Diving and Hyperbaric Medicine Volume 43 No. 2 June 2013 63 Original articles Oxidative stress in breath-hold divers after repetitive dives Sigrid Theunissen, Nicola Sponsiello, Miroslav Rozloznik, Peter Germonpré, François Guerrero, Danilo Cialoni and Costantino Balestra Abstract (Theunissen S, Sponsiello N, Rozloznik M, Germonpré P, Guerrero F, Cialoni D, Balestra C. Oxidative stress in breath-hold divers after repetitive dives. Diving and Hyperbaric Medicine. 2013 June;43(2):63-66.) Introduction: Hyperoxia causes oxidative stress. Breath-hold diving is associated with transient hyperoxia followed by hypoxia and a build-up of carbon dioxide (CO 2 ), chest-wall compression and significant haemodynamic changes. This study analyses variations in plasma oxidative stress markers after a series of repetitive breath-hold dives. Methods: Thirteen breath-hold divers were asked to perform repetitive breath-hold dives to 20 metres’ depth to a cumulative breath-hold time of approximately 20 minutes over an hour in the open sea. Plasma nitric oxide (NO), peroxinitrites (ONOO – ) and thiols (R-SH) were measured before and after the dive sequence. Results: Circulating NO significantly increased after successive breath-hold dives (169.1 ± 58.26% of pre-dive values; P = 0.0002). Peroxinitrites doubled after the dives (207.2 ± 78.31% of pre-dive values; P = 0.0012). Thiols were significantly reduced (69.88 ± 19.23% of pre-dive values; P = 0.0002). Conclusion: NO may be produced by physical effort during breath-hold diving. Physical exercise, the transient hyperoxia followed by hypoxia and CO 2 accumulation would all contribute to the increased levels of superoxide anions (O 2 2– ). Since interaction of O 2 2– with NO forms ONOO – , this reaction is favoured and the production of thiol groups is reduced. Oxidative stress is, thus, present in breath-hold diving. Key words Freediving, breath-hold diving, hyperoxia, free radicals, nitric oxide, exercise Introduction At the end of the dive, breath-hold diving may result in hypoxia/hypercapnia, where alveolar oxygen partial pressure can be as low as 20–30 mmHg and arterial oxygen saturation around 50%. 1 Moreover, in order to increase the maximal breath-hold time, some divers perform hyperventilation, which reduces the partial pressure of carbon dioxide (PCO 2 ) before breath-holding and delays the urge to breathe. This procedure increases the risk of a hypoxic event during the dive. Whilst breath-hold divers may experience hypoxia during the ascent, the compression phase of the dive is associated with the reverse, hyperoxic conditions, because of increasing hydrostatic pressure leading to reduction of the intra-pulmonary gas volume and compression of the chest wall (Boyle’s Law). 2 This, in turn, induces a reduction of cardiac output during the dive. 3 Oxidative stress has also been involved in cardiovascular pathologies and hypertension. 4–6 In a previous study, decreased flow-mediated dilatation (FMD) of the brachial artery was observed after a series of breath-hold dives. 6 Interestingly, circulating nitric oxide (NO) was increased in these conditions. It was hypothesised that the production of reactive oxygen species (ROS) was increased during breath-hold diving, and that these ROS react with nitric oxide (NO) to form peroxinitrites (ONOO – ) which, in turn, reduce the ability of NO to achieve vasodilation. 6 The increased NO production could be due to physical exercise. 7 Different factors can be involved in the increased oxidative stress, including transient hyperoxia followed by hypoxia and CO 2 build up and/or an impaired tetrahydrobiopterin bioactivity, as found in hypertension- induced endothelial dysfunction. 8 Alternatively, in the absence of oxidative stress, a decreased FMD could result from alterations in cardiovascular function and autonomic control. 9 The aim of this study was to observe whether oxidative stress markers are increased in breath-hold diving in order to confirm/refute the hypothesis that NO reacts with ROS to form ONOO – leading to a decrease in its bioactivity. Methods STUDY POPULATION After written informed consent and institutional ethics committee approval (B200-2009-39), 13 non-smoking, experienced (at least four years of experience) male breath- hold divers volunteered for the study. Prior to entering the study, they were assessed as fit to dive by a qualified diving physician. None of the subjects had a history of previous cardiac abnormalities and none of them was on any cardio- active medication. All participants were asked to refrain from performing strenuous exercise for 48 hours before testing. For all the subjects, the daily diet was controlled by a medical nutritionist (author NS), avoiding nitrate-rich