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* From Apex (altitude physiology expeditions) [Drs. Baillie, Bates, Thompson, Partridge, Schnopp, and Simpson], College of Medicine & Veterinary Medicine; and Clinical Pharmacology Unit (Drs. Waring, Maxwell, and Webb, and Ms. Gulliver-Sloan), Centre for Cardiovascular Science, University of Edinburgh, The Queen’s Medical Research Institute, Edinburgh, UK.
Correspondence to: J. Kenneth Baillie, BSc, MBChB, MRCP, Apex (altitude physiology expeditions), c/o College of Medicine & Veterinary Medicine, University of Edinburgh, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK; e-mail: j.k.baillie{at}doctors.org.uk
Abstract
Background: Both tissue hypoxia in vitro, and whole-body hypoxia in vivo, have been found to promote the release of reactive oxygen species (ROS) that are potentially damaging to the cardiovascular system. Antioxidant systems protect against oxidative damage by ROS and may exhibit some degree of responsiveness to oxidative stimuli. Production of urate, a potent soluble antioxidant, is increased in hypoxic conditions. We aimed to determine whether urate is an important antioxidant defense in healthy subjects exposed to hypoxia.
Methods: We conducted a cohort study of 25 healthy lowland volunteers during acute exposure to high altitude (4 days at 3,600 m, followed by 10 days at 5,200 m) on the Apex high-altitude research expedition to Bolivia. We measured markers of oxidative stress (8-isoprostane F2), serum urate concentration, and total plasma antioxidant activity by two techniques: 2,2'-amino-di-[3-ethylbenzthiazole sulfonate] spectrophotometry (total antioxidant status [TAS]) and enhanced chemiluminescence (ECL).
Results: On ascent, F2-isoprostane levels were significantly elevated compared with those at sea level (p < 0.01). After 1 week at high altitude, plasma antioxidant capacity (AOC) by both TAS and ECL, and serum urate concentration were significantly elevated (each p < 0.01 vs sea level), and F2-isoprostane levels were reduced to values at sea level. There was a highly significant correlation between plasma urate and AOC at this stage (ECL, r2 = 0.59, p = 0.0001; TAS, r2 = 0.30, p = 0.0062).
Conclusions: Our results support the hypothesis that urate may act as a responsive endogenous antioxidant in high-altitude hypoxia.
Key Words: altitude • free radicals • hypoxia • oxidative stress • uric acid
Hypoxia at high altitude is associated with a number of consequences that are potentially damaging to the vascular endothelium. In response to hypoxia, reactive oxygen species (ROS) are generated from the mitochondrial electron transfer chain, hemoglobin autooxidation, and xanthine oxidase and cause irreversible damage to proteins, lipids, and DNA.1 ROS release in hypoxia can alter vascular endothelial permeability, which may be a key component of the pathogenesis of high-altitude pulmonary edema and high-altitude cerebral edema.2
Endogenous antioxidant defenses may exhibit a degree of responsiveness to oxidative stimuli. The enzyme antioxidant superoxide dismutase is induced following exposure to hypobaric hypoxia in rats3 and humans at moderate4 and extreme5 altitudes. Urate is responsible for up to two thirds of the antioxidant capacity (AOC) of human blood,6 and urate is released from hypoxic tissues following the breakdown of adenosine diphosphate.7 Elevated urate levels have been demonstrated following tissue hypoxia such as coronary angioplasty and ischemia-reperfusion in limb surgery,8 and may be a protective response to excess free-radical production. A study by Jefferson et al9 shows that urinary urate excretion is elevated, consistent with increased production of urate, in humans after 48 h of exposure to high altitude, although plasma urate levels did not change during this short exposure. Although plasma urate levels are significantly elevated after 3 months at high altitude, the net antioxidant effect is offset by a decrease in other soluble antioxidants after this duration of exposure.10
Oxidative stress at high altitude in humans has been reported in several studies111213 conducted at relatively moderate altitudes, < 3,000 m, in which subjects have participated in strenuous exercise, which is associated with free-radical generation.14 Other work5 has found evidence of oxidative stress in climbers returning from 7,000 m, and in healthy lowlanders following acute exposure to an altitude of 4,300 m.15 Interestingly, this study15 also found a small increase in plasma glutathione during altitude exposure that may have a protective effect. The balance between oxidative stress and endogenous antioxidant function may be a key determinant of endothelial function in humans at high altitude. We sought to determine the biochemical significance of urate as an antioxidant in healthy subjects exposed to hypoxia. In particular, we hypothesized that an increase in serum urate would occur, and that this would lead to augmented AOC. We further hypothesized that increased AOC may mitigate oxidative stress.
