Free Radical Biology & Medicine 52 (2012) 1123–1134

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Free Radical Biology & Medicine

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Review Article

Nitric oxide in adaptation to altitude

Cynthia M. Beall a,⁎, Daniel Laskowski b, Serpil C. Erzurum b

a Department of Anthropology, Case Western Reserve University, Cleveland, OH 44106, USAb Department of Pathobiology, Cleveland Clinic, Cleveland, OH 44195, USA

Abbreviations: d, effect size; FAD, flavin adenine dinuphosphate; NOS, nitric oxide synthase; RSNO, S-nitroso⁎ Corresponding author. Fax: +1 216 368 5334.

E-mail address: cmb2@case.edu (C.M. Beall).

0891-5849/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.freeradbiomed.2011.12.028

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 October 2011Revised 29 December 2011Accepted 29 December 2011Available online 20 January 2012

Keywords:AcclimatizationAdaptationNitric oxideHypoxiaAltitudeTibetanPulmonary artery pressureHigh-altitude pulmonary edemaFree radicals

This review summarizes published information on the levels of nitric oxide gas (NO) in the lungs and NO-derived liquid-phase molecules in the acclimatization of visitors newly arrived at altitudes of 2500 m ormore and adaptation of populations whose ancestors arrived thousands of years ago. Studies of acutelyexposed visitors to high altitude focus on the first 24–48 h with just a few extending to days or weeks.Among healthy visitors, NO levels in the lung, plasma, and/or red blood cells fell within 2 h, but then returnedtoward baseline or slightly higher by 48 h and increased above baseline by 5 days. Among visitors ill withhigh-altitude pulmonary edema at the time of the study or in the past, NO levels were lower than those oftheir healthy counterparts. As for highland populations, Tibetans had NO levels in the lung, plasma, andred blood cells that were at least double and in some cases orders of magnitude greater than other popula-tions regardless of altitude. Red blood cell-associated nitrogen oxides were more than 200 times higher.Other highland populations had generally higher levels although not to the degree shown by Tibetans. Over-all, responses of those acclimatized and those presumed to be adapted are in the same direction, although theTibetans have much larger responses. Missing are long-term data on lowlanders at altitude showing howsimilar they become to the Tibetan phenotype. Also missing are data on Tibetans at low altitude to see theextent to which their phenotype is a response to the immediate environment or expressed constitutively.The mechanisms causing the visitors’ and the Tibetans’ high levels of NO and NO-derived molecules at alti-tude remain unknown. Limited data suggest processes including hypoxic upregulation of NO synthase geneexpression, hemoglobin–NO reactions, and genetic variation. Gains in understanding will require integratingappropriate methods andmeasurement techniques with indicators of adaptive function under hypoxic stress.

© 2012 Elsevier Inc. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124Background on nitric oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124

Selection of studies for review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124Technical considerations for measurement of NO in exhaled gas at high altitudes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124Sample collection for NO analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1125Units of measurement and reporting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1125Sources of NO apart from synthesis in the body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1129

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1129Acute exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1129

NO therapy for failure to acclimatize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1129Acute exposure of 3 to 48 h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1130Acute exposure of 2 to 30 days . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1131

Chronic exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1131Gas-phase measurements of NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1131Circulating nitrogen oxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1131

cleotide; FMN, flavin mononucleotide; HAPE, high-altitude pulmonary edema; NADPH, nicotinamide adenine dinucleotideprotein.

rights reserved.

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Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1132Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1133References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1133

Introduction

This review summarizes and evaluates published information onlevels of nitric oxide (NO) in the lungs and circulation of people at al-titudes above 2500 m and, when available, the causal mechanisms orfunctional consequences. It discusses methodological issues and de-scribes the effects of acute exposure on measures of NO among visi-tors and among populations indigenous to high altitude. The aim isto determine if there is a scientific consensus on the effect of high al-titude on levels of NO and to identify research needed to discover itsroles in offsetting the severe stress of high-altitude hypoxia.

In 1990, Gustafsson, Persson, Moncada, and collaborators reportedthat hypoxia decreased pulmonary NO and caused vasoconstriction inisolated rabbit lungs. They suggested their findings could account forthe puzzling, yet well-known, hypoxic pulmonary vasoconstrictionresponse to high altitude [1,2]. In 1996, Scherrer and collaboratorsreported that inhaled NO reduced pulmonary artery pressure andimproved oxygen saturation among patients ill with high-altitudepulmonary edema (HAPE), a maladaptation characterized by exag-gerated hypoxic pulmonary vasoconstriction [3]. Since then, an accu-mulating body of data has demonstrated that NO in various forms andlocations in the body plays roles at all levels of the oxygen deliverycascade, from the pulmonary to the cardiovascular, hematological,and mitochondrial [4–11]. The role of NO in oxygen delivery underthe stress of high-altitude hypoxia is an area of active investigationbecause of the potential for improving understanding of human biol-ogy and health. A first step toward understanding is establishing howmuch is available, where it is located, and in what form.

Background on nitric oxide

Nitric oxide, originally described as endothelium-derived relaxa-tion factor, is a product of the NO synthases (NOSs), which convertL-arginine to NO and L-citrulline in a reaction that requires oxygen,NADPH, and cofactors FAD, FMN, and tetrahydrobiopterin. NOS en-zymes include neuronal, inducible, and endothelial forms (nNOS,iNOS, and eNOS, respectively) [12]. nNOS and eNOS are generallyexpressed in the brain and the vascular endothelium, whereas iNOSis constitutively expressed in respiratory epithelium and is upregu-lated in other tissues in response to numerous factors, including in-flammation and hypoxia [13]. An increased expression of iNOS andeNOS under hypoxia through the activation of gene expression byhypoxia-inducible transcription factors (HIF1 and HIF2) leads to theexpectation of more NO, but NO production in the lung decreases im-mediately under acute hypoxia, which may be due to limitation of theoxygen substrate for the reaction [14]. A NOS-independent pathwayfor generating NO operates through the reduction of nitrate and ni-trite [11,15,16]. Thus, a priori, the effect of high hypoxia on nitricoxide synthesis, metabolism, and clearance is uncertain.

