectrometer with a 110TM cavity. All samples were vacuum degassed using liquid nitrogen and a turbo molecular pump (West Technologies). ESR spectral peak height (average of six peaks) was used as a measure of PBN spin adduct concentration present in the biological sample. PBN contains a hydrogen atom β, which forms due its interaction with an unpaired electron in a manner that causes each of the three nitrogen lines to split into doublets, resulting in a six line spectrum. The distance between the two lines of the first doublet is termed the β-hydrogen split (aHβ). The distance between the first and second doublets (nitrogen component) is termed aN. Nitrone values for aN and aHβ are called hyperfine coupling constants and aid in the identification of the type of free radical species present in the biological sample. Results are expressed in arbitrary units. Intra-assay CV (coefficient of variation) at 1795 arbitrary units was 5.2%.

MDA was measured by HPLC in EDTA plasma using a modified method as described by Young and Trimble [17]. Intra- and inter-assay CV at 0.56 μmol/l was 6.2 and 9.1% respectively. LH was measured spectrophotometrically in serum using a modified method as described by Wolff [18]. Intra- and inter-assay CV at 0.57 μmol/l was 4.6 and 6% respectively.

Antioxidant status

Ascorbic acid was measured in EDTA plasma with 5% metaphosphoric acid using the fluorimetric method as described by Vuilleumier and Keck [19]. The interassay CV at a concentration of 51.1 μmol/l was 0.72%. The HPLC method as described by Thurnham et al. [20] was used to simultaneously determine plasma lipid soluble antioxidant status. Intra- and inter-assay CV were both <5%.

Muscle damage

Total CK (creatine kinase) was determined using a diagnostic kit and measured on a Vitros 750 analyser (Ortho-Clinical Diagnostics), with an intra- and inter-assay CV of 4.4%. Myoglobin was analysed using an automated chemiluminescene immunoassay (ACS 180; Bayer-Chiron Immunodiagnostics), with an intra- and inter-assay CV of 3.5% and 1.3% respectively.

White blood cells

Whole blood leucocytes and neutrophils cells were analysed using a COULTER® GEN.S™ automated haematology analyser (Coulter).

Statistical analysis

Statistical analysis was performed using the SPSS social statistics package (version 9.0). Prospective power calculations were calculated as described by Altman [21]. Retrospective power calculations were performed using SPSS. Data were analysed using parametric statistics following mathematical confirmation of a normal distribution by Shapiro–Wilks tests. Independent sample Student t tests were used to compare physiological responses to incremental exercise between groups. A one-way ANOVA was used to compare subject characteristics. Experimental resting and exercise data were analysed using two-way split plot [A×(B)] mixed ANOVA, which incorporated one between [group: normoxia compared with hypoxia (relative) compared with hypoxia (absolute)] and one within (state: rest compared with exercise) subjects factor. Following a significant interaction effect (state×group), within-subject factors were analysed using Bonferroni-corrected paired samples Student t test. Between-subject differences were analysed using a one-way ANOVA with a posteriori Tukey HSD (honestly significant difference) test. The α value was set at P<0.05, and all values are reported as means±S.D.

RESULTS

Dietary status

There was no significant difference in caloric intake and macronutrient composition between groups, and all values were within recommended UK daily range.

Preliminary tests

There was a difference in relative and absolute V̇O2peak and SaO2 between groups (P<0.05) as shown in Table 2.

Table 2
Physiological responses to incremental exercise in acute hypoxia

Values are means±S.D. *P<0.05 compared with the normoxia group. [La]B, corrected whole-blood lactate.

ParameterNormoxia (FiO2=0.20)Hypoxia (FiO2=0.16)
n 30 30 
V̇O2peak (ml·kg−1·min−147.8±7 30±8* 
V̇O2peak (litres/min) 3.49±0.4 2.18±0.5* 
Maximum workload (kg) 3.5±0.4 3.32±0.3 
Maximum HR (beats/min) 195±9 193±8 
[La]B (mmol/l) 7.5±2 7.7±2 
SaO2 (%) 96±3 88±5* 
ParameterNormoxia (FiO2=0.20)Hypoxia (FiO2=0.16)
n 30 30 
V̇O2peak (ml·kg−1·min−147.8±7 30±8* 
V̇O2peak (litres/min) 3.49±0.4 2.18±0.5* 
Maximum workload (kg) 3.5±0.4 3.32±0.3 
Maximum HR (beats/min) 195±9 193±8 
[La]B (mmol/l) 7.5±2 7.7±2 
SaO2 (%) 96±3 88±5* 

Experimental tests

As shown in Table 3, V̇O2 and HR increased (P<0.05) during submaximal exercise. SaO2 was lower in the hypoxic groups (P<0.05 compared with normoxia).

