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BASIC SCIENCES: Original Investigations

Effects of Maximal Static Apnea on Antioxidant Defenses in Trained Free Divers


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Medicine & Science in Sports & Exercise: July 2008 - Volume 40 - Issue 7 - p 1307-1313
doi: 10.1249/MSS.0b013e31816a7188
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Periods of breath holding with the head immersed in water elicits the well-known, primordial, diving reflex (7). The body's reliance upon a continual supply of oxygen is thought to drive this response, characterized by pronounced bradycardia, peripheral vasoconstriction, increased blood pressure, and red blood cell (RBC) expulsion from the spleen (22,25,26). Face immersion apnea results in hemoglobin desaturation and systemic hypoxia, the extent of which is related to apnea training status, lung volume, and breath hold duration (5,14,25). In trained divers, we have observed hemoglobin saturation values below 50%, exemplifying the hypoxic response (25). Upon rebreathing, reactive hyperemia and systemic reoxygenation result in increased reactive oxygen species (ROS) production and a state of oxidative stress where the ratio of reducing species (antioxidants) to oxidizing species is decreased (13).

Understanding the antioxidant and the oxidative stress responses to apnea is important because it may improve our understanding of the possible health consequences of competitive apneic sports, including free diving and spear fishing, and disorders including sleep apnea. The effects ofdynamic apnea (apnea including an exercise component) on antioxidant defenses and oxidative stress are equivocal (21,27-29). Research on static apnea (apnea with no exercise component) may provide a clearer picture of the effects of apnea without the confounding effects of exercise. In addition, using a static apnea protocol would provide valuable information on the effects of static apnea training and competition in groups of amateur free divers, who currently number in the thousands, worldwide.

Two studies have compared the effects of apnea in trained divers and untrained individuals (10,11). Maximal static apnea decreased -circulating antioxidant concentrations (ascorbate and glutathione) and induced oxidative damage, measured by thiobarbituric acid reactive substances (TBARS), in untrained individuals. In contrast, trained divers had preserved antioxidant status and no oxidative stress response (10). In a following study, apnea training protected from antioxidant depletion and oxidative stress compared with baseline, suggesting an apnea-induced training effect on reduction-oxidation physiology (11). These findings have provided an initial examination of the antioxidant and the oxidative stress response to static apnea; however, more comprehensive measures of antioxidant status and oxidative stress are required to establish the mechanisms responsible for the training effect. Currently, no studies have compared the activities of antioxidant enzymes in trained versus untrained individuals or in response to static apnea, which might explain the previously cited observations. In addition, this is the first study to measure the endogenous antioxidants uric acid and bilirubin and global measures of antioxidant capacity in response to apnea. Finally, in this study, oxidative stress was measured by quantifying plasma malondialdehyde (MDA) using high-performance liquid chromatography (HPLC) in comparison to TBARS. TBARS has previously been criticized for its lack of specificity in quantifying oxidative stress responses (8).

Therefore, the aim of this study was to investigate the effects of static apnea, between groups and over time, on previously unmeasured plasma antioxidant variables [bilirubin, uric acid, ferric reducing ability of plasma (FRAP), and trolox equivalent antioxidant capacity (TEAC)], the oxidative stress marker MDA, and antioxidant enzyme activities [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)]. It was hypothesized that trained free divers would possess greater antioxidant enzyme activities and therefore would be resistant to apnea-induced antioxidant depletion. Secondly, we hypothesized that controls would have depleted antioxidant concentrations after repeated bouts of apnea and that both groups would have increased antioxidant enzyme activities after five static apneas.



Eight trained free divers (seven males and one female; age = 38 ± 12 yr; height = 182 ± 7 cm; weight = 83 ± 10 kg) with 17 yr average training and experience in national and international competition and 11 control subjects matched for age, weight, and height (nine males and two females; age = 31 ± 10 yr; height = 173 ± 8 cm; weight = 74 ± 12 kg) received a verbal description of the experiment and completed a written informed consent form, which was approved by the human research ethics committee.