The vascular effects of NO are generally attributed to eNOS-derived NO production and its reaction products. Once produced inthe endothelium, NO gas is freely diffusible and enters smoothmusclecells to activate soluble guanylate cyclase to produce 3′, 5′-cyclicmonophosphate (cyclic GMP), which leads to vasodilatation. Nitro-gen oxides measurable in blood, saliva, urine, and broncholavagecan be biologically active. They include nitrite (NO2

−), nitrate(NO3

−), iron nitrosyls (FeNO), and S-nitrosothiols (SNO) such asSNO–hemoglobin. Hemoglobin allostery can regulate interconversionof nitrogen oxide forms and their biological activity [8–11]. NO

metabolism also leads to nitration of tyrosine in proteins in tissueand blood [17]. Methods to measure nitrogen oxides have been welldescribed [11,18–22]. Methods to measure gas-phase NO are dis-cussed in some detail because measurement of gases requires instru-ments validated for use at altitude.

Materials and methods

Selection of studies for review

Database searches linking altitude and nitric oxide and humanidentified 32 published articles (Table 1). The databases were AnnualReviews, Article First, BIOSIS, CINAHL, ClinicalTrials.gov, DissertationAbstracts, PubMed (Medline), Science Direct, SCOPUS, SPORTDiscus,TOXNET, Worldcat, and Web of Science. Abstract screening eliminat-ed articles using other organisms or conducted in hypoxia chambersor tents. Studies at altitudes above 2500 m were included based onthe evidence that many responses to acute exposure are first manifestabove that altitude [23] and on convention in the field of high-altitude human biology [24]. Table 1 summarizes the altitude expo-sure, sample, and measurement method of the studies retained forreview.

Study outcomes are summarized for comparison using a calculat-ed variable called an effect size, d. The advantage of this approach isthat d can compare studies with different designs, outcome variables,and sample sizes to estimate the magnitude of the treatment effect(altitude, for example) on the outcome variable (total lung nitricoxide or pulmonary artery pressure, for example). Cohen's d is calcu-lated as the difference between two means divided by the “squareroot of the mean of the two variances” [25, p. 44]. That is, d quantifiesthe size of the difference between two means in terms of the numberof their pooled standard deviations. For example, an effect size d of1.0 describes a situation in which fewer than half the observationsin two samples overlap, whereas an effect size d of 0.2 describes asituation in which more than 80% of the observations overlap. By con-vention, d's of 0.8 or more are considered large, d's of 0.5 are consid-ered medium effects, and those of 0.2 are considered small effects.Because there are no conventions for effect sizes in the range from 1to more than 5 that are encountered here, this review providesthose effect size values. Positive effects indicate that the characteristicwas higher at high altitude; negative effects indicate that the charac-teristic was lower at high altitude. An effect size calculator is availableat http://www.uccs.edu/~faculty/lbecker/ (accessed 1 October 2011).

Technical considerations for measurement of NO in exhaled gas at highaltitudes

Detecting gas-phase molecules requires instruments that providevalid measurements at the generally lower humidity, temperature,and barometric pressure found at altitudes above 2500 m than inthe low-altitude laboratory or clinic for which most instruments aredesigned. Ambient conditions at morning calibrations in one of ourstudies illustrate the contrast: 14.4 °C average temperature, 52% rela-tive humidity, and 464 Torr barometric pressure at 4200 m in thefield laboratory in Tibet compared with warmer and drier conditionsof 23 °C, 29% relative humidity, and 754 Torr barometric pressure in aclimate-controlled room in Cleveland, Ohio, USA [26,27]. These envi-ronmental contrasts can confound interpretations.

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Five sensor technologies detect gas-phase molecules of NO:chemiluminescence, optical spectroscopy, mass spectrometry, chro-matography, and electrochemistry. Two technologies are commer-cially available and have been used to measure NO at high altitude:the Sievers NOA (GE Analytical, Boulder, CO, USA) and the NIOXMINO (Aerocrine, New Providence, NJ, USA). The Sievers NOA uses achemiluminescence sensor and the NIOX MINO uses disposable elec-trochemical sensors. The Sievers NOAi analyzer is based on a gas-phase chemiluminescence photomultiplier tube that detects photonemission from electronically excited nitrogen dioxide producedfrom the reaction between nitric oxide and ozone in the reactionchamber. Although the design of the photomultiplier housing systemand reaction chamber compensates for any change in external baro-metric pressure, variation in the atmospheric pressure can influencethe sampling flow rate through the restrictor of the sensor and thuslead to lower levels of the NO signal. To avoid this artifact, calibrationat the prevailing barometric pressure with standard NO gas is re-quired so that any change in the sampling flow rate is accounted forin the collection and measure of NO at altitude. The Sievers NO ana-lyzer measures gas-phase NO with a sensitivity of less than 1 ppband liquid-phase NO-derived molecules with a sensitivity of 1 pmol[28].

The NIOX MINO sensor is electrochemical. Details of the electro-chemistry are not available; however, this type of sensor reacts withgas-phase molecules that diffuse into a container in which electrodessit in a solution designed to detect NO. Because electrochemical sen-sor technology depends on diffusion to drive the gas into the reactionchamber, changes in atmospheric pressure influence NO detection.Lower air pressure at altitude results in less gas diffusion into the re-action chamber and reduces the accuracy of the measurement. Thetechnical specifications of the instrument list an operating range ofbarometric pressures from 700 to 1060 hPa or from about 3100 mto below sea level. Yet a 2009 paper identified factors that cause thereadings to be inaccurate at high altitudes and warned against usingthe device above 1000 m [29]. That paper suggested using two correc-tion factors based on measurement of nine people in a temperature-controlled hypobaric chamber. The manufacturer describes accuracyas ±5 ppb or max 10% (http://www.aerocrine.com/en-us/niox-mino/Specifications/ accessed 27 June 2011). In addition, relative hu-midity and temperature can affect the accuracy of some electrochem-ical reactions.

Calibration is essential when equipment is moved from one altitudeto another. The Sievers NOA calibration and measurement of NO occurin the reaction chamber containing the photomultiplier tube main-tained at constant pressure, humidity, and temperature. The manufac-turer recommends two point calibrations using air free of NO (obtainedwith a filter that removes NO) and a high NO concentration chosen bythe investigator (such as 45 ppm). In contrast, the NIOXMINO does notcalibrate. The company recommends an external quality control usingan individual with steady and stable exhaled nitric oxide levels. Thisis problematic for investigations designed to test hypotheses aboutchanges in exhaled NO at different altitudes. Thus, the NIOX MINO isnot recommended for studies across altitudes.

Because of these considerations, this review reports only gas-phase measures obtained by chemiluminescence. For the future, itwill be important to (a) publish details of electrochemical analysesand their sensitivities to temperature and other relevant environ-mental factors and (b) simultaneously compare electrochemical andchemiluminescence measures under field conditions.