Materials and Methods
High-Altitude Medical Research Expedition to Bolivia
All high-altitude time points in this report are counted from the day of arrival at La Paz, Bolivia. Twenty-five healthy lowland natives traveled from London (40 m) by air to La Paz, Bolivia (3,600 m). Sea level control samples were obtained 1 month before departure. Subjects stayed in La Paz for 4 days (during which one blood sample was obtained at day 2) before traveling by bus to Chacaltaya Laboratory (5,200 m). They remained at this altitude for 10 days. Daily measurements were made of BP using a manual sphygmomanometer, and heart rate and arterial oxygen saturation using a portable pulse oximeter (N20; Nellcor Puritan Bennett; Pleasanton, CA). All subjects consumed the same diet based on cooked meat, potatoes, and boiled vegetables. Acute mountain sickness symptoms were recorded using the Lake Louise consensus scoring system.16 Subjects abstained from chocolate and beverages containing caffeine or cocaine, and were receiving no medications for the duration of the study. Isoprostane sea level control samples were obtained 1 week after return to sea level. This study was approved by the Lothian Research Ethics Committee, and informed consent was obtained from all participants.
Blood samples were obtained at 5,200 m on days 6, 9, and 13 of the expedition. After the subject rested for 10 min, 35 mL of blood was removed from an antecubital fossa vein into potassium ethylenediamine tetra-acetic acid (F2-isoprostanes), Li-Heparin (total AOC), or serum (urate) tubes (Monovette; Sarstedt, Germany). After centrifugation in these tubes at 3,000g for 10 min (Labofuge 200; Kendro Laboratory Services; Herts, UK), the supernatant was pipetted into 2-mL screw-top containers (Camlab; Cambridge, UK) and immediately frozen in dry ice. High-altitude samples were transported on dry ice by international courier and stored in a – 40°C freezer until analysis.
Biochemical Assays
Two measures of AOC were employed. The enhanced chemiluminescent (ECL) assay was performed as previously described.17 Briefly, light emission is produced when luminol (Amersham; Buckinghamshire, UK) is oxidized in an ECL reaction catalyzed by horseradish peroxidase (Amersham) and is measured with a BioOrbit 1251 luminometer (BioOrbit; Turku, Finland). Light emission is suppressed during a lag period, the duration of which is proportional to the antioxidant content of added serum. Measurements are calibrated using an aqueous vitamin E analog (Trolox; Sigma; Dorset, UK), and AOC expressed as a Trolox equivalent. Total antioxidant activity was also assayed using the total antioxidant status (TAS) spectrophotometric assay employing the chromogen, 2,2'-amino-di-[3-ethylbenzthiazole sulfonate], as described by Miller et al.18 Urate was assayed by an automated method based on the uricase peroxidase system, and concentrations of 8-isoprostane F2 using a commercial enzyme immunoassay (Cayman Chemical; Ann Arbor, MI), both using previously described methods.1920
Predicted AOC
IV infusion of urate in healthy volunteers leads to an increase in the plasma AOC measured by both the TAS and ECL assays. In a previous study,17 we quantified the rise in total AOC that is brought about by a given increase in plasma urate concentration. Using a best-fit equation from this sea level infusion data, we predicted the change in AOC (ECL) expected from the measured changes in serum urate on ascent to high altitude. This is best predicted by a linear model: AOC (ECL) = 1.3942 [urate].