Subjects had a cannula inserted into a prominent vein within the antecubital fossa of their nondominant arm. They assumed a prone position on a plinth with their head resting on the cover of a container filled with cold water (∼10°C). A water temperature of 10°C was chosen because it stimulates the diving reflex and prolongs breath hold duration (19). Arterial oxygen saturation (SaO2) was assessed using pulse oximetry of the ear lobe (RAD 9; Masimo, Irvine, CA) with a 2-s averaging function. After a 15-min rest, a baseline blood sample was taken. Trained divers and control subjects were then instructed to hold their breath underwater for as long as possible. Individuals in both groups tended to hyperventilate before face immersion. Normalization of the predive ventilation was not undertaken because the ritual is quite idiosyncratic; however, all subjects were instructed to undertake the same procedure before each apnea. Five maximal static apneas, with face immersion, were separated by 2-min recovery intervals as previously described (25). Blood (10 mL) was drawn from the cannula at baseline (B) and within 30 s after apneas 1 (A1), 3 (A3), and 5 (A5) and transferred into vacutainers containing ethylenediamine tetraacetic acid (EDTA). The blood was centrifuged at 800g for 10 min (CentraMP4R; International Equipment Company, Neadham, MA), and the plasma was aliquoted and frozen at −80°C until analysis. The RBC from the baseline and A5 blood samples were washed three times in an antioxidant buffer containing butylated hydroxytoluene (1 mM), tris(hydroxymethyl)-aminomethane (50 mM), and hydrochloric acid (pH 7.6) and were then hemolyzed in cold, distilled water and frozen at −80°C until analysis.

Plasma antioxidant and other physiological measures.

TEAC and FRAP were determined via the methods of Miller et al. (17) and Benzie and Strain (2), respectively. Uric acid and total bilirubin were measured using kits (Thermo Electron Corp, Noble Park, Victoria, Australia). The coefficient of variation for these assays were as follows: TEAC 1.2%, FRAP 0.5%, uric acid 3.0%, and bilirubin 3.4%. Circulating triglycerides and glucose were measured using kits (Thermo Electron Corp) to assess whether apnea caused lipid and glucose mobilization. Human C-reactive protein (CRP) was measured using an immunoturbidimetric kit (Kamiya Biomedical Company, Seattle, WA) to assess whether static apnea caused a systemic inflammatory response. All assays were conducted in duplicate on a Cobas Mira auto analyzer (Roche Diagnostics, Basel, Switzerland).

RBC antioxidant enzyme activities.

SOD activity was measured in erythrocytes according to the method of Madesh and Balasubramanian (15). One unit of SOD activity was defined as the concentration of SOD that inhibits formazan crystal formation from the reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide in the presence of O2 generators by 50%.

CAT activity was determined by the rate of breakdown of hydrogen peroxide (H2O2) to form water and molecular oxygen according to the method of Slaughter and O'Brien (24), adapted for the Cobas Mira (Roche Diagnostics). One unit of CAT activity is defined as the concentration of CAT that inhibits quinonimine color formation from the production of H2O2 by uricase-uric acid by 50%.

GPx activity was measured via the oxidation of NADPH to NADP2+, according to the method of Wheeler et al. (31), modified for the Cobas Mira (Roche Diagnostics). One unit of GPx activity was defined as 1 μmol NADPH oxidized per minute. All erythrocyte antioxidant enzyme levels were measured in duplicate and were expressed relative to hemoglobin concentration. In our laboratory, the coefficients of variation for SOD, CAT, and GPx assays are 7%, 2%, and 4%, respectively.

Total glutathione.