Table 3
Physiological responses to sustained exercise in acute hypoxia

Values are means±S.D. for subjects in the normoxia (n=10), hypoxia relative (n=10) and hypoxia absolute (n=10) groups. Main effect for state indicates a difference (P<0.05) between 5 min of exercise compared with 110 min of exercise (pooled normoxia and hypoxia values). Main effect for group indicates a difference (P<0.05) between normoxia and hypoxia groups.

NormoxiaHypoxia (relative)Hypoxia (absolute)
ParameterTime…5 min100 min5 min100 min5 min100 min
V̇O2 (litres/min) Main effect for state  1.6±02 1.9±0.3 1.5±0.3 1.9±0.2 1.4±0.2 2±0.3 
HR (beats/min) Main effect for state  112±8 137±16 111±12 142±10 123±19 151±23 
NormoxiaHypoxia (relative)Hypoxia (absolute)
ParameterTime…0 min90 min0 min90 min0 min90 min
SaO2 (%) Main effect for group  97±1 96±1 93±3 90±3 92±3 87±4 
NormoxiaHypoxia (relative)Hypoxia (absolute)
ParameterTime…5 min100 min5 min100 min5 min100 min
V̇O2 (litres/min) Main effect for state  1.6±02 1.9±0.3 1.5±0.3 1.9±0.2 1.4±0.2 2±0.3 
HR (beats/min) Main effect for state  112±8 137±16 111±12 142±10 123±19 151±23 
NormoxiaHypoxia (relative)Hypoxia (absolute)
ParameterTime…0 min90 min0 min90 min0 min90 min
SaO2 (%) Main effect for group  97±1 96±1 93±3 90±3 92±3 87±4 

Oxidative stress indices

Figure 1 shows that exercise itself, but not selectively in hypoxia, increased the concentration of free radicals in venous blood (pooled rest compared with exercise data, P=0.004; state×group interaction, P=0.12). Typical ESR spectra of the PBN adduct before and following aerobic exercise are shown in Figure 2. For all ESR spectra of the PBN adduct detected ex vivo in human sera, the hyperfine coupling constants were measured at aN of 13.8 Gauss and aHβ of 1.9 Gauss [where 1 Gauss=10−4 T (telsa)]. Based on these coupling constants, the free radical species were identified as being secondary oxygen-centred lipid radicals. A positive correlation was observed between the delta (exercise−rest) PBN adduct and delta V̇O2 (r=0.48, P<0.05). Exercise increased circulating LH concentration (pooled rest compared with exercise data, P=0.01), but not selectively between groups (state×group interaction, P=0.27). This latter finding supports the supposition that the free radicals detected are lipid in origin. In contrast, no between- or within-group differences were observed for MDA concentration (state×group interaction, P=0.71).

Rest and exercise PBN adduct concentration in the normoxia and hypoxia groups

Figure 1
Rest and exercise PBN adduct concentration in the normoxia and hypoxia groups

Main effect for state (pooled rest compared with exercise data, P=0.004).

Figure 1
Rest and exercise PBN adduct concentration in the normoxia and hypoxia groups

Main effect for state (pooled rest compared with exercise data, P=0.004).

Typical rest (A) and (hypoxia) exercise (B) ESR spectra of PBN adducts in serum

Figure 2
Typical rest (A) and (hypoxia) exercise (B) ESR spectra of PBN adducts in serum
Figure 2
Typical rest (A) and (hypoxia) exercise (B) ESR spectra of PBN adducts in serum

Antioxidants

As shown in Table 4, exercise selectively decreased venous lycopene concentration by 18.5% in the normoxia group only (P=0.05). Both α- and β-carotene concentrations increased following exercise (pooled rest compared with exercise data, P=0.004 and 0.01 for α- and β-carotene respectively). No between- or within-group differences were observed for any other antioxidant parameter (state×group interaction, P=0.06, 0.43 and 0.97 for ascorbic acid, α-tocopherol and retinol respectively).