Sample collection for NO analysis

NO is present in the exhaled breath of humans [3]. The use ofchemiluminescence analyzers allowed for the detection of NO in ex-haled breath in the early 1990s and ultimately the use of exhaledNO as a clinical test [30–32]. The methods and equipment for

measuring NO in exhaled breath were standardized for clinical pul-monary function laboratories. The standardized “online” measure ofNO is useful for clinical testing for airway disease, but other experi-mental methods remain in place and are optimal for study of NO athigh altitudes.

The online technique measures airway NO in the early part of ex-halation during a 10-s exhalation at 50 ml/s that is the standardmethod recommended for clinical pulmonary function laboratories.Thus the measurements represent the level of NO in the first500–750 ml of exhaled gases from the conducting airways [33]. Incontrast, the technique of offline collection of exhalate in nonperme-able Mylar balloons measures exhaled NO in the vital capacity lungvolume, including NO derived from alveolar spaces and vascularbed, and is recommended for evaluation of NO at altitude. A breath-hold maneuver increases the sensitivity for detection of vital capacityNO [14,34,35].

The online–offline distinction is important because the twomethods inform about different locations. Studies may reach oppositeconclusions depending on the method of collection. Fig. 1A comparesthe cumulative frequency distributions of exhaled NO measured on-line at a rate of 50 ml/s for a U.S. sample at low altitude and two Tibet-an samples at different high altitudes. Roughly half the low-altitudesample exhaled 15 nm Hg or more compared with about 20% of theTibetans at 4200 m and none of the Tibetans at 4700 m. The data re-flect less NO production in the conducting airway of the respiratorytract among high-altitude Tibetans that decreases with altitude.Data on low-altitude Tibetans are needed to determine airway pro-duction in normoxia.

The relative positions of the samples are reversed in Fig. 1B compar-ing offline collection of exhaled breath NO measurements after a 15-sbreath hold. Offline measurements represent the equilibration of thepools of NO in the pulmonary vascular bed, alveolar space, and conduct-ing airways and therefore provide insight into the total NO of the lung[14,34,35] and its conduction and transfer [36]. Roughly half the Ti-betans at 4200 m exhaled more than 10 nm Hg NO compared withroughly 10% of the Tibetans at 4700 m and none of the lowlanders atlow altitude. The lower values for the Tibetan sample at 4700 m reflectoxygen sensitivity of the NOS or substrate limitation [37].

The contrast in Figs. 1A andB starkly illustrates that sample collectiontechniques can lead to different conclusions. This paper uses the term“airway NO” for measures obtained using online techniques and theterm “vital capacity NO” for measures obtained using offline techniques.Airway NO among Tibetans at 4200 m is lower than among low-altitudecontrols, whereas vital capacity NO is higher. Regional heterogeneity ofNO levels in the lung probably accounts for these findings [36].

Units of measurement and reporting

NO analyzers report measures in parts per billion in the exhaledbreath. However, converting to and reporting exhaled NO as the par-tial pressure of exhaled gas are appropriate for comparing levelsacross altitudes in which barometric pressures affect the measure ofgas in a volume. For example, we published concentration in NOppb in a paper comparing Tibetan and Andean highlanders withlow-altitude controls [37]. Because the Tibetan and Andean sampleswere collected at roughly the same altitude (4200 and 3900 m withsimilar barometric pressures of 467 and 484 mm Hg, respectively)the Tibetan–Andean comparison of vital capacity NO reported therehad a straightforward interpretation: Tibetans had higher vital capac-ity NO than Andean highlanders. The barometric pressure differencesconfound interpretation of high- and low-altitude differences. Al-though Tibetans’ vital capacity NO in mm Hg was higher than thatof lowlanders, Bolivian Aymara exhaled significantly more than low-landers in ppb but there was only a trend toward differences in nmHg. Figs. 2A and B illustrate this with a comparison of plots in unitsof ppb and nm Hg.

Table 1Summary of studies reviewed.

Source Altitude and lengthof exposure

Sample composition Measurement technique

Ethnicity or country Health status Male(n)

Female(n)

Total No.of people

Age (mean)or age range

Acute exposure b24 hBailey et al. [46] 3 and 20 h after travel

from 110 to 4559 mEthnicity unspecified, Germany Healthy, nonsmoking 32 6 38 37 years NO-derived products (NOx) in

plasma and red blood cellsa

Berger et al. [74] 3 and 20 h after travelfrom 110 to 4559 m

Ethnicity unspecified, Germany Healthy, nonsmoking,including some HAPE-S

29 5 34 37 years Plasma NOxa

Brown et al. [73] 4200 m, 3 h Ethnicity unspecified, USA Healthy, nonsmoking 24 23 47 ~45 years Offline exhaled breatha

Acute exposure 24–48 hDuplain et al. [49] 4559 m, 48 h Ethnicity unspecified, Switzerland HAPE-S mountaineers 18 10 28 40 years Offline, exhaled NOa

Ethnicity unspecified, Switzerland HAPE-R mountaineers 15 9 24 38 yearsMansoor et al. [50] 4342 m, 24 h Ethnicity unspecified, USA Healthy, active 7 0 7 37 years L-Arginine supplement, breath

condensate, NO2−, NO3

− a

Sartori et al. [71] 4559 m for 48 h Ethnicity unspecified, Switzerland Experienced perinatal insult 10 21 years Breathed 40 ppm NO for 20 min,change in ΔPAPEthnicity unspecified, Switzerland No history of perinatal

complications10 21 years

Scherrer et al. [3] 4559 m, 48 h Ethnicity unspecified, Switzerland HAPE-S mountaineers 15 3 18 42 years Breathed 40 ppm NO for 15 min,ΔPAPEthnicity unspecified, Switzerland HAPE-R mountaineers 13 5 18 42 years

Schneider et al.[75]

4350 m, 36 h after travelfrom 35 m

Ethnicity unspecified, France Healthy 8 3 11 28 years L-Arginine infusion, ΔL-citrullineand cGMPb

Swenson et al. [53] 4559 m, 48 hours Lowlanders, Switzerland HAPE-S 12 24–52 years BAL, NOxa

Lowlanders, Switzerland 6 HAPE-R 10 24–52 years

Acute exposure 2 to 30 daysDonnelly et al. [76] 0 to 5050 m over an est.