Statistical Analysis
Unless otherwise stated, results are expressed as mean ± 95% confidence interval. Nonparametric data are presented as median ± interquartile range (IQR). For paired comparison of change in variables over time at altitude, a Dunnett test was applied after significance at the level p < 0.05 was confirmed using repeated-measures analysis of variance. A Pearson correlation coefficient is quoted as r2, and significance was determined by applying the Bonferroni correction for eight repeated tests with significance at p < 0.05. Statistical analysis was performed (Graphpad Prism version 3.00; GraphPad Software for Windows; San Diego, CA; and Excel 2000; Microsoft; Redmond, WA). Throughout this report, day 1 is taken as the first day at 3,600 m; day 5 is the first day at 5,200 m.
Results
Basic Physiologic Measurements
No significant relationship was detected in this study between any antioxidant measure or evidence of oxidative stress and basic physiologic measurements including BP, heart rate, and arterial oxygen saturation, or symptoms of acute mountain sickness (Lake Louise score) [Table 1
]. Sixty percent of the subjects were male. The median age of subjects was 21 years (IQR, 20 to 22 years), and mean weight was 67 kg (SD, 9.7 kg). Activity levels were strictly limited throughout the time at altitude; all subjects spent the majority of time within the laboratory, and no subject walked > 10 km or 500 m ascent on any 1 day.
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Urate
Urate concentration was significantly elevated at day 6 (0.28 ± 0.03 mmol/L vs 0.24 ± 0.03 mmol/L at sea level, p < 0.01) and day 9 (0.32 ± 0.03 mmol/L, p < 0.01). The pattern of change in serum urate was similar to the pattern of change in AOC (Fig 1) by both the TAS and ECL assays. Furthermore, plasma urate correlated significantly with measured AOC by both the ECL and the TAS assays at high altitude (Table 2
).
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These results demonstrate that urate concentration and two measures of plasma AOC are significantly elevated during acclimatization to high altitude. In most high-altitude samples, there is a highly statistically significant correlation between serum urate and both measures of AOC (Table 2). The isoprostane levels, which were initially elevated indicating oxidative stress, normalize as the AOC rises. This, combined with the close temporal relationship between change in urate and total AOC (Fig 1), is consistent with the hypothesis that the endogenous antioxidant response described above is a protective response mediated by an increase in serum urate concentration.
Increased serum urate has been found in a variety of other conditions associated with hypoxia. Urate production is elevated in patients with obstructive sleep apnea due to tissue hypoxia, and normalizes following continuous positive airway pressure therapy.21 High serum urate concentration has been independently associated with increased risk of cardiovascular morbidity including hypertension, myocardial infarction, coronary artery disease,22 new stroke, and poor outcome following stroke.23 It is still not known if the association of urate with cardiovascular disease is causal or a protective response. During exercise, plasma urate increases14 and exogenous urate administration reduces oxidative stress.24 If, as our results suggest, urate is a key component of a protective antioxidant response to hypoxia, then urate itself may have a protective role in these conditions.
Jefferson et al9 showed that urate production is elevated but serum urate concentration is unchanged in healthy subjects after 48 h of exposure to an altitude of 4,300 m. Our study extends this work by demonstrating an increase in serum urate concentration in healthy subjects after 9 days at a similar altitude. A comparable increase in urate concentration after 3 months at high altitude has also been demonstrated, although decreases in the concentration of other soluble antioxidants, in particular ascorbate, prevented a corresponding increase in plasma antioxidant activity.10 In order for the serum urate concentration to rise, release of urate into the blood must have exceeded its removal. After 9 days at high altitude, the hematopoietic response to hypoxia has begun,25 and recent data from a similar population with the same ascent profile show a 14.5% rise in hematocrit at this time point (J.K. Baillie; unpublished data; September 2006). Likely sources of urate in these subjects include muscle catabolism,26 increased purine metabolism due to cell turnover, and adenosine diphosphate breakdown.27 Renal urate excretion is reduced by organic anions including lactate28 and sympathetic activation.29 In the context of increased urate production, these factors, both present in hypoxia,30 may have acted to prevent a compensatory rise in fractional urate excretion, permitting the accumulation of urate in blood.