Total hemolysate glutathione was determined by HPLC, in duplicate, according to the method of Katrusiak et al. (12), with modification. Twenty microliters of a 20-mM dithiothreitol solution was added to thawed RBC aliquots (200 μL). After 5 to 10 min, 100 μL of a 0.125-M 5,5′-dithiobis-(2-nitrobenzoic acid) solution, in 0.5-M phosphate buffer (pH 8), was added. The samples were mixed, and after 5 min, 200 μL of sulfosalicylic acid (9% in 0.02-M EDTA) was added. The samples were vortex mixed and centrifuged at 8500g for 10 min (Biofuge A; Heraeus Sepatech, Osterode, Germany). Ten microliters of the supernatant was injected into the HPLC for separation. Glutathione concentrations were determined at 330 nm using HPLC (Shimadzu, Kyoto, Japan) with a LiChrospher C18 column (250 × 4 mm, 5 μm; Merck, Darmstadt, Germany). Gradient elution, as per Katrusiak et al. (12), separated glutathione from the other hemolysate constituents. Reduced glutathione (>98%; Sigma-Aldrich, Castle Hill, Australia) solutions were used as external standards. The coefficient of variation of this assay was <5%.


The plasma MDA concentration was determined in duplicate by HPLC according to the method of Sim et al. (23) with slight modification. Samples were hydrolyzed in 1.3 mM NaOH to a final concentration of 1 mM NaOH, incubated for 60 min at 60°C, and cooled on ice for 5 min. Proteins were precipitated by adding 35% perchloric acid, cooled on ice, and centrifuged for 5 min at 10,000g (Biofuge A; Heraeus Sepatech). The supernatant was added to 2,3-dinitrophenylhydrazine and incubated for 10 min at room temperature in the dark. The aqueous phase was extracted with hexane and evaporated with the dry extract reconstituted in mobile phase containing 45% acetonitrile and 0.2% glacial acetic acid. MDA concentrations were determined at 310 nm using HPLC (Shimadzu) with a LiChrospher C18 column (250 × 4 mm, 5 μm; Merck). MDA solutions (97%; Sigma-Aldrich) were used as external standards. The coefficient of variation for this assay was <5%.

Statistical analysis.

All data are described as mean ± SD. Repeated-measures factorial ANOVA were used to test for group and apnea effects in the variables. When significant F-ratios were observed, Scheffe tests were applied post hoc to determine where the differences occurred. A correlation analysis between the independent variables of hypoxemia (minimum hemoglobin O2 saturation) and breath hold duration (maximum and cumulative breath hold duration) versus the relative change, from baseline to apnea 5, in the dependent antioxidant and oxidative stress variables was performed. The level of significance was set at P < 0.05.


Table 1 shows that apneic time increased with successive attempts, with the trained (Tr) group achieving significantly (P < 0.01) longer maximal apneas than controls (Con, 120 ± 40 s; Tr, 242 ± 36 s). Trained subjects attained significantly (P < 0.01) lower hemoglobin O2 saturation levels than the controls during the third (Con, 89.6 ± 5.8%; Tr, 74.9 ± 9.6%) and fifth apneas (Con, 86.4 ± 5.7%; Tr, 69.3 ± 10.0%). There were no group or apnea effects for circulating glucose or triglycerides.

Performance and general physiological variables in controls (n = 11) and apnea-trained (n = 8) subjects at baseline and after the first (A1), third (A3), and fifth (A5) apneas.