20 daysNew Zealand Healthy nonsmokers 7 4 11 (9 analyzed) 32 years Online, exhaled NOc

Janocha et al. [55] 1300 to 5050 m over19 days

Italy, USA Not stated 10 8 18 Not stated Serum, saliva, urine NOxa

Levett et al. [21] 75 to 5300 m over30 days

European Healthy 18 6 24 19–59 years Plasma NOxa,d

Liu et al. [77] 3658 m, 5 days comparedwith lowlanders

Ethnicity unspecified, China Healthy 60 18–21 years Plasma NOxe

Ethnicity unspecified, China Healthy 70 18–21 yearsSchena et al. [54] 3100 m, 8 days European lowlanders: trained skiers Active, physically trained,

nonsmokers9 0 9 26 years Plasma NOx

e

European lowlanders: untrained men Active nonsmoker 6 0 6 32 yearsVinnikov et al. [78] 4000 m, 14 or 21 days Kyrgyz and European lowlanders, Kyrgyzstan 73 81 126 (81

analyzed)32 years Online, exhaled NOc

Acute exposure unspecified time, patientsAhsan et al. [52] 3500 m, unspecified and

sea levelEthnicity unspecified, India HAPE patients 72 0 72 30–40 years Plasma NOxe

Same unstated ethnicity, India HAPE-R 60 0 60 30–40 yearsAnand et al. [70] 3600 m, unspecified India HAPE 14 0 14 29 years Breathed NO at 15 ppm for 30 min,

ΔPAP

High-altitude residents and nativesAhsan et al. [61] 3400 m and low, native

residentsEthnicity unspecified highlander monks, India 131 131 46 years Plasma NOx

e

Ethnicity unspecified highlanders, India Control 136 136 21 yearsEthnicity unspecified lowlanders at low, India Control 170 170 30 years

Ahsan et al. [60] 3400 m, native residents Ethnicity unspecified lowlanders, India HAPE 59 59 30–40 years Plasma NOxe

Ethnicity unspecified highlanders, India Control 136 136 30–40 years

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Table 1 (continued)

Source Altitude and lengthof exposure

Sample composition Measurement technique

Ethnicity or country Health status Male(n)

Female(n)

Total No.of people

Age (mean)or age range

Ethnicity unspecified lowlanders same ethnicity,India

Control 170 170 30 years

Appenzeller et al.[67]

3622 m residents and794 m for 24 h

High-altitude Amhara, Ethiopia No history of chronicdisease

9 9 36 years Sublingual pharmaceutical NOdonor: isosorbide dinitrate inPeru, nitroglycerin in Ethiopia4388 m residents and

150 m for 24 hHigh-altitude residents, Peru 5 healthy, 4 with CMS 9 9 37 years

Beall et al. [37] 4200 m Tibetan Healthy, nonsmokers 45 62 105 8–56 years Offline, exhaled NOa

3900 m Bolivian Aymara Healthy, nonsmokers 67 78 144 8–56 years272 m Native residents, USA Healthy, nonsmokers 13 20 33 32/31?

yearsde Bisschop et al.[79]

4000 m lifelong, sea levellifelong, 3750 m 4 days

Highlanders, Bolivia 10 with CMS, 8 healthy 14 4 18 45 years Pulmonary NO diffusing capacity,NO/CO transfer techniqueEuropean Healthy 8 8 16 36 years

Droma et al. [62] 3440 and 1300 m lifelong Sherpas, Nepal Normal blood pressure 44 61 105 ~31 years Serum NOxb

Non-Sherpa Nepalis Normal blood pressure 53 58 111 30 yearsErzurum et al. [27] 4200 m, lifelong, indigenous

populationTibetan Healthyf 25 63 88 31 years Plasma and red blood cells, NO2

−,NO3

−, FeNO, and RXNOe,gLowlanders, USA 23 27 50 37 yearsGao et al. [80] 3658 lifelong and long-term

residentsTibetan and Han Chinese Women at full-term delivery 0 Not stated Not stated Not stated Umbilical vein endothelial cells,

HIF-1A, VEGF, eNOS, and iNOSmRNAh

Ge et al. [81] 4300 m, some lifelong,some 10 years

Tibetan CMS 8 42 years Serum eNOSi

Han Chinese 16 42 yearsTibetan Healthy 40 39 yearsHan Chinese 10 39 years

Gonzales et al. [56] 4340 and 150 m High-altitude residents for 37–38 years, Peru Excessive erythrocytosis 33 33 43 years Serum NOxe

Healthy 29 29 45 yearsLow-altitude residents for 34 years, Peru Healthy 30 30 43 years

Hoit et al. [26] 4200 m, lifelong, indigenouspopulation

Tibetan Healthyf 20 37 57 31 years Online, exhaled NOa

Lowlanders, USA Healthy 7 13 20 47/39 yearsHoit et al. [59] 3700, 1200, 282 m lifelong Highland Amhara, Ethiopia Healthyf 53 22 76 31 years Urinea

Lowland Amhara, Ethiopia Healthyf 45 9 54 30 yearsLowlanders, USA Healthyf 21 25 46 35 years

Jayet et al. [48] 3600 m lifetime Aymara highlanders, Bolivia Mothers were preeclamptic 24 24 48 13 years Inhaled 40 ppm NO for 20 min,ΔPAPMothers had normal

pregnancy55 35 90 14 years

Julian et al. [69] 3100, 1600 m, resident High-altitude residents, USA Pregnant and postpartum 0 25 25 26 years Serum NO, NO2−, NO3

−, andnitrosothiolaLow-altitude residents, USA Pregnant and postpartum 0 18 18 28 years

Schwab et al. [82] 3600–4000 m lifelongresidents or lowlanderswith 6 years of residence

Aymara highlanders, Bolivia Healthy 24 10 34 36 years Online, exhaled NOc

European lowlanders, Bolivia Healthy 21 13 34 38 years

Stuber et al. [83] 3600–4000 m lifelong residentsor upward migrants with 2+years of residence and 450 m

Aymara highlanders, Bolivia Healthy and free of infirmity 105 95 200 9.5 years Online, exhaled NOc

European highlanders, Bolivia Healthy and free of infirmity 42 35 77 9.5 yearsLowlanders, Switzerland Healthy and free of infirmity 16 13 21 8.8 years

Teran et al. [68] 2800 m, resident High-altitude residents, Ecuador Healthy pregnant term 0 30 30 ~20 years Plasma, placenta NOxa

High-altitude residents, Ecuador Preeclamptic 0 33 33 ~20 yearsLow-altitude residents, Ecuador Healthy pregnant term 0 30 60 ~20 years

Preeclamptic 0 30

HAPE-R, HAPE-resistant; HAPE-S, HAPE-sensitive; ΔPAP, pulmonary artery pressure; BAL, bronchoalveolar lavage; RSNO, nitrosoproteins; CMS, chronic mountain sickness.a NO measured with chemiluminescence.b Measurement by chromatography for L-citrulline, radioimmunoassay for cGMP.c NO reported using NIOX MINO.d Measurement by high-pressure liquid chromatography (HPLC).e NO measured using nitrate reductase and the Griess reaction.f Normal blood pressure, nonsmoking, not pregnant.g Amperometric, HPLC, chemiluminescence.h Measurement by Western blot.i Measurement by enzyme immunoassay.