Production of urate is catalyzed by xanthine oxidase, and this reaction is itself associated with release of ROS.31 Hence, the increase in urate may not be a protective response to hypoxia-induced oxidative stress but rather another direct consequence of the same underlying biochemistry. However, the time course of the increase in urate, occurring after, not concurrent with, a significant oxidative insult argues in favor of a protective response. Finally, the theory that urate itself may have an important protective role is supported by the observation that humans lost the enzyme uricase, which breaks down urate, relatively recently during evolution.32
The initial slight fall in AOC reflected in the ECL and urate assays coincides with increased isoprostane concentration compared with sea level (Fig 1). As the antioxidants measured by the ECL assay are by definition inactivated by free radicals, the slight fall in measured AOC shortly after the onset of hypoxia may represent antioxidant consumption by endogenous free radicals. Importantly, after the AOC has increased from baseline, isoprostane levels fall, consistent with the hypothesis that the rise in AOC is a protective response.
The differences between the TAS and ECL measurements plasma AOC (Fig 1) may reflect the limited degree of overlap in the specific plasma antioxidants detected by these different systems.6 Differences in the profile of antioxidants detected by each assay, for example, the presence of an unknown antioxidant, detected by one method but not by the other, may account for the observed differences in the pattern of change in AOC immediately after exposure to hypoxia. In common with all assays of total AOC, ECL has a bias toward some antioxidants over others. In healthy humans at sea level, urate is responsible for 65% of the AOC by ECL, with the remainder supplied by other soluble antioxidants including vitamins C and E.6
In addition to exposure to profound hypoxia, the subjects in this study may have been exposed to other environmental stimuli. Uncontrolled environmental factors include hypobaria, cosmic radiation, potential exposure to foreign organisms, communal living, alterations in diurnal rhythm associated with moving to a different time zone, and the physiologic effects of a long-haul flight. Dietary antioxidants can cause small alterations in antioxidant function measured by ECL and TAS.14 It is conceivable that dietary antioxidants or purines had a detectable impact on AOC. However the magnitude of the rise in AOC was much greater than we have previously observed with high-dose antioxidant vitamin supplementation14; and in a previous experiment,30 an artificially high dietary purine intake produced only a modest rise in plasma urate concentration. Although a reduction in plasma urate levels has been found in military jet pilots following prolonged missions, these pilots were exposed to mild hypoxia and several drugs including amphetamines and barbiturates.33 Although we are not aware of any previous studies to determine the effects of commercial long-haul air travel on antioxidant function, other studies have demonstrated an acute increase in markers of oxidative stress following ascent from sea level to high altitude in native populations traveling only a few hundred miles15 or within a hypobaric chamber.34 Ideally, a control group would have been studied at low altitude in circumstances that precisely mimicked the conditions of the high-altitude group. However, this was found to be prohibitively expensive and impractical: for example, second to hypoxia, cosmic radiation may be the most profound physiologic insult at 5,200 m, and mimicking the effects of this at low altitude would be difficult and ethically questionable.35
Although the data presented here are consistent with the hypothesis that urate may act as an inducible antioxidant in response to hypoxia, there is no clear evidence that this has evolved as a protective response, rather than a biochemical coincidence. Further work is necessary to investigate the importance of urate in oxidant/antioxidant balance in sea level conditions complicated by tissue hypoxia, such as obstructive sleep apnea and cardiovascular disease.
Acknowledgements
The authors would like to express their continuing gratitude to the Apex Bolivia 2001 expedition team. We also thank World Courier for generously transporting our samples, and Sarstedt, Camlab, and Kendro Laboratory Services for donations of research equipment.
Footnotes
Abbreviations: AOC = antioxidant capacity; ECL = enhanced chemiluminescent; IQR = interquartile range; ROS = reactive oxygen species; TAS = total antioxidant status
This research was funded by a project grant from the British Heart Foundation (PG/2001067), a small project grant from Chest, Heart, and Stroke Scotland (230103), and in part by the Scottish Charity Apex (Altitude Physiology Expeditions).
None of the authors have any conflicts of interest to declare.
Received for publication September 12, 2006. Accepted for publication December 16, 2006.
References
excretion in man. J Pharmacol Exp Ther 1995;275,94-100
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