Table 4
Effect of acute hypoxia on oxidative stress, muscle damage and antioxidant indices

Values are means±S.D.; n=10 per group. Interaction effect (state×group) indicates a difference (P<0.05) within group as a function of time; †within group difference (P<0.05). Main effect for state indicates a difference (P<0.05) between rest compared with exercise (pooled normoxia and hypoxia values).

MetaboliteGroupRestExerciseMain effectInteraction effect
LH (μmol/l) Normoxia 0.59±0.06 0.7±0.16 State – 
 Hypoxia (relative) 0.62±0.16 0.62±0.07   
 Hypoxia (absolute) 0.57±0.07 0.65±0.07   
MDA (μmol/l) Normoxia 0.48±0.1 0.5±0.2 – – 
 Hypoxia (relative) 0.56±0.2 0.55±0.2   
 Hypoxia (absolute) 0.65±0.2 0.68±0.2   
Myoglobin (units/l) Normoxia 36±6 37±14 – – 
 Hypoxia (relative) 43±16 46±23   
 Hypoxia (absolute) 43±15 59±26   
Ascorbic acid (μmol/l) Normoxia 60.8±10 60.9±16 – – 
 Hypoxia (relative) 62.2±10 61.5±8   
 Hypoxia (absolute) 51±18 61.6±1   
α-Tocopherol (μmol/l) Normoxia 19.7±2 19.9±4 – – 
 Hypoxia (relative) 17.7±3 19.1±3   
 Hypoxia (absolute) 18.2±1 18.8±3   
α-Carotene (μmol/l) Normoxia 0.02±0.01 0.02±0.01 State – 
 Hypoxia (relative) 0.01±0.01 0.02±0.01   
 Hypoxia (absolute) 0.02±0.01 0.02±0.01   
β-Carotene (μmol/l) Normoxia 0.06±0.04 0.09±0.08 State – 
 Hypoxia (relative) 0.04±0.02 0.05±0.02   
 Hypoxia (absolute) 0.07±0.05 0.07±0.05   
Lycopene (μmol/l) Normoxia 0.32±0.08 0.27±0.06† – State×group 
 Hypoxia (relative) 0.27±0.10 0.30±0.19   
 Hypoxia (absolute) 0.35±0.12 0.37±0.13   
Retinol (μmol/l) Normoxia 0.74±0.11 0.73±0.22 – – 
 Hypoxia (relative) 0.80±0.15 0.80±0.15   
 Hypoxia (absolute) 0.76±0.14 0.73±0.15   
MetaboliteGroupRestExerciseMain effectInteraction effect
LH (μmol/l) Normoxia 0.59±0.06 0.7±0.16 State – 
 Hypoxia (relative) 0.62±0.16 0.62±0.07   
 Hypoxia (absolute) 0.57±0.07 0.65±0.07   
MDA (μmol/l) Normoxia 0.48±0.1 0.5±0.2 – – 
 Hypoxia (relative) 0.56±0.2 0.55±0.2   
 Hypoxia (absolute) 0.65±0.2 0.68±0.2   
Myoglobin (units/l) Normoxia 36±6 37±14 – – 
 Hypoxia (relative) 43±16 46±23   
 Hypoxia (absolute) 43±15 59±26   
Ascorbic acid (μmol/l) Normoxia 60.8±10 60.9±16 – – 
 Hypoxia (relative) 62.2±10 61.5±8   
 Hypoxia (absolute) 51±18 61.6±1   
α-Tocopherol (μmol/l) Normoxia 19.7±2 19.9±4 – – 
 Hypoxia (relative) 17.7±3 19.1±3   
 Hypoxia (absolute) 18.2±1 18.8±3   
α-Carotene (μmol/l) Normoxia 0.02±0.01 0.02±0.01 State – 
 Hypoxia (relative) 0.01±0.01 0.02±0.01   
 Hypoxia (absolute) 0.02±0.01 0.02±0.01   
β-Carotene (μmol/l) Normoxia 0.06±0.04 0.09±0.08 State – 
 Hypoxia (relative) 0.04±0.02 0.05±0.02   
 Hypoxia (absolute) 0.07±0.05 0.07±0.05   
Lycopene (μmol/l) Normoxia 0.32±0.08 0.27±0.06† – State×group 
 Hypoxia (relative) 0.27±0.10 0.30±0.19   
 Hypoxia (absolute) 0.35±0.12 0.37±0.13   
Retinol (μmol/l) Normoxia 0.74±0.11 0.73±0.22 – – 
 Hypoxia (relative) 0.80±0.15 0.80±0.15   
 Hypoxia (absolute) 0.76±0.14 0.73±0.15   