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Fig. 2. Units of reporting change comparisons of NO across altitudes. The top comparison leads to the inference that Tibetans have uniquely high values, whereas the bottom leads tothe inference that highlanders generally have high values. (Top) Tibetans at 4200 m exhaled more NO than Bolivian Aymara at 3900 m, who did not differ significantly from a U.S.low-altitude sample when reported as partial pressure of NO in vital capacity exhalate. The respective geometric means were 8.7 nm Hg with a range of 2.6 to 26 among Tibetans,4.6 nm Hg with a range of 1.3 to 14.7 among Aymara, and 5.5 nm Hg with a range from 3.3 to 10.8 among lowlanders. (Bottom) Tibetans at 4200 m exhaled more NO than BolivianAymara at 3900 m, who in turn exhaled more than a U.S. low-altitude sample when reported as the concentration of NO in exhalate. The respective geometric means were 18.6 ppbwith a range of 5.5 to 55.7 among Tibetans, 9.5 ppb with a range of 2.7 to 30.3 among Aymara, and 7.5 ppb with a range from 4.5 to 14.6 among lowlanders. Redrawn from [37] withpermission of the publisher.

Fig. 1.Measures of NO in exhaled breath from the airway or the vital capacity can lead to different inferences about altitude effects. Tibetans at 4200 m had less NO in gases collectedfrom the proximal conducting airways and substantially more in gases collected from the entire exhaled breath than a low-altitude sample. (A) A low-altitude U.S. sample exhaledmore NO and a wider range of values from the conducting airways (measured online at a flow rate of 50 ml/s into the NO analyzer) than a Tibetan sample at 4200 m, which in turnexhaled more than a Tibetan sample at 4700 m. The x axis marks intervals of 5 nm Hg NO and the y axis the percentage of observations in the interval or lower. (B) A Tibetan sampleat 4200 m contained more NO in the vital capacity volume collected from exhaled breath and a wider range of values in the total lung exhalate (measured in the total volume ofbreath exhaled into a Mylar collection bag after a 15-s breath hold, i.e., offline) than a Tibetan sample at 4700 m, which in turn exhaled more than a low-altitude U.S. sample.

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This review converts values published in ppb to nm Hg if the authorsreported barometric pressure. Partial pressure of NO in nm Hg is cal-culated as follows: reported NO in ppb multiplied by ambient baro-metric pressure in mm Hg and divided by 1000. Using thiscalculation, a reading of 10 ppb obtained at sea level with barometricpressure 760 mm Hg and at 4200 m with barometric pressure464 mm Hg corresponds to a partial pressure of NO of 7.6 nm Hg atsea level and 4.6 nm Hg at 4200 m, i.e., there are fewer NO moleculesin the measured volume at high altitude owing to the lower baromet-ric pressure. Reports of studies at high altitude should include baro-metric pressure [38].

Sources of NO apart from synthesis in the body

Oral bacteria can produce NO3−, whereas food and drink may con-

tribute NO2− and NO3

− to circulating levels of biologically active com-pounds [39]. Guidelines for exhaled NO collection recommend thatstudy participants refrain from food and drink an hour before provid-ing samples [32].

These two potential sources of NO could contribute to total NOmeasures yet are rarely taken into account [40,41]. Dietary sourcesof NO among Tibetans at 4200 mwere evaluated by collecting dietaryinformation in a rural agropastoral district of the Tibet AutonomousRegion [27,37,42]. Five men and five women volunteered to providesamples of exhaled breath and urine. They also reported their dietaryintake in the previous 24 h and provided aliquots of the foods, whenavailable, and of the household water supply. Beverages—water, tea,and home-brewed barley beer—from volunteers’ own meals con-tained levels ranging from undetectable to b15 μM NO2

− andb70 μM NO3

−. None of the food contained green vegetables or othersources of NO3

− such as cured meat; typical meals contained lowlevels of NO2

− (b0.4 mg/kg) and NO3− (b125 mg/kg). Overall, the av-

erage daily consumption of NO3− was not at a level expected to signif-

icantly alter circulating NO3− or NO2

− levels [43]. Excluding dietarysources for high levels of NO strengthens the inference of high endog-enous production in this high-altitude sample with a diet typical ofhighland Tibet.

Fig. 3. Inhalation of NO lowers pulmonary artery pressure at high altitude and shows tha(systolic except for sample 1, which is mean arterial pressure) fell after breathing 30–40 pof Indian soldiers at 3600 m with HAPE [70]; 2 was HAPE-susceptible lowlanders with HAPEall at 4559 m [3]; 5 was lowlanders, age 21 years, who suffered perinatal hypoxia and 6 waswhose mothers had preeclampsia; and 8 was those mothers that had normal pregnancies atafter NO inhalation at 3600–4559 m (shown on y axis) declines from high-altitude baselinelevels and the decline across studies is 28% based on linear regression analyses.

Results

Adaptation is a concept with many meanings sharing the sense ofresponse that improves function under a stress. Modes of adaptationmay be distinguished on a time scale ranging from rapid and revers-ible acclimatization to evolutionary adaptation in the gene pool overgenerations [44,45]. Samples of people with different high-altitudeexposures, for example, acute exposure for hours or chronic over life-times or generations, can offer insights into the different modes of ad-aptation to high altitude. Studies of NO response to high-altitudehypoxia have evaluated function many ways, for example, thepresence or absence of acute or chronic mountain sickness or high-altitude pulmonary edema. Because NO is a vasodilator and intimate-ly involved in oxygen delivery to tissues, some studies have quanti-fied pulmonary artery pressure; blood flow in the brain, lungs,uterus, and forearm; and exercise capacity. Genotype, experience ataltitude, physical fitness, ethnicity, and other individual factors canmodify NO response.

Acute exposure

NO therapy for failure to acclimatizeAcute exposure to hypoxia engages short-term responses. Accli-

matization is usually successful in the sense of preserving function al-though not always at normal low-altitude baseline levels. However,some people do not acclimatize successfully. Evidence suggests thatfailure to acclimatize may be related to insufficient NO production.A potentially fatal response is high-altitude pulmonary edema(HAPE), characterized by an exaggeration of the normal constrictionof pulmonary blood vessels in response to hypoxia that causes an ex-treme elevation of pulmonary blood pressure. For example, pulmo-nary artery systolic pressure increased by an average of 13 mm Hgin a healthy low-altitude sample at 4559 m for 20 h; it increasedtwice as much (27 mm Hg) in a subsample that developed HAPE [46].