Table 2 shows the plasma antioxidant and oxidative stress markers for the individual groups before and after repeated static apnea. There were no significant differences between groups in circulating bilirubin (P = 0.60), uric acid (P = 0.85), FRAP (P = 0.94), or hemolysate total glutathione (P = 0.96) values. TEAC was significantly (P < 0.05) greater in the trained subjects after the first (Tr, 1.91 ± 0.10 mEq·Trolox−1·L−1; Con, 1.76 ± 0.18 mEq·Trolox−1·L−1) and third apnea (Tr, 1.91 ± 0.11 mEq·Trolox−1·L−1; Con, 1.76 ± 0.17 mEq·Trolox−1·L−1). With no other group differences, data from both groups were combined so that the effects of repeated apnea could be analyzed. Bilirubin decreased significantly from baseline (14.1 ± 5.7 μmol·L−1) after apnea (A1, 11.5 ± 5.1 μmol·L−1; A3, 11.6 ± 4.8 μmol·L−1; A5, 11.8 ± 4.9 μmol·L−1; group data combined; P = 0.01). Concordantly, FRAP decreased significantly from baseline (0.94 ± 0.17 mEq·(Fe2+)−1·L−1) after apnea (A1, 0.92 ± 0.16 mEq·(Fe2+)−1·L−1; A3, 0.91 ± 0.16mEq·(Fe2+)−1·L−1; A5, 0.92 ± 0.17 mEq·(Fe2+)−1·L−1; group data combined; P = 0.03). Uric acid decreased significantly from baseline (295 ± 52.6 μmol·L−1) after the first and the third apnea (A1, 287 ± 47.5 μmol·L−1; A3, 288 ± 49.0 μmol·L−1; group data combined; P = 0.01). Apnea had no effect (P = 0.74) on TEAC, and there was no significant (P = 0.32) apnea effect for plasma MDA.

Plasma antioxidant status and oxidative stress variables in controls (n = 11) and trained (n = 8) subjects at baseline and after the first (A1), third (A3), and fifth (A5) apneas.

Figure 1 shows the antioxidant enzyme activities of SOD,GPx, and CAT between the control and the apnea-trained groups. There were no significant group effects (P = 0.94 and 0.98, respectively) for GPx and CAT activities. However, SOD activity was significantly higher in the trained divers (Con, 43.2 ± 10.1 U·g Hb−1; Tr, 50.1 ± 7.3 U·g Hb−1; baseline and A5 data combined; P = 0.04). With no other group differences, data from both groups were combined so that the effects of repeated apnea could be analyzed. Table 3 shows the antioxidant enzyme activities before and after five maximal static apneas. Significant increases in GPx (B, 60.5 ± 14.9 U·g Hb−1; A5, 70.1 ± 16.0 U·g Hb−1; P = 0.02) and SOD (B, 44.1 ± 11.1 U·mg Hb−1; A5, 48.1 ± 7.5 U·mg Hb−1; P = 0.05) activity were noted between baseline and A5. A significant decrease in CAT activity occurred after five maximal apneas (B, 5.25 ± 0.59; A5, 5.00 ± 0.53 U·mg Hb−1; P = 0.03).

The activities of antioxidant enzymes SOD, GPx, and CAT in the control (n = 11) and apnea-trained (n = 8) groups (data from baseline and A5 combined). *P < 0.05 between groups.
RBC antioxidant enzyme activities and RBC total glutathione content in controls (n = 8) and trained (n = 11) persons at baseline and after five apneas.

Table 4 shows a correlation analysis of the relationships between the minimum hemoglobin O2 saturation, cumulative and maximum breath hold duration versus the relative changes in antioxidant and oxidative stress markers. A significant positive correlation between the antioxidant measure TEAC and the cumulative breath hold duration time (r = 0.467; P < 0.05) was apparent. These data suggest that individuals who had greater cumulative apnea times had preserved TEAC after five maximal static apneas (see Fig. 2).

Correlation analysis between minimum O2 saturation and measures of apnea duration versus the percent change, from baseline to apnea 5, in antioxidant and oxidative stress markers.
The relationship between cumulative apnea duration and percentage change from baseline to after apnea five in TEAC (r = 0.467; P < 0.05).


This is the first study to explore the effects of apnea training status and repeated static apnea on the activity of circulating antioxidant enzymes, SOD, GPx, and CAT. Furthermore, this study investigated the effects of multiple static apneas on the concentrations of endogenous antioxidants (uric acid and bilirubin), measures of plasma total antioxidant capacity (TEAC and FRAP), and oxidative stress (MDA). Trained free divers possessed greater SOD enzyme activity compared with controls throughout the diving protocol, and the activities of SOD and GPx increased after five maximal static apneas. The TEAC measure was preserved in the trained group when compared with untrained controls after repeated static apnea. Generally, repetitive static apnea decreased the individual circulating antioxidant concentrations (bilirubin and uric acid) and augmented antioxidant enzyme activity without causing an increase in the oxidative stress marker MDA in either group. These data suggest that antioxidant enzyme activities increase in response to acute periods of apnea and protect from hypoxia or reoxygenation-induced free radical production and oxidative stress. These conclusions may differ from those of dynamic apnea studies (21,29) due to the confounding effects of exercise on antioxidant and oxidative stress responses (30).

