Beet on Alps: Time-course changes of plasma nitrate and nitrite concentrations during acclimatization to high-altitude

Highlights

• Time-course changes of plasma NO₃⁻ and NO₂⁻ concentrations were evaluated during 1 week at 3269 and 4554 m.

• Possible confounding factors such as dietary NO₃⁻ intake, physical activity and altitude changes were controlled.

• NO metabolites were significantly higher at altitude than at sea level.

• The higher the altitude reached, the longer the time taken to peak in plasma NO₃⁻ and NO₂⁻ concentrations.

Abstract

Nitric oxide seems to be involved in the altitude acclimatization process due to its ability to regulate pulmonary, cardiovascular and muscular responses to hypoxia. In this study, we investigated the plasma nitrate (NO₃⁻) and nitrite (NO₂⁻) response to hypobaric hypoxia in two groups of lowlanders exposed at different altitudes.

For seven days, fourteen subjects were evaluated at Casati Hut (3269 m a.s.l. M.CEVEDALE) and eleven individuals were studied at Capanna Regina Margherita (4554 m a.s.l. M.ROSA). Before expeditions and at different time points during high-altitude sojourn, plasma NO₃⁻ and NO₂⁻ concentrations were measured by chemiluminescence. Resting peripheral arterial oxygen saturation (SpO₂), heart rate (HR) and mean arterial blood pressure (MAP) were monitored during the experimental period. Possible confounding factors such as dietary NO₃⁻ intake, physical activity and altitude changes were controlled.

Sea level plasma NO₃⁻ and NO₂⁻ concentrations significantly increased at altitude in both M.CEVEDALE group (+26.2 μM, p ≤ 0.0001, 95% CI [+17.6, +34.8] and +559.2 nM, p ≤ 0.0001, [+332.8, +785.6]) and M.ROSA group (+18.7 μM, p ≤ 0.0001, [+10.8, +26.5] and +463.7 nM, p ≤ 0.0001, [+314.3, +613.0]). Average peak value in NO metabolites concentration occurred earlier in M.CEVEDALE group vs M.ROSA group (NO₃⁻, day 3 vs day 5, p = 0.007; NO₂⁻, day 3 vs day 5, p = 0.019). In both groups, resting SpO₂, HR and MAP values changed according to altitude levels.

This study shows that exposure to hypobaric hypoxia affects nitric oxide metabolites, resulting in a significant increase in plasma NO₃⁻ and NO₂⁻ concentrations from sea level values. Interestingly, the higher the altitude reached, the longer the time taken to reach a peak in plasma concentrations of nitric oxide metabolites.

Introduction

Hiking, mountaineering and skiing are only a few of the many activities that millions of people perform in the mountains, exposing themselves to a challenging environment. Moving from sea level to altitude implicates exposure to hypoxic conditions, since the partial pressure of oxygen (PO₂) in ambient air progressively decreases alongside barometric pressure. The reduction in PO₂ in the environment impairs adequate oxygen supply to peripheral tissues by causing decreased PO₂ throughout the entire oxygen cascade in the human body, from the lungs to peripheral organs and tissues. Fortunately, the human body is able to adapt to hypoxic environments and counteract/overcome possible life-threatening conditions using a process termed “acclimatization” [1].

Acclimatization to hypoxia is a complex phenomenon characterized by several physiological responses at renal, cardio-pulmonary and hematological levels that act to increase oxygen delivery to tissues. It is known that at the molecular level, the response to hypoxia is largely governed by hypoxia-inducible factor-1 (HIF-1), a transcription factor that binds to hypoxia-response promoter regions and activates different genes regulating cellular oxygen homeostasis [2]. The activity of the α subunit of HIF-1 seems to be regulated by several molecules including nitric oxide (NO) that elicits a regulatory feedback mechanism in HIF-1α degradation [3].

NO is a gaseous signaling molecule produced endogenously, starting from the amino acid l-arginine in an oxygen-dependent pathway catalyzed by NOS enzymes [4]. NO is a very reactive molecule; it cannot be stored in free form and is generally synthesized with specific physiological effects [5]. In the plasma, NO is highly unstable and is almost immediately oxidized to nitrite (NO₂⁻) and nitrate (NO₃⁻). In the past, these metabolites were considered inert compounds. Recently, it has been demonstrated that NO₃⁻ and NO₂⁻, besides having intrinsic effects in blood flow regulation and mitochondrial activity, can be reverted to NO [6,7]. Indeed, NO₃⁻ can be converted in NO₂⁻ in the oral cavity by anaerobic bacteria present on the tongue surface, following which NO₂⁻ is reduced to NO in peripheral tissues [8]. This alternative NO₃⁻-NO₂⁻-NO pathway has been demonstrated to be more effective in conditions of low PO₂ and low pH [9].

Even though it is still under debate, NO seems to play a key role in the physiological responses to hypoxia and there is a growing body of evidence suggesting that NO and its metabolites are directly involved in the acclimatization process [10,11]. The first evidence comes from Himalayan populations, who have lived at high altitudes for generations and are well-adapted to hypoxia (i.e. Tibetans and Ladakhi natives). It was reported that they show 10-fold higher plasma NO₃⁻ and NO₂⁻ levels than lowlanders [12,13]. This difference in circulating concentrations of bioactive NO products is associated to the higher resting forearm blood flow observed in Tibetans compared to Caucasians [14] and is considered an adaptive response to cope with O₂ reduction at the tissue level [12].

Interestingly, recent evidence suggests that NO and its metabolites may be involved in the physiological responses to hypobaric hypoxia not only in high altitude populations, but also in lowlanders exposed to altitude. In 2011, two independent research groups reported lowlanders ascending to altitude demonstrated increased levels of NO metabolites compared to sea level during a trek in Nepal [10,15]. Both studies reported comparable peak values of plasma NO₃⁻ and NO₂⁻ concentrations occurring at similar altitudes (3440 m [15] and 3500 m [10]), although the timing of exposure to hypoxia was different (5 days [15] and 9 days [10]).

However, this evidence was questioned by recent work that reported no significant change in plasma NO₃⁻ concentration and a reduction in plasma NO₂⁻ concentration in lowlanders exposed for 5 days at 4559 m [16]. This mixed evidence might be due to potential confounding factors on plasma NO₃⁻ and NO₂⁻ concentrations following hypoxic exposure such as physical activity, diet and altitude changes.

The aim of the present study was to investigate the response of NO metabolites to hypobaric hypoxia in two groups of subjects exposed at two different altitudes by assessing plasma NO₃⁻ and NO₂⁻ concentrations at sea level and during hypoxic exposure whilst minimizing the effects of confounding factors. We tested the hypothesis that, once controlling for dietary NO₃⁻ intake, physical activity and altitude changes, plasma NO₃⁻ and NO₂⁻ levels would increase significantly in response to altitude exposure. Moreover, we explored whether different altitudes may affect the magnitude and time-course changes of these metabolites.