NO can counteract hypoxic pulmonary vasoconstriction by caus-ing vasodilation. Inhaling a gas mixture of 15–40 ppm NO for15–40 min lowers pulmonary artery pressure in every samplereported. Fig. 3A shows extremely large effect sizes ranging from

t hypoxic pulmonary vasoconstriction is vasoreactive. (A) Pulmonary artery pressurepm NO for 15–40 min at 3600–4665 m altitude [3,48,70,71]. Sample 1 was composed; 3 was HAPE-susceptible lowlanders without HAPE; 4 was HAPE-resistant lowlandersthose who did not at 4559 m [71]; 7 was Bolivian Aymara highlanders, age 13–14 years,3600 m [48]. Effect size, d, of NO on pressures is shown. (B) Pulmonary artery pressure(shown on x axis). The posttreatment levels are directly associated with pretreatment

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−2 to −5 pooled standard deviations (d) among six samples ofpeople acutely exposed to altitudes ranging from 3600 to 4559 m.The average fall in pulmonary artery pressure was 15±7 mm Hg or28%. Pre- and post-NO treatment pressures were highly correlated(Fig. 3B, r=+0.93). The falls show that high-altitude pulmonary hy-pertension was vasoreactive and related to insufficient levels of NO tomaintain pulmonary vasodilation [47].

Perinatal stress, postnatal hypoxia, or gestation by a mother withpreeclampsia was associated with stronger hypoxic pulmonary vaso-constriction later in life. The top two bars of Fig. 3A compare 13- to14-year-old Bolivian Aymara highlanders at 3600 m whose mothershad normal pregnancies with those whose mothers had mild-to-moderate preeclampsia [48]. The next two bars contrast 21-year-oldlow-altitude Europeans who had a normal transition to extrauterinelife with those who received oxygen therapy for transient perinatalhypertension. Samples 5 and 7 suffered an insult and had higherbaseline pulmonary artery pressure at altitude and larger falls afterbreathing NO than their controls (samples 6 and 8). This occurredamong individuals exposed chronically (Bolivian Aymara) and thoseexposed acutely (21-year-old Europeans). There may be perinatal or-igins of vulnerability to HAPE later in life that are NO-dependent.

Acute exposure of 3 to 48 h

Gas-phase measurements of NO. A fall in NO due to less synthesis of NOdue to oxygen substrate limitation or oxygen sensitivity of the NOScould explain the immediate rise in pulmonary artery pressureupon arrival at altitude as a consequence of a reduced capacity tovasodilate. Reports of lowlanders’ first 3 to 48 h of exposure to alti-tudes of 4200 and 4559 m reveal a dynamic situation.

Fig. 4 shows that vital capacity NO fell slightly at 3 h and substan-tially at 12 and 24 h at 4559 m. HAPE-resistant study volunteersreturned to baseline during the second 24 h. HAPE-sensitives didnot; instead their vital capacity NO fell by more than 2 SD at 12 hand remained at least 1 SD below baseline for the full 48 h of expo-sure [49].

NO is synthesized from oxygen, in short supply at high altitude,and L-arginine. Therefore one study provided additional L-argininesubstrate before and during exposure to 4342 m in an attempt todrive greater NO synthesis. Lung NO trended upward for 12 h butnot significantly so, perhaps because the time was too short, as sug-gested by Fig. 4, or the changes in NO occurred in locations that do

Fig. 4. HAPE-resistant people, whose previous exposure to altitude did not incur HAPE, havHAPE-susceptible people. (A) Vital capacity NO falls in the first hours at altitudes above 400uals and returns to baseline by 38 h among HAPE-resistant individuals [72,73]. (B) Healthy H(BAL) fluid at 12 h. In contrast HAPE-susceptible individuals had reductions in nitrogen oxide[53].

not accrue benefit, or oxygen limitation could not be overcome [50].Exercise capacity did not change, and unfortunately, L-arginine sup-plementation increased high-altitude headache. The latter suggestsNO and/or NO-derived species might have increased in locations ofno benefit to acclimatization and, if so, perhaps added to adverseeffects.

Circulating nitrogen oxides. NO gas is a free radical that diffuses intothe blood where it may form NO2

−, NO3−, and S-nitrosoproteins

(RSNO), all of which are measurable in the plasma and serum.Allostery of hemoglobin determines formation of many of the NOproducts. Oxyhemoglobin reacts with NO to form NO3

−. Deoxyhemo-globin reacts with NO2

− to form NO, RSNO, and iron nitrosyls in heme(FeNO) [51]. Because deoxyhemoglobin levels are much higher at highaltitude, this almost certainly leads to changes in NO–hemoglobin reac-tions and levels of NO-derived products. For example, Tibetan men at4200 mwith an oxygen saturation of 85% had 1.6 mmol/L deoxyhemo-globin compared with men at 282 m with an oxygen saturation of 97%who had 0.3 mmol/L deoxyhemoglobin. The influence of deoxyhemo-globin on circulating nitrogen oxides has received little considerationin studies despite accumulating evidence of the fundamental role ofthe NO–hemoglobin interactions on blood flow and oxygen delivery.

Intravascular levels of plasma NO2−, RSNO, and red blood cell NO in

arterial and venous blood during the first 20 h at altitude provide in-sight into the changes in NO production and chemistry that comprisethe acclimatization response. Fig. 5A shows large and unequal falls ofarterial and venous NO2

− resulting in a smaller arterial–venous differ-ence by 20 h at 4559 m. Reporting only those measures would havebeen misleading because a very large increase in arterial nitrosopro-teins also occurred. Both arterial and venous red blood cell nitrogenoxides increased more than a standard deviation and resulted in alarger arterial–venous difference. The finding of increases in nitrogenoxides in the blood was in contrast to lower bioavailable NO in thelung [46]. The large increase in red blood cell-associated nitrogen ox-ides was interpreted as evidence of reapportionment of NO to longer-lived forms [46, p. 4844]. Red blood cell nitrogen oxides accounted forabout 22% of total arterial NO at high altitude compared with about11% at low. The red cell-associated NO could be released locallyto cause vasodilation, greater blood flow, and improved oxygendelivery.