In this study, the antioxidant capacity of the blood was assessed by measuring the concentration of endogenous antioxidants (bilirubin and uric acid), TEAC, and FRAP. The concentrations of the antioxidants bilirubin and uric acid decreased after the first apnea, and bilirubin remained depressed throughout the apnea protocol (Table 2). In agreement with the individual antioxidant measurements, FRAP decreased significantly after the first apnea in both groups (Table 2). The decrease in FRAP cannot be explained entirely by the decrease in the bilirubin and uric acid concentrations, implying that other antioxidants were also consumed after the first apnea. Our data support those of Joulia et al. (10,11) who showed that multiple static apneas also decrease the reduced ascorbic acid plasma concentration in controls. Interestingly, TEAC was not affected by apnea but was higher in the trained group at apneas 1 and 3 (Table 2). These data could reflect the consumption of different antioxidants in the control and trained groups, that is, ascorbic acid and glutathione (10,17).

Although changes in antioxidant capacity (i.e., the reduced or oxidized glutathione ratio) can be used to quantify red-ox status, specific measures of cell oxidative modification are required to confirm a state of oxidative stress. Previously, oxidative stress has been quantified by measuring TBARS (10,11), MDA (28), and protein carbonyls (28,29) in the blood of apneic subjects. Joulia et al. (10) found an increase in plasma TBARS after controls performed static apnea. However, the use of plasma TBARS, as a marker of lipid peroxidation, has been questioned due to the nonspecific nature of the assay (8). We measured plasma MDA, a specific product of lipid peroxidation, and found no effect of apnea on the concentration of this oxidation product (Table 2). Our data are in agreement with Sureda et al. (28,29) who reported no change in plasma MDA and protein carbonyls after chronic periods of dynamic apnea. Interestingly, significant increases in the oxidative stress markers were noted the following day after the dynamic protocol of Sureda et al. (28), and it is possible that this was caused by an acute reactive inflammatory response to dynamic apnea (28,29). We found no group or apnea effects for CRP (Table 1), a marker of tissue inflammation, suggesting that static apnea does not induce an inflammatory state, at least in the short term. We did not measure MDA and CRP 24 h after the final apnea as the subjects were unavailable for blood collection.

A novel aspect of this study was the measurement of SOD, CAT, and GPx before and after repeated static apnea in persons of differing apnea training status. The activity of SOD was higher in divers versus controls with no group differences in GPx or CAT (Fig. 1). The increased SOD activity might reflect an adaptive mechanism that detoxifies superoxide during hypoxia or reoxygenation (18,34,35). These data are in agreement with results that show increased erythrocyte SOD activity in diving marine mammals compared with terrestrial animals (4,33). That there were no differences in the activity of GPx between the groups is consistent with previous findings in trained and untrained apnea divers (21).

This study was specifically designed to investigate the effects of static apnea (similar to that used in competition) on antioxidant enzyme activities by removing the dynamic, exercise component. Interestingly, the response of different antioxidant enzyme activities to static apnea was inconsistent. The activity of SOD and GPx increased significantly after five apneas; however, the activity of CAT decreased (Table 3). The disparate results probably reflect the complex interactions of antioxidant enzymes in controlling the accumulation of ROS and oxidants (for a review, see Ref. (16)). Only two other studies have assessed these antioxidant enzyme activities before and after repeated apnea, and their experimental designs used dynamic apnea protocols (29). Sureda et al. (29) reported that erythrocyte SOD activity remained unchanged after 4 h of dynamic apnea, although lymphocyte SOD activity increased. Our results also differ to those of Rousseau et al. (21) who measured the effects of a single dynamic apnea on erythrocyte enzyme SOD activity and found no change pre-versus postdive.

