Positive correlation between plasma nitrite and performance during high-intensive exercise but not oxidative stress in healthy men

Abstract

Several studies suggest that exercise is associated with elevated oxidative stress which diminishes NO bioavailability. The aim of the present study was to investigate a potential link between NO synthesis and bioavailability and oxidative stress in the circulation of subjects performing high-intensive endurance exercise. Twenty-two male healthy subjects cycled at 80% of their maximal workload. Cubital venous blood was taken before, during and after exercise, and heparinized plasma was generated. Plasma concentrations of nitrite and nitrate were quantified by GC–MS and of the oxidative stress biomarker 15(S)-8-iso-PGF_2α by GC–MS/MS. pH and pCO₂ fell and HbO₂ increased upon exercise. The duration of the 80% phase (d80) was 740 ± 210 s. Subjects cycled at 89.2 ± 3.3% of their peak oxygen uptake. Plasma concentration of nitrite (P < 0.01) and 15(S)-8-iso-PGF_2α (P < 0.05) decreased significantly during exercise. At the end of exercise, plasma nitrite concentration correlated positively with d80 and performed work (w80) (each P < 0.05). Changes in nitrate concentration also correlated positively with d80 (P < 0.05) and w80/kg (P < 0.01). These findings provide evidence of a favorable effect of nitrite on high-intensive endurance exercise. The lack of association between 15(S)-8-iso-PGF_2α and NO bioavailability (nitrite concentration) and NO biosynthesis (nitrate concentration) suggest that oxidative stress, notably lipid peroxidation, is not linked to the l-arginine/NO pathway in healthy male subjects being on endurance exercise.

Introduction

During strenuous exercise there may be a 10- to 20-fold increase in molecular oxygen (O₂) uptake. It is therefore reasonable to assume that exercise may increase the production of reactive oxygen species (ROS) which may damage various cell constituents. ROS are produced in intermediate metabolism [1]. Most of O₂ is utilized to produce ATP in the mitochondria. During oxidative phosphorylation incomplete reduction of O₂ and leak out of the electron transfer chain in the mitochondria produce different ROS including superoxide (${\text{O}}_2^{-}$), hydrogen peroxide (H₂O₂) and highly toxic hydroxyl radicals (OH) [2]. Irreversible oxidative damage of certain vulnerable molecules by ROS is thought to contribute to the degenerative process associated with cell breakdown and aging [3]. Prevention of potential harmful effects of ROS in the healthy human body is ensured by various antioxidative defense mechanisms including superoxide dismutase, catalase, glutathione peroxidase and many non-enzymatic antioxidants, notably reduced glutathione [3].

Several studies have reported that intense physical activity may shift the balance between ROS production and ROS inactivation in favor of oxidative stress [3]. ROS can alter cell functions by multiple ways, for instance by causing lipid peroxidation of polyunsaturated fatty acids in cell membranes [4]. The F₂-isorostane 8-iso-prostaglandin F_2α (8-iso-PGF_2α) belongs to the most frequently and best studied biomarkers of oxidative stress [5]. 8-iso-PGF_2α and other isoprostanes are generally thought to be specific end-products of non-enzymatic free radical-catalyzed oxidation of arachidonic acid esterified to lipids [6]. Many studies, e.g. [1], [7], have reported increased levels of lipid peroxidation products in tissues and plasma during exercise and that exercise volume and intensity may influence the extent of lipid peroxidation. However, most of these studies did not investigate the extent of lipid peroxidation during exercise but only before the start and after completion of the trial. Furthermore, most of the performed exercises were prolonged but of a low intensity [4], [8], [9], [10].

ROS produced with elevated formation rates during physical exercise may decrease the bioavailability of nitric oxide (NO) [11], one of the most potent endogenous vasodilatators. NO is enzymatically synthesized from l-arginine by different isoforms of NO synthases. In some circumstances, nitrite, the autoxidation product of NO, can be reduced back to NO in the circulation [12]. Hence, circulating nitrite could be regarded as a NO reservoir. Within erythrocytes nitrite and NO are oxidized to nitrate [13]. NO has been suggested to play a role in adaptation to physical exercise by modulating blood flow, muscular contraction, glucose uptake, glycolysis, and cellular respiration. Interestingly, it has been reported that more efficient energy production during exercise can be induced by a dietary nitrate/nitrite supplementation [14], [15]. It has been assumed that NO is involved in endothelium-mediated vasodilatation and that this is one of the regulatory mechanisms by which substrate supply to working muscles is increased, thus allowing prolonged exercise [16]. Because NO is highly reactive towards oxidants such as superoxide, an association between NO bioavailability and oxidative stress is reasonable and has been proposed. High-intensive exercise has been reported to be associated both with elevated oxygen uptake and ROS production [17], [18]. NO reacts very rapidly by superoxide to form peroxynitrite (ONOO⁻) [11]. As peroxynitrite most likely does not release NO any more but degrades to nitrate and nitrite, the reaction of superoxide with NO would diminish favorable effects of NO such as increase in muscle perfusion and reduction of oxygen cost during exercise [19].

It is currently discussed that both NO and oxidative stress play major roles in adaptation to physical exercise. The aim of the present study was to examine a potential link between NO synthesis/bioavailability and oxidative stress in healthy young male subjects during and after high-intensive exercise on a cycle ergometer. One aim of the present study was to monitor potential changes in the status of lipid peroxidation. For this, we measured the total plasma concentration of the F₂-isoprostane 15(S)-8-iso-PGF_2α. The second aim of the study was to examine potential effects of intense physical exercise not only on NO bioavailability but also on NO biosynthesis. For this, we measured plasma concentrations of nitrite and nitrate, respectively. We have assumed that measurement of these biochemical parameters by thoroughly validated and frequently proven GC–MS and GC–MS/MS methods would allow identify a possible link between oxidative stress and NO during a high-intensive endurance exercise.