Another study reported that plasma nitrogen oxides obtained ret-rospectively at low altitude from individuals who had been resistant

e generally less reduction of NO or even an increase when reexposed to altitude than0 m and then trends slightly toward baseline values among HAPE-susceptible individ-APE-resistant individuals showed increased nitrogen oxides in bronchoalveolar lavages at altitude regardless of whether they suffered from HAPE during this particular study

Fig. 6. Tibetan and Andean highlanders show higher vital capacity NO levels comparedwith sea level controls. Tibetans at the same altitude have lower airway NO levels.Among Tibetans, samples at 4700 m have lower NO values in both compartmentsthan those at 4200 m. The lower values of airway NO may be related to decreasedlocal production of NO by airway epithelial type 2 NO synthase due to limitation of ox-ygen substrate to the enzyme, whereas higher total lung NO may be related to increas-ing values of circulating nitrogen products. Source [27,37].

Fig. 5. Plasma NO2− fell as nitrogen oxide-derived products (NO) in plasma and associated with erythrocytes increased over 20 h at 4559 m [46]. (A) Arterial and venous plasma

NO2− and venous S-nitrosothiol (RSNO) concentrations among healthy people generally fell at 3 and 20 h at 4559 m but arterial RSNO values increased strikingly, which may reflect

synthesis and/or greater hemoglobin–NO interactions [46]. Arterial–venous differences uniformly decreased. (B) Arterial and venous erythrocyte (RBC) nitrogen oxides (nitrite,nitrosyl hemoglobin, and S-nitrosohemoglobin) and total nitrogen oxides (sum of those in plasma plus those associated with the erythrocytes) at 3 and 20 h at 4559 m amonghealthy people [46] are presented. Erythrocyte-associated nitrogen oxides increased and plasma NO2

− fell. The arterial–venous differences in erythrocyte nitrogen oxides increased,suggesting greater offloading of NO to tissues at altitude.

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to HAPE were much higher than levels among individuals with HAPEat 3400 m [52]. The meaning of that finding is difficult to interpret be-cause the samples differ in health, altitude, and response. Further,other studies reported that HAPE-resistant and HAPE-sensitive indi-viduals did not differ before exposure [49,53]. Two prospective com-parisons of HAPE-sensitive and HAPE-resistant individuals comparedpreexposure baseline measures and found no differences in total lungNO [49] or NO2

− or NO3− in bronchoalveolar lavage fluid (BAL) [53].

However, BAL from a single sample of HAPE-resistant individuals ataltitude showed a very large increase in nitrogen oxides (Fig. 4B)[53], whereas BAL from HAPE-sensitive individuals had marked de-creases in nitrogen oxides of nearly 4 SD. Fitness or activity mayalso play a role in the size of the response to altitude. Plasma nitrogenoxides increased more among untrained men than among athleteswho continued to train for 8 days at 3100 m [54]. Thus, the magni-tude of the NO acclimatization response may partly depend on phys-ical fitness.

Acute exposure of 2 to 30 daysTwo studies of trekkers and climbers describe processes over days

of hypoxia. One provided values at a 1300 m baseline, 3400 m on day5, and 5050 m on day 13 of the trek [55]. The other plotted trendsover roughly the same route and schedule [21]. Both reported a mod-erate increase in serum or plasma NO2

− at 3440 m and then a slightdecrease at 5050 m. In contrast, hemoglobin-associated nitrogen ox-ides including S-nitrosohemoglobin (SNO:Hb) and FeNO hemoglobinincreased moderately at 3440 m and more at 5050 m. Higher levels ofSNO:Hb and hemoglobin were both associated directly with exerciseperformance. Thus, hemoglobin-associated nitrogen oxides and NO2

−

are part of the biological response to altitude.Although details of study design varied enormously and NO was

measured in different locations, overall, studies of acute exposurereported that higher NO levels were associated with less illness andbetter function at altitude, confirming that NO is important in the ac-climatization to altitude.

Chronic exposure

Less is known about NO levels among long-term high-altitude res-idents and natives. High-altitude native refers to someone from apopulation on the Tibetan, Andean, or East African plateau with along history and prehistory of high-altitude residence and the

opportunity for natural selection to work. Most of the informationwas provided by Tibetan highlanders. Whereas acutely exposed low-landers usually serve as their own controls, so far studies of high-landers compare them with low-altitude samples of low-altitudeancestry. This confounds altitude and ancestry. There is evidencethat both influence NO location in the body and the relative amountsof NO-derived products.

Gas-phase measurements of NOBolivian Aymara had a small elevation and Tibetan highlanders

had a very large elevation of vital capacity NO (Fig. 6). Relievinghypoxia with 20 min of 50% oxygen did not elicit a response in theBolivian Aymara sample, but raised vital capacity NO by more than1 nm Hg among the Tibetans at 4200 m [37]. That result plus lowervital capacity NO among more hypoxic Tibetans at a higher altitudesuggested that greater NO production was due to greater NO synthe-sis, but maximal production of NO was limited by oxygen availability.

Circulating nitrogen oxidesTibetans had much higher levels of circulating nitrogen oxides

than low-altitude samples at low altitude (Fig. 7). High-altitude na-tives and residents of India at 3400 m and Peru at 4350 m had small

Fig. 7. Tibetans have very high and other highlanders have high levels of plasma orserum nitrogen oxides compared with lowlanders at low altitude [27,56,60,61].

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to large elevations. Peruvians with more symptoms of a maladapta-tion (such as palpitations, cyanosis, and headaches) known as chronicmountain sickness had lower levels of serum nitrogen oxides thantheir healthy counterparts [56]. The lower levels of circulating nitro-gen oxides in the Nepalese Sherpa sample is puzzling because theyemigrated from eastern Tibet beginning in the early 1500s [57] andwould therefore be expected to resemble Tibetans. Consistent withFig. 7 is the finding that red blood cell-associated nitrogen oxideswere more than 200 times higher among Tibetans at 4200 m com-pared with lowlanders at low altitude [27].

The whole-body production of NO quantified as urinary nitrogenoxides [58] was higher in Tibetans than in low-altitude populationsand consistent with the total lung and nitrogen oxide measurements,suggesting greater synthesis of NO. Tibetans at 4200 m had a 10-foldelevation of urinary nitrate compared with a U.S. low-altitude sample[27]. Interestingly, Tibetans have slightly more deoxyhemoglobinthan other populations at comparable altitudes. For example, Tibetanmen at 4200 m have an average of 1.6 mmol/L deoxyhemoglobincompared with 1.2 mmol/L among Andean Aymara men at 3900 m.The comparable figures for Tibetan and Andean Aymara women are1.3 and 1.1 mmol/L deoxyhemoglobin. Perhaps NO–deoxyhemoglo-bin interactions contribute to the greater circulating nitrogen oxidesof Tibetans. However, Ethiopian Amhara at 3700 m had a 3-fold ele-vation of urinary nitrate, but so did Amhara at 1200 m, suggestingthat hypoxia did not account for the elevated NO levels in that case[59]. Perhaps genetic variation predisposes some populations togreater NO production or perhaps dietary differences play a role.

In this context, the responsible NOS or NOS-pathway gene orchemical reaction is not known. Candidate gene analyses focused onendothelial NOS, the constitutively expressed form. Highlandersfrom Ladakh, India, and Sherpas from Nepal had more than 10%higher allele frequencies at two polymorphic NOS3 sites associatedwith higher nitrogen oxide levels [52,60–62]. Selecting appropriatecontrol samples is difficult because allele frequencies of low-altitudepopulations vary widely [63]. A genome scan for signals of natural se-lection found preliminary evidence of selection on the inducible NOS(NOS2) in an Andean sample but not a Tibetan sample [64,65].

Regardless of the mechanisms leading to greater NO, functionalbenefits accompany the Tibetans’ high levels. Higher levels of totallung NO were associated with lower pulmonary artery systolicpressure and more pulmonary blood flow and oxygen delivery at4200 m [26]. The higher plasma and red cell nitrogen oxides amongTibetans accompanied more than twofold higher forearm blood flowand oxygen delivery [27]. Notably, gas-phase measurements did notassociate with forearm blood flow and plasma measurements didnot associate with pulmonary artery systolic pressure. Those findingsemphasize the importance of studying local concentrations andeffects.

However, findings in Tibetans cannot be uniformly applied. Differ-ent high-altitude native populations have unique NO responses. For

example, cerebral blood vessels normally dilate in response to hypox-ia and in response to NO [66]. Neither Peruvian nor Ethiopia residentsof 4340 or 3700 m had the expected fall in cerebral blood flow whenacutely removed from hypoxia by visiting low altitude, implying thatneither had hypoxic cerebral vasodilation at altitude. However, re-sponses to pharmaceutical NO donors among Peruvian Quechua andEthiopian Amhara highlanders identified population differences inNO-mediated vasoreactivity of cerebral blood vessels. AmongPeruvians, pharmaceutical NO donors caused a medium fall in cere-bral blood flow at altitude and a large fall at sea level. That is,Peruvians’ cerebral blood vessels had greater NO-mediated cerebralvasoreactivity under normoxia. Among Ethiopian Amhara, pharma-ceutical NO donors caused a large fall in cerebral blood flow at alti-tude and a medium fall at low altitude, i.e., more pronounced NOmediation under hypoxia. The authors concluded that these findingsreflect acclimatization among the Peruvians and genetic adaptationamong the Amhara [67]. Alternatively, these results may be inter-preted to reflect differences in reactive oxygen species production,which will affect NO availability [46].

Pregnancy adds to oxygen requirements and the stress of high-altitude hypoxia, which offers another opportunity to understandthe role of NO in thriving under conditions of hypoxia. A study of pre-eclamptic women at 2800 m in Ecuador found higher plasma and pla-cental nitrogen oxides than in healthy pregnant women. That wasone of the few instances in which higher NO levels associated withpoorer function. In comparison, preeclamptic women at sea levelhad lower plasma and placental nitrogen oxides than healthy preg-nant women [68]. Another study considered the balance of vasocon-strictors and NO-derived vasodilators on blood flow to the uterus.Pregnant high-altitude residents at 3100 m in Colorado had lowerserum nitrogen oxides than their counterparts at 1600 m, and theratio of the vasoconstrictor endothelin relative to nitrogen oxideswas higher at 3100 m. The higher ratios were associated with loweruterine artery diameter and blood flow and lower birth weight [69].These studies point out the importance of studying NO in the contextof other counteractive biologic mediators of altitude effects.

Overall, Tibetans had exceptionally high levels of NO at all loca-tions except for the conducting airways. High levels of circulating ni-trogen oxides were associated with high blood flow and oxygendelivery. The few measurements available for Andean Aymara andEthiopian Amhara showed trends in the same direction, suggestingthat some degree of elevated NO is important for all populations athigh altitude.

Conclusion

Studies of NO in humans at high altitude, where all individuals areunavoidably exposed to low ambient oxygen, cumulatively identify arole for NO in many beneficial adaptive responses. Unfortunately, nosingle study provides comprehensive information about all biologicallocations, forms of NO, or their functional outcomes, which is neededto provide a clear view to the mechanisms of production and effect.This review begins to outline a model of high levels of pulmonaryNO and NO-derived molecules providing functional benefits includinglimiting the extent of hypoxic pulmonary hypertension and sustain-ing high systemic blood flow for greater oxygen delivery. Much ofthis review deals with measurement and data reporting issues. Stud-ies at high altitudes have the challenges of appropriate sample collec-tion and measurement under field conditions very different fromthose of low-altitude laboratories and research institutes. Chemilumi-nescence sensors are appropriately designed for the conditions, al-though they are heavy and bulky. Electrochemical sensors areconvenient, but their accuracy at high altitudes and cold tempera-tures has not been established. There are reasons to doubt the validityof their measurements and they cannot be calibrated. Until that infor-mation is available, chemiluminescence sensors should be used.

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Otherwise, scientists and study volunteers will continue to devote re-sources to unproven technology.

Early studies simply asked if NO is higher or lower at high altitude.Studies affirm that high levels of NO are associated with function ben-efits and the avoidance of illness. However, many questions remain tobe answered. Questions include whether NO remains elevated in low-landers who successfully acclimatize, or if the increases are transient,i.e., is NO an early but unsustained response? Similarly, informationon Tibetans at low altitude is necessary to understand whether thehigh levels of NO at altitude are maintained when hypoxia is re-moved. Other questions remain to be answered regarding the chem-istry of NO-derived products. Which nitrogen oxides are importantfor functional benefits and what are the mechanisms? Functionalbenefits come to those with high nitrogen oxides; however, we donot yet know how the high levels are achieved. Genetic polymor-phisms in the many pathways that influence NO synthesis areunstudied.

Future human studies that integrate samples of lung, plasma/serum, and red cell nitrogen oxides along with relevant functionalmeasures will begin to answer some of these questions.

Acknowledgments

S.C.E. received support from NIH HL60917. C.M.B. received supportfrom NSF 0924726, 0452326, and 021547. D.L. has consulted for GEAnalytics. We thank our colleagues for their insights and supportiveenthusiasm, our study participants for their willingness to volunteer,and our reviewers for their helpful suggestions.

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