Although investigating the mechanisms of antioxidant enzyme activity up-regulation was not an aim of this particular study, a brief discussion of the possible mechanisms is warranted. Two explanations exist for the increased antioxidant enzyme activity between the groups and in response to apnea. The first suggests de novo synthesis of antioxidant enzymes, and the second suggests increased activity of existing antioxidant enzymes. De novo synthesis could possibly account for the increased activity of SOD in the trained group. As yet, total SOD protein in trained apnea divers versus controls has not been measured. De novo synthesis of SOD and GPx is unlikely to account for the increased enzyme activity after acute apnea. The entire apnea protocol was conducted in less than 30 min, and several hours or days would be required to synthesize new RBC containing greater concentrations of antioxidant enzymes. The increased activity of existing antioxidant enzymes, in response to apnea, seems more likely based upon increased abundance of antioxidant enzyme substrates, superoxide, and H2O2 after hypoxia or reoxygenation (13). Hypoxia alone up-regulates GPx and SOD (but interestingly, not CAT) activity (1,32), a probable consequence of increased hexose monophosphate shunt and glucose-6-phosphate dehydrogenase (G6PDH) activity (32). G6PDH provides reducing equivalents for glutathione recycling enzymes (glutathione reductase), improving the bioavailability of reduced glutathione, which is required for GPx activity. GPx and SOD, which have peroxidase activity, could compete with CAT for its substrate (H2O2) and explain the decreased activity of CAT. Furthermore, the increased SOD activity might be explained by increased HCO3 and lactate concentrations in response to the respiratory and metabolic acidosis of prolonged apnea. Bicarbonate and lactate have been shown to inhibit H2O2-induced SOD deactivation (9). Increased ROS production could also provide a novel hypothesis for the regulation of hypoxia inducible factor-1 (6,20) and therefore its rapid downstream effects on vascular endothelial growth factor and erythropoietin synthesis (3). Whether hypoxia inducible factor-1 activation increases antioxidant enzyme activities is currently unknown.

The global measure of total antioxidant capacity, TEAC, was positively correlated with the cumulative breath hold duration in this study (Table 4). This relationship suggests that persons with a greater cumulative apnea duration (i.e., trained individuals) had preserved TEAC values, and those with lower cumulative apnea times (i.e., untrained individuals) tended to have depleted TEAC status (Fig. 2). This result further supports the findings of Joulia et al. (10) and the hypothesis that the apnea training status affects the resistance to static apnea-induced antioxidant consumption. We propose that the trained subjects' higher SOD activity is responsible for this protection. That the antioxidant and oxidative stress markers were not correlated with the minimum hemoglobin O2 saturation levels suggests the severity of hypoxemia is not related to antioxidant depletion and oxidative stress in this study (Table 4).

In conclusion, these data have shown that trained apnea divers possess greater SOD antioxidant enzyme activity and resistance to antioxidant depletion (TEAC) in response to five maximal static apneas when compared with untrained subjects. The elevated SOD activity in trained divers might explain their resistance to hypoxia or reoxygenation-induced free radical production. Novel findings presented here also suggest repetitive static apnea depletes circulating antioxidants (uric acid and bilirubin) and increases SOD and GPx antioxidant enzyme activity but does not cause oxidative stress in either trained divers or controls. In the absence of an oxidative stress response, the modulation of the antioxidant enzyme system suggests an adequate biological response exists for combating free radical production during repetitive maximal static apnea.

Andrew Bulmer, who is a 2001 Centenary Scholarship recipient, would like to acknowledge the generous financial assistance of the Foundation for Young Australians and the Australian Commonwealth Government. Mr Gary Wilson and Ms Natalie Strobel are acknowledged for their invaluable laboratory assistance. The authors would like to thank Ms Lynne Ridgway for her assistance in recruiting the trained divers. The results of the present study do not constitute an endorsement by the American College of Sports Medicine.


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