Olivier Chedeville a
email: olivier.chedeville@univ-orleans.fr

Marie Debacq

Catherine Porte

Removal of phenolic compounds present in olive mill wastewaters by ozonation

Keywords: Wastewater treatment, ozone, kinetic, margines

The aim of this work is to study a pre-treatment process of olive mill wastewaters based on ozonation. The efficiency of the process depends on the removal of pollutants and on ozone mass transfer performance. In order to choose an appropriate gas / liquid contactor, the rate constants of three phenolic compounds (gallic acid, phydroxybenzoic acid and p-coumaric acid) were determined by using competition kinetic model. These constants, obtained at pH 5, were found to be high (from 3.8x10 4 L /mol s to 2.9x10 5 L/mol s), inducing a diffusion controlled regime (Ha > 3). Thus, to obtain an efficient ozonation process, gas/liquid contactor should be adapted to this regime. An ejector was chosen as gas/liquid contactor. In a first time, treatment of synthetic effluent containing the three phenolic compounds was performed to evaluate efficiency of the process. Experimental conditions were chosen to obtain a diffusion controlled regime (Ha > 3). It appeared that this gas/liquid contactor permit to obtain complete and fast removal of pollutants with a very efficient ozone mass transfer (up to 90 % during removal of phenolic compounds). So, this process was used to perform the ozonation treatment of olive mill wastewaters from Sfax (Tunisia). It was proved to be very efficient: up to 80 % of phenolic compounds were removed and ozone mass transfer reached 95 % during this oxidation.

Introduction

Wastewaters resulting from olive oil production, called margines, are a serious environmental problem in Mediterranean countries, where this activity is located [START_REF] Paraskeva | Membrane processing for olive mill wastewater fractionation[END_REF][START_REF] Khoufi | Extraction of antioxidants from olive mill wastewater and electro-coagulation of exhausted fraction to reduce its toxicity on anaerobic digestion[END_REF].

The content of olive mill wastewaters (OMW) depends on a number of parameters, such as geographic location, season, soil and weather. Generally acid (pH 4.5 to 5), they present a high organic and inorganic pollutant load [START_REF] Sayadi | Screening of white rot fungi for the treatment of olive mill waste-waters[END_REF][START_REF] Mellouli | The use of olive mill effluents ("margines") as soil conditioner mulch to reduce evaporation losses[END_REF][START_REF] Ginos | Treatment of olive mill effluents by coagulation-flocculation-hydrogen peroxide oxidation and effect on phytotoxicity[END_REF]. In particular, these wastewaters contain high amounts of phenolic compounds, in concentrations varying from 6 to 20 g/L [START_REF] Amat | Ozonisation coupled with biological degradation for treatment of phenolic pollutants: a mechanistically based study[END_REF].

Biological degradation methods were tested to reduce pollution load of these wastewaters. Fezzani et al. [START_REF] Fezzani | Optimisation of the mesophilic anaerobic codigestion of olive mill wastewater with olive mill solid waste in a batch digester[END_REF] tested the codigestion of OMW with olive mill solide waste containing high level of nitrogen source and obtained COD removal between 16,7% and 58,4%. Ammary et al [START_REF] Ammary | Treatment of olive mill wastewater using an anaerobic sequencing batch reactor[END_REF] studied the treatment of OMW by using an anaerobic treatment and obtain total phenol removal of 63 %. These methods were limited by the high phenolic compounds concentration of OMW. The bactericidal properties these compounds do not allow OMW to be treated in the traditional way, as they inhibit the activity of microorganisms during the biological treatment step [START_REF] Turhan | Removal of phenol from water using ozone[END_REF][START_REF] Drouiche | A compact process for the treatment of olive mill wastewater by combining OF and UV/H2O2 techniques[END_REF].

A specific pre-treatment is therefore needed to remove their toxicity before they are introduced into a treatment plant. Different treatment methods have been tested (adsorption on activated carbon, ultrafiltration, and ion exchange resins) but do not present a high efficiency [START_REF] Monteagudo | Photo-Fenton-assisted ozonation of p-Coumaric acid in aqueous solution[END_REF]. In contrast, ozonation and advanced oxidation processes seem to lead to interesting results [START_REF] Vassilakis | Sonolysis of natural phenolic compounds in aqueous solutions: degradation pathways and biodegradability[END_REF][START_REF] Amat | p-Coumaric acid photodegradation with solar light, using a 2,4,6-triphenylpyrylium salt as photosensitizer: A comparison with other oxidation methods[END_REF]. According to Rivas et al. [START_REF] Rivas | two-step wastewater treatment: sequential ozonationaerobic digestion[END_REF], ozonation is a suitable treatment to obtain more biodegradable effluent and permit to use a biological treatment step. Some studies showed that process based on ozone or advanced oxidation process allow high COD reductions and a nearly complete removal of aromatic compounds [START_REF] Beltrán | Effects of single and combined ozonation with hydrogen peroxide or UV radiation on the chemical degradation and biodegradability of debittering table olive industrial wastewaters[END_REF][START_REF] Cañizares | Advanced oxidation processes for the treatment of olive-oil mills wastewater[END_REF]. Moreover, Beltran et al. showed that in these processes, much of ozone fed in is adsorbed and reacted [START_REF] Beltrán | Industrial wastewater advanced oxidation. Part 2. Ozone combined with hydrogen peroxide or UV radiation[END_REF]. So, ozonation process of these effluents is controlled by ozone mass transfer, and process efficiency depends on the choice of an appropriate gas/liquid contactor.

The aim of the present study is to design a pre-treatment process using ozone, which removes phenolic compounds and transfers ozone efficiently into water. Firstly, in order to determine the phenomenon limiting ozone mass transfer (reaction or diffusion) and to choose an appropriate gas / liquid contactor, the ozonation kinetics of three phenolic compounds (gallic acid, p-coumaric acid and p-hydroxybenzoic acid) were determined.

Secondly, treatment of synthetic effluent containing these compounds was performed in order to evaluate process efficiency in diffusion controlled regime (Ha >3). Finally, the treatment of olive mill wastewaters from Sfax (Tunisia) was performed in the selected contactor.

Experimental

Competition kinetics model

It is admitted that ozonation is a second order reaction [START_REF] Hoigné | Rate constants of reactions of ozone with organic and inorganic compounds in water-II: Dissociating organic compounds[END_REF][START_REF] Dantas | Sulfamethoxazole abatement by means of ozonation[END_REF]. To determine the rate constants for the direct oxidation of phenolic compounds with ozone, the competition kinetics model was used [START_REF] Deborde | Kinetics of Aqueous Ozone-Induced Oxidation of Some Endocrine Disruptors[END_REF]. In this method, a phenolic compound is introduced with a reference compound whose ozonation kinetics constant is known. In this study, the reference compound was phenol, whose ozonation kinetics rate constant was determined by Hoigné et al. [START_REF] Bader | Determination of ozone in water by the indigo method[END_REF]. To avoid radical reaction, tBuOH was used as a radical scavenger. The kinetics equations can therefore be written:

] ].[ .[ ] [ 3 / 3 PC O k dt PC d PC O   (1) ] ].[ .[ ] [ 3 / 3 phenol O k dt phenol d phenol O   (2)
where [PC] and [phenol] are respectively the phenolic compound and phenol concentration (mol/L), t the time (s), kO3/PC and kO3/phenol represent respectively the ozonation kinetics constant of the phenolic compound and the phenol (L/mol s), and

[O3] is the dissolved ozone concentration (mol/L). The ratