Using an integrated multi-biomarker/whole-body physiology approach in humans, we here demonstrate that NO formation increases in lowlanders ascending to high altitude, in agreement with a role in adaptation to hypoxia. Our findings are consistent with earlier observations of elevated NO metabolite concentrations in the blood of Tibetan highlanders18
and show that enhanced NO production is not unique to this particular population (perhaps as a result of evolutionary selection pressure over millennia) but an integral physiological response to hypoxic stress in humans. In spite of a parallel increase in oxidative stress with hypoxia, in well acclimatised lowlanders not only the production but also the availability of NO is enhanced at all altitudes. Furthermore, we show that enhanced NO levels are associated with changes in microcirculatory blood flow which may affect local tissue oxygen delivery. Contrary to our expectations, we found no evidence for a change in the oxygen cost of exercise. Unchanged oxygen cost with physiological elevation of nitrite in lowlanders at altitude contrasts with reduced oxygen cost of work shown in studies of dietary nitrate supplementation at sea level20,21
and in Tibetans at altitude.23
However, nitrite levels in Tibetan highlanders exceed those observed in our subjects by an order of magnitude,18
raising the possibility that improved mechanical efficiency may have evolved as an adaptation to residence in a hypoxic environment in this population.
In many cases, the biochemical changes in response to high altitude exposure are so striking that almost everything is statistically significant when compared to baseline. However, uncovering the biological relevance of those alterations remains a challenge. Relatively small (and statistically
insignificant) changes in the steady-state concentration of biomarkers and signalling molecules may be associated with biologically
significant changes in physiology, in particular when regulatory circuits are operating at their limits. In other cases, statistically significant changes from baseline may simply indicate that the system is operating at a different setpoint (which may still be well within the regulatory range). Given the significance of NO in the regulation of so many vital bodily functions, including vascular tone and mitochondrial activity, an enhanced NO availability would seem to be important for sustained local NO signalling under conditions of globally elevated reactive oxygen species production, as we here document to occur on sojourn to high altitude. This seems to be accomplished by a combination of factors, including elevated de novo
synthesis by NOS (resulting in higher levels of circulating nitrite and nitrate), release of NO by redox-activation of plasmatic storage forms (as evidenced by the initial drop in S-nitroso species), and reduction of nitrite (indicated by gradual directional changes in association between plasma nitrite concentrations and some of the physiological parameters on exposure to increasing levels of hypoxia). Reduction of nitrite (and possibly also nitrate)16
may also account for our observation that peak nitrite and nitrate levels were not sustained on prolonged stay at high altitude; in addition, it may have limited the magnitude of the increases measured. That elevated NO production indeed translates into enhanced NO bioavailability (in spite of concomitantly elevated reactive oxygen species production) is suggested by the increases in circulating cGMP concentrations we detected at high altitude; in the absence of major changes in natriuretic peptide levels, which could also have contributed to those changes via stimulation of particulate guanylyl cyclase, and no reason to suspect an inhibition of phosphodiesterase activity24
, this signature is consistent with an enhanced NO activity. Although elevations in circulating cGMP levels appear to be rather robust this does not necessarily translate into increases in blood flow, a prerequisite for enhanced convective delivery of oxygen through the blood. This conclusion is supported by the contrasting associations of microvascular blood flow with circulating nitrite and cGMP levels. Pharmacological doses of nitrite can lower blood pressure,25,26
but whether or not this extends all the way down to physiological levels is currently unknown. Our observation that physiological nitrite concentrations, at sea level and across altitudes, are negatively correlated with blood flow in the microcirculation was unexpected but is consistent with (i) its lack of direct vasodilator activity at low-to-moderate concentrations27
and (ii) the increase in blood pressure at low nitrite doses observed in animals28
Thus, while the positive correlation of microvascular blood flow with cGMP at 5,300m suggests that NO formation (from NOS or nitrite reduction) is enhanced, this appears to be insufficient to normalize reduced microcirculatory blood velocity. An alternative or additional explanation, given that all metabolites were measured in venous plasma, is that nitrite present in arterial blood was metabolized to NO in upstream capillary beds and that the cGMP and blood flow changes are reflections of that process. Paradoxically, if diffusion limitation of oxygen is a critical factor with respect to performance, slower blood flow at high altitude19
may be a beneficial adaptation allowing increased time for oxygen unloading in tissues. Alterations in microcirculatory function have been associated with morbidity and mortality in human sepsis.30
It would seem to be important, therefore, to clarify whether these alterations are indeed unfavourable perturbations of normal physiology or perhaps an intentional feature in tissue hypoxemia, and whether or not therapeutic enhancement of microvascular blood flow under these conditions has merit.