White blood cells and muscle damage markers

Peripheral leucocyte and neutrophil cell number increased following sustained exercise (pooled rest compared with exercise data, P=0.000; state×group interaction, P=0.72 and 0.34 for leucocytes and neutrophils respectively); however, there were no differences between normoxia and hypoxia groups (Table 5). Total CK activity increased following exercise (pooled rest compared with exercise data, P=0.02; state×group interaction, P=0.50) as shown in Figure 3. No differences were observed within or between groups for myoglobin activity (state×group interaction, P=0.13) as shown in Table 4.

Table 5
Effect of acute hypoxia on white blood cell numbers

Values are means±S.D.; n=10 per group. Main effect for state indicates a difference (P<0.05) between rest compared with exercise (pooled normoxia and hypoxia values).

Blood cellsGroupRestExerciseMain effect
Leucocytes (103 cells/μl) Normoxia 5.6±1.2 12.7±5.4 State 
 Hypoxia (relative) 5.6±1.0 10.7±4.5  
 Hypoxia (absolute) 5.7±1.3 12.6±7.5  
Neutrophils (103 cells/μl) Normoxia 2.8±0.7 8.7±4.5 State 
 Hypoxia (relative) 2.8±0.8 6.8±4  
 Hypoxia (absolute) 3.1±1.0 9.8±5.0  
Blood cellsGroupRestExerciseMain effect
Leucocytes (103 cells/μl) Normoxia 5.6±1.2 12.7±5.4 State 
 Hypoxia (relative) 5.6±1.0 10.7±4.5  
 Hypoxia (absolute) 5.7±1.3 12.6±7.5  
Neutrophils (103 cells/μl) Normoxia 2.8±0.7 8.7±4.5 State 
 Hypoxia (relative) 2.8±0.8 6.8±4  
 Hypoxia (absolute) 3.1±1.0 9.8±5.0  

Rest and exercise CK concentration in the normoxia and hypoxia groups

Figure 3
Rest and exercise CK concentration in the normoxia and hypoxia groups

Main effect for state (pooled rest compared with exercise data, P=0.02). CPK, CK.

Figure 3
Rest and exercise CK concentration in the normoxia and hypoxia groups

Main effect for state (pooled rest compared with exercise data, P=0.02). CPK, CK.

DISCUSSION

Recent literature postulates that exercise performed in hypoxia causes oxidative stress [8,9]. The present study used ESR spectroscopy to measure free radicals directly, and demonstrates for the first time that a single bout of moderate aerobic exercise in hypoxia (of an absolute and relative nature) does not selectively (against normoxia) increase free radical concentration. This finding is at variance with previous reports that have quantified free radical generation by measuring the by-products of molecular oxidation, and may be due, in part, to a limited statistical power. In addition, other hypoxia studies have largely used short-term exhaustive protocols [i.e. at or near V̇O2max (maximum V̇O2)] to generate an oxidative stress [8,9], whereas the exercise intensity was much lower in the present study. Nevertheless, pooled group data would suggest that 2 h of exercise at 55% V̇O2peak generates free radical species within the systemic circulation to levels beyond the capacity of the antioxidant defence system.

