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Oct 17, 2014 - Email: [email protected]. Introduction. The origins .... and gas exchange was measured using a semi-automated. Douglas bag system. .... hypoxia did not alter the metabolic response at rest or during exercise.
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Research Paper

Systemic oxidative–nitrosative–inflammatory stress during acute exercise in hypoxia; implications for microvascular oxygenation and aerobic capacity John D. S. Woodside1 , Mariusz Gutowski2 , Lewis Fall3 , Philip E. James4 , Jane McEneny5 , Ian S. Young5 , Shigehiko Ogoh6 and Damian M. Bailey3 1

Vascular Physiology Unit, Institute of Cardiovascular Science, University College London, London, UK Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 3 Neurovascular Research Laboratory, Faculty of Life Sciences and Education, University of South Wales, Pontypridd, UK 4 Wales Heart Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, Pontypridd, UK 5 Centre for Public Health, Nutrition and Metabolism Group, Queen’s University Belfast, Belfast, UK 6 Department of Biomedical Engineering, Toyo University, Kawagoe-Shi, Saitama, Japan

Experimental Physiology

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New Findings r What is the central question of this study? Exercise performance is limited during hypoxia by a critical reduction in cerebral and skeletal tissue oxygenation. To what extent an elevation in systemic free radical accumulation contributes to microvascular deoxygenation and the corresponding reduction in maximal aerobic capacity remains unknown. r What is the main finding and its importance? We show that altered free radical metabolism is not a limiting factor for exercise performance in hypoxia, providing important insight into the fundamental mechanisms involved in the control of vascular oxygen transport.

Exercise performance in hypoxia may be limited by a critical reduction in cerebral and skeletal tissue oxygenation, although the underlying mechanisms remain unclear. We examined whether increased systemic free radical accumulation during hypoxia would be associated with elevated microvascular deoxygenation and reduced maximal aerobic capacity (V˙ O2 max ). Eleven healthy men were randomly assigned single-blind to an incremental semi-recumbent cycling test to determine V˙ O2 max in both normoxia (21% O2 ) and hypoxia (12% O2 ) separated by a week. Continuous-wave near-infrared spectroscopy was employed to monitor concentration changes in oxy- and deoxyhaemoglobin in the left vastus lateralis muscle and frontal cerebral cortex. Antecubital venous blood samples were obtained at rest and at V˙ O2 max to determine oxidative (ascorbate radical by electron paramagnetic resonance spectroscopy), nitrosative (nitric oxide metabolites by ozone-based chemiluminescence and 3-nitrotyrosine by enzyme-linked immunosorbent assay) and inflammatory stress biomarkers (soluble intercellular/vascular cell adhesion 1 molecules by enzyme-linked immunosorbent assay). Hypoxia was associated with increased cerebral and muscle tissue deoxygenation and lower V˙ O2 max (P < 0.05 versus normoxia). Despite an exercise-induced increase in oxidative–nitrosative–inflammatory stress, hypoxia per se did not have an additive effect (P > 0.05 versus normoxia). Consequently, we failed to

J. D. S. Woodside and D. M. Bailey contributed equally to this work. DOI: 10.1113/expphysiol.2014.081265

 C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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Free radicals and oxygen transport

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observe correlations between any metabolic, haemodynamic and cardiorespiratory parameters (P > 0.05). Collectively, these findings suggest that altered free radical metabolism cannot explain the elevated microvascular deoxygenation and corresponding lower V˙ O2 max in hypoxia. Further research is required to determine whether free radicals when present in excess do indeed contribute to the premature termination of exercise in hypoxia. (Received 10 June 2014; accepted after revision 10 October 2014; first published online 17 October 2014) Corresponding author D. M. Bailey: Neurovascular Research Laboratory, Faculty of Life Sciences and Education, University of South Wales, South Wales CF37 4AT, UK. Email: [email protected]

Introduction The origins of fatigue and precisely why maximal exercise performance is limited during acute exposure to hypoxia remain unclear. In combination with peripheral mechanisms that involve biochemical changes within skeletal muscle (Allen et al. 2008), attention has been drawn towards more central processes resulting from failure of the CNS to provide adequate drive to the motoneurones (i.e. central fatigue; Gandevia, 2001). Emerging evidence suggests that a critical, albeit undefined reduction in the cerebral P O2 may constitute the upstream signal that serves to limit central motor command, resulting in the premature decision to terminate exercise (Noakes et al. 2001; Calbet et al. 2003; Amann et al. 2006; Subudhi et al. 2007, 2008). Near-infrared spectroscopy (NIRS) is an optical technique that provides continuous, non-invasive monitoring of local tissue deoxygenation and thus reflects the balance between local tissue O2 delivery (Q˙ O2 T ) and utilisation (V˙ O2 T ) within a given region of interest (Ferrari et al. 2011). Studies employing NIRS have consistently demonstrated a reduction in prefrontal cortical deoxygenation during submaximal and maximal whole-body exercise in hypoxic conditions compared with normoxia (Verges et al. 2012). More recently, this was shown to be associated with lower prefrontal cortical blood flow and a corresponding reduction in cortical oxygen delivery (Vogiatzis et al. 2011), which may contribute to the lower (calculated) cerebral mitochondrial P O2 previously documented (Rasmussen et al. 2007; Bailey et al. 2011b). While current interests continue to focus primarily on the link to exercise performance per se, the fundamental mechanisms that serve to limit tissue oxygenation remain to be established. While acute exercise is an established pro-oxidant stimulus (Powers & Jackson, 2008), human studies have demonstrated that hypoxia can equally serve to promote systemic free radical accumulation due to independent contributions from the muscle (Bailey et al. 2004b), brain (Bailey et al. 2009d, 2011b) and lungs (Bailey et al. 2010). Spin-trapping studies have shown hypoxia to compound exercise-induced lipid-derived alkoxyl  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

radical accumulation (Bailey et al. 2001; Davison et al. 2006) despite a comparatively lower maximal (absolute) power output. We have reasoned that a reduction in mitochondrial P O2 even in the face of a comparatively lower O2 flux is potentially one of several as yet unidentified mechanisms, underpinning the synergistic (pro-oxidant) effects of hypoxic exercise (Bailey et al. 2004b). In terms of exercise performance, free radicals when in excess have the potential to promote fatigue by directly interfering with the contractile components of skeletal muscle (Ferreira & Reid, 2008; Powers & Jackson, 2008) and increasing the central perception of fatigue (Mantovani et al. 2006). Furthermore, inflammatory stress in combination with nitrosative stress caused by the oxidative inactivation of the vasodilator molecule NO have equal capacity to limit tissue oxygen transport subsequent to impaired vascular endothelial function (Bailey et al. 2004a, 2006, 2013; Richardson et al. 2007; Eltzschig & Carmeliet, 2011). In support, a reduction in the vascular bioavailability of NO using L-NAME has been associated with a reduction in microvascular P O2 in contracting skeletal muscle (Ferreira et al. 2006). In light of these findings, the present study was designed to examine the potential relationships between systemic free radical metabolism, NIRS-derived indices of local deoxygenation and exercise performance during hypoxia. Given our previous findings (Bailey et al. 2001; Davison et al. 2006), we hypothesised that compared with normoxia, hypoxia would compound exercise-induced oxidative–nitrosative–inflammatory stress and that this would be associated with a more pronounced increase in microvascular deoxygenation and corresponding reduction in V˙ O2 max .

Methods Ethics

The study was approved by the University of South Wales Human Research Ethics Committee (UK). All procedures were carried out in accordance with the Declaration of Helsinki of the World Medical Association (Williams,

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2008), and written informed consent was obtained from all participants. Design

The present study adopted a randomised, single-blind design as outlined in Fig. 1. All measurements were performed in an environmental chamber (120 m3 ) maintained at 21°C and 50% relative humidity (Design Environmental, Ebbw Vale, UK). Participants attended the laboratory following a 12 h overnight fast and, following cannulation, rested in normoxia for 30 min in a semi-recumbent position to establish baseline control NIRS measurements. Formal data collection commenced following 30 min passive exposure and at the point of volitional exhaustion during a cycling test performed in both normoxia (O2 = 21%) and (normobaric) hypoxia (O2 = 12%). For the hypoxia trial, an N2 -rich gas mixture was delivered at a high flow rate from molecular sieves at the prevailing barometric pressure. Ambient O2 and CO2 concentrations were monitored continuously using fast-responding paramagnetic and infrared analysers (Servomex 1400 Series Analyser; Servomex, Crowborough, UK), respectively, and maintained via a servo-controlled system that allowed for the addition of N2 or air through solenoid valves. Each trial was separated by a 7 day recovery period. Participants

Eleven healthy, physically active male participants were recruited into the study. They were 23 ± 5 years old, with a body mass index of 26 ± 2 kg m−2 . All participants were sea-level residents, non-smokers and abstained from taking nutritional supplements, such as oral antioxidants, and anti-inflammatories. Participants were specifically asked to refrain from physical activity, caffeine and alcohol for a period of 48 h prior to formal experimentation, consistent with our previous approaches designed to minimise the biological variation associated with free radical metabolism (Davison et al. 2012). They were also encouraged to follow a low-nitrate/nitrite (NO3 − /NO2 − ) diet for 96 h prior to the study, with specific instructions to avoid fruits, salads and cured meats (Wang et al. 1997). Maximal exercise test

Following two familiarisation sessions, each participant was seated on an electronically braked, semi-recumbent cycle ergometer (Corival; Lode BV, Groningen, The Netherlands), with the backrest maintained at 70 deg. The test commenced with a 4 min (steady-state) workload at 60 W (80 r.p.m.), before increasing by 30 W every 2 min until volitional exhaustion. Each participant was

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instructed to signal clearly to the investigators when they considered they could continue at the specified power output for no longer than 60 s, as previously described (Bailey et al. 2011a). Cardiorespiratory measurements Pulmonary gas exchange. Subjects wore a leak-free mask, and gas exchange was measured using a semi-automated Douglas bag system. Expired gas fractions were measured using fast-responding paramagnetic O2 and infrared CO2 analysers (Servomex 1400 Series Analyser, Servomex, Crowborough, UK). The volume of expired gas for determination of ventilation (V˙ E ) was measured using a dry gas meter (Harvard Apparatus, Ltd, Edenbridge, UK), and oxygen uptake (V˙ O2 ) and carbon dioxide output (V˙ CO2 ) were calculated via the Haldane equation. Heart rate (HR) and arterial oxyhaemoglobin saturation (SaO2 ). Heart rate was recorded using ECG-calibrated

bipolar telemetry (Vantage; Polar Electro, Oy, Finland) and S aO2 via finger-tip pulse oximetry (515C; Novametrix Medical Systems, Wallingford, CT, USA). Metabolic measurements

Overnight fasted blood samples were obtained from a cannula located in a forearm antecubital vein following 30 min of rest and timed to coincide with the termination of exercise. Blood was centrifuged at 600 g (4°C) for 10 min and the supernatant immediately snap-frozen and stored under liquid nitrogen prior to batch analysis. Oxidative stress: ascorbate free radical (A•− ). Plasma

(1 ml) was injected into a high-sensitivity multiple-bore sample cell (AquaX; Bruker Daltonics Inc., Billerica, MA, USA) housed within a TM110 cavity of an electron paramagnetic resonance (EPR) spectrometer operating at X-band (9.87 GHz). Samples were recorded 12 min after the end of plasma recovery by signal averaging three scans with the following instrument parameters: resolution, 1024 points; microwave power, 20 mW; modulation amplitude, 0.65 G; receiver gain, 2 × 105 ; time constant, 40.96 ms; scan rate, 0.25 G s−1 for scan width, 15 G. Spectra were filtered identically using WINEPR (Version 2.11; Bruker, Karlsruhe, Germany), and the double integral of each doublet was calculated using Origin software (OriginLabs, Northampton, MA, USA). The intra- and interassay coefficients of variation (CV) were both