Although our results suggest that several different pathways and sources of NO cooperate to enhance NO availability in response to hypoxia, the precise mechanisms that confer enhanced hypoxia tolerance remain unclear. Our study is limited inasmuch as it does not provide information about accompanying changes in NO metabolite levels in vascular tissue, skeletal muscle, or red blood cells, compartments also expected to contribute to the physiological adaptations observed. Moreover, it remains unclear at this stage whether NO itself or a change in NO-related post-translational modification (such as S-nitrosation) of proteins or transcription factors, or all of the above, contribute(s) to beneficial adaptation to hypoxia. Of note, S-nitrosothiols transduce the ventilatory response to hypoxia31
and HIF-1α itself is a target for S-nitrosation.32
Thus, nitrosothiols may not only act as sources of NO (as indicated by the initial drop in S-nitrosoalbumin in the present study) but also as signal transducers,33
and changes in S-nitrosation status may affect many other downstream processes. Although the main objective of our study was to assess the temporal and quantitative changes of the NO pathway in adaptation to hypoxia during ascent to high altitude several intriguing associations between biochemical parameters and physiological variables (e.g., NO production with oxygen consumption at rest, S-nitrosothiols with blood oxygen content, and the inverse relationship between nitrate and haemoglobin) at sea-level are worthy of further investigation. Clearly, additional mechanistic investigations are warranted to identify the sources of NO and reactive oxygen species production (i.e. whether they originate in blood cells, vascular tissue, and skeletal muscle or are derived from other organs), the sequence of signalling pathways involved, and their relationship with tissue phenotypic changes (e.g. microcirculatory blood flow). Nevertheless, our results suggest that the observed changes in microvascular function are likely to involve cGMP-independent effects13
mediated by longer-lived NO metabolites such as nitrosothiols, nitrite and nitrate. Considering the rich chemical biology of NO in relation to its reaction with oxygen and reactive oxygen species34
it is likely that in addition to HIF-1α several other redox-sensitive signalling nodes are involved in the acclimatisation process.
Our findings may be of relevance not only to healthy subjects exposed to hypobaric hypoxia, but also to patients in whom oxygen delivery is limited through disease affecting the heart, lung or vasculature,8
and to the field of developmental biology.35
Cellular hypoxia with consecutive organ dysfunction/damage is a near universal problem in critical illness. Patient management decisions with large resource implications are often based on very limited ‘hard data', and few biomarkers other than indices of inflammation or acute tissue damage are available to guide this process. Cellular hypoxia may result from anemia, from pulmonary, cardiac micro-and macrovascular pathology, or from abnormal oxygen handling. Whilst tolerance to hypoxia has traditionally been considered to depend on increased convective delivery, efforts to maximize oxygen supply or utilization either provide no benefit or are harmful.36,37
Mechanisms underpinning inter-individual differences in the ability to cope with hypoxia in human (patho)physiology, which ultimately translate into differences in outcome, are largely unexplored - in part because the complex (often multi-system) nature of critical illness represents a major obstacle to mechanistic investigation. The search for prognostic markers in critical illness is particularly challenging due to the multitude of derangements in signalling/metabolic pathways that characterize conditions involving multiple organ failure. Moreover, many of the classical markers are affected by the often aggressive treatment and invasive procedures applied, posing a challenge to untangling cause/effect relationships. An alternative approach might begin with the detailed study of healthy individuals exposed to profound and prolonged hypoxia, where functional heterogeneity can be linked to biological diversity by use of an integrated biochemical/whole-body physiology assessment, and translation through application of derived data to the patient population. Thus, systematic identification and study of the molecular signatures of pathways involved in hypoxic adaptation in healthy
individuals may hold the key to understanding adaptation to reduced oxygen availability in critically ill patients
, and help identify novel therapeutic targets. In this regard, our data suggest that individuals unable to mount an adequate intrinsic response to hypoxia may benefit from direct (nitrite, nitrate) or indirect (e.g., small-molecule modulators of NOS expression/activity) manipulation of NO metabolism to activate the physiological program that confers tolerance to hypoxia. The results of the current study also make us wonder whether the findings of greater mortality in septic shock patients treated with a NOS inhibitor a decade ago38
might now perhaps be interpreted as proof-of-concept that survival in stress/hypoxia depends on a sufficient production of NO. Finally, our study raises the intriguing possibility that maximizing arterial oxygen levels (PaO2
) in patients who have been exposed to sustained hypoxia may be harmful, if cellular energetics and organ function have become reliant on reductive rather than oxidative pathways of NO generation. This may be particularly pertinent to the neonatal intensive care setting where, after months of embryonic development under conditions of physiological hypoxia, our youngest patients are likely to suffer disproportionately from additional oxidative damage due to a low-capacity anti-oxidative defence system.