The hyperfine coupling constants of all ex vivo PBN-trapped free radicals were consistently the same and are suggestive of secondary oxygen-centred lipid-derived alkoxyl radicals [10,11]. ESR spectra from the auto-oxidation of α-linolenic acid show similar hyperfine coupling constants (aN=13.8 Gauss, and aHβ=1.9 Gauss) to those of human blood in the present study, supporting our observation that the free radical species detected are lipid in origin [11]. Coupled with the fact that LH concentration increased following exercise, it is reasonable to suggest that the primary source of the alkoxyl radical observed in the present study is via initial primary oxygen-centred radical attack and subsequent PUFA (polyunsaturated fatty acid) decomposition [22]. Lipid peroxidation is a self-perpetuating chain reaction, which generates oxidation by-products and free radical intermediates. In support, Ashton et al. [10] suggest that the alkoxyl radical results from lipid peroxidation of cellular membranes by primary free radical attack resulting in increased levels of plasma LH post-exercise. Direct free radical detection, in addition to a rise in LH concentration in the present study, provides evidence that the damage inflicted to cellular membranes during aerobic exercise may well be free radical mediated. In the present study, we suggest that either muscle mitochondria or extracellular leucocytes had a role to play in the generation of primary free radicals and lipid peroxidation during aerobic exercise. The mitochondrial electron transport chain within a muscle fibre has long been considered the major site of ROS (reactive oxygen species) production at rest and during exercise [23]. Studies on isolated mitochondria suggest that between 2–5% of total electron flux through the cytochrome chain may undergo one electron univalent reduction with the formation of superoxide and H2O2 [24,25]. Given the positive association between V̇O2 and the PBN adduct and the significant rise in V̇O2 from rest to post-exercise in the present study, it is postulated that aerobic exercise increased tissue oxygen flux, causing an increase in electron flux within the mitochondrial respiratory chain with rapid formation of primary oxygen-centred radical species (e.g. superoxide anion) [26]. As supported by the work of Davies et al. [27], rodents performing endurance exercise produce ESR-detected semiquinone radicals derived from an inner mitochondrial membrane ‘leakiness’. More recent evidence using a microdialysis probe has demonstrated a rise in the reduction of cytochrome c, suggesting an increase in superoxide production within the interstitial space of contracting mouse muscle [28]. These data provide evidence that free radicals are formed in exercising skeletal tissue and supports the hypothesis that mitochondria may induce the formation of free radical species. Although muscle mitochondria are generally regarded as the main site of intracellular free radical generation during exercise, it must be taken into account that other tissues, such as liver, have the potential to produce free radical species [27]. It is implied that the observed increase in PBN adduct concentration after exercise is due to the primary radical species attacking either intracellular or extracellular PUFAs, which would decompose LH in the presence of iron, yielding a rapid rise in alkoxyl radical formation and allowing subsequent detection via ESR spectroscopy [11]. Evidence is provided for the presence of free iron to aid in such a reaction by Jenkins et al. [29], who have demonstrated that iron becomes more loosely bound to transferrin during exercise.

LH concentration throughout was shown to increase after exercise and, to our knowledge, this is the first evidence of an increase in LH after 2 h of moderate aerobic exercise at 55% V̇O2peak. Since there is evidence of membrane peroxidation, perhaps the integrity of the muscle cell membrane was compromised, particularly as an increased release of the glycolytic protein CK was observed. The presence of systemic CK may suggest increased sarcolemmal permeability, which during prolonged exercise predominately rises from sarcolemmal membrane rupture [30], possibly as a result of free radicals produced during exercise [31].

Although aerobic exercise in hypoxia did not increase free radical generation, other studies with hypoxia have demonstrated an increase in lipid peroxidation. The interpretation of these findings may have some relevance to the overall increase in oxidative stress in the present investigation, as it can be observed from Figure 1 that hypoxia plus exercise causes the greatest contribution to free radical generation. Simon-Schnass and Pabst [32] observed a significant increase in lipid peroxidation in hypobaric hypoxia, and Simon-Schnass [4] suggests further that hypoxic cells are particularly susceptible to oxidative stress due to an accumulation of electrons that cannot be transferred to oxygen at the level of mitochondrial cytochrome oxidase, but transferred to other low-molecular-mass molecules which, in turn, induce radical chain reactions. Notwithstanding the fact that overall V̇O2 increased in the present study, there was a selective decrease in SaO2 between normoxia and both hypoxia groups. This may suggest a decrease in diffusive oxygen delivery to active skeletal myocytes, causing a decrease in the V̇max of cytochrome oxidase and an increase in univalent reduction [33]. Work by Bailey and co-workers [8] has shown an increase in LH concentration following exhaustive exercise in normobaric hypoxia (FiO2=0.16). This increase in lipid peroxidation was associated with a decrease in SaO2 (LH, r=−0.61, P<0.05; MDA, r=−0.50, P<0.05) as opposed to V̇O2max. Bailey et al. [9] suggest that an exercise-induced mass action effect of mitochondrial oxygen flux is not the exclusive mediator of ROS production and postulate that a decrease in mitochondrial PO2 (partial pressure of oxygen) may be a contributor to ROS generation.

It is clear from Figure 1 that the main effect observed for free radical concentration is largely due to a change from rest to exercise in the hypoxia subgroups. The lack of a selective difference between normoxia and relative and absolute hypoxia exercise may in part be due to the consequence of limited statistical power. Retrospective calculation of power (power=0.42) shows that, for a possible interaction effect to occur, any future study using the same methodological design would need approximately double the existing number of subjects. Furthermore, although it is known that a decrease in intracellular PO2 can influence free radical exchange [34], perhaps a more mechanistic reason for the lack of a hypoxic effect may be due to an insufficient decrease in intracellular PO2 as a consequence of using a 16% FiO2 at 55% V̇O2peak. It can also be observed in Table 3 that V̇O2 and work rate were similar across all groups, and perhaps this also contributed to the lack of an interaction effect.

Activated vascular leucocyte and neutrophil cells are known to produce ROS [35] and both were increased following exercise in the present study. As V̇O2 increased during exercise, it is possible that phagocytosis activated membrane-bound NADPH oxidase, which reduced molecular oxygen to superoxide [36]. As erythrocyte membranes are rich in PUFAs and highly susceptible to oxidative damage [37], it is possible that the increase in LH and lipid alkoxyl radicals originate from this extracellular source.

The lack of change in MDA observed as a function of exercise may possibly be due to a number of factors. Because MDA levels were not measured continuously during exercise, we cannot exclude the possibility of MDA redistribution between plasma and exercising tissue [11]. Moreover the lack of change in MDA may be as a consequence of free radical and lipid peroxidation termination by available circulating antioxidants. An increased mobilization of important carotenoids as a function of exercise and a selective decrease in lycopene would suggest an increase in plasma antioxidant activity and perhaps a stabilization of lipid-derived free radicals, thereby inhibiting the production of MDA. It is also conceivable that perhaps MDA was taken from an inappropriate sampling site at an inappropriate time following exercise. Jenkins [38] suggest that the failure of some investigators to detect a rise in MDA that was actually produced include potential factors such as a failure to look for a marker at the right time or place.

In conclusion, the present study has measured and quantified oxidative stress in human blood following a single bout of moderate aerobic exercise and demonstrates that exercise performed at 55% of V̇O2peak can increase (pooled group data) free radical concentration as measured by ESR spectroscopy. Exercise performed in absolute and relative hypoxia did not selectively (against normoxia) increase oxidative stress, due in part to limited statistical power. In addition to an exercise-induced increase in the PBN adduct, exercise of this duration and intensity increased LH and CK production, which suggests cell membrane damage and increased sarcolemmal permeability.

Abbreviations

     
  • CV

    coefficient of variation

  •  
  • ESR

    electron spin resonance

  •  
  • FiO2

    inspired fraction of oxygen

  •  
  • HR

    heart rate

  •  
  • LH

    lipid hydroperoxide

  •  
  • MDA

    malondialdehyde

  •  
  • PBN

    α-phenyl-tert-butylnitrone

  •  
  • PO2

    partial pressure of oxygen

  •  
  • PUFA

    polyunsaturated fatty acid

  •  
  • ROS

    reactive oxygen species

  •  
  • SaO2

    arterial oxygen saturation

  •  
  • V̇O2

    oxygen uptake

  •  
  • V̇O2max

    maximum V̇O2

  •  
  • V̇O2peak

    peak V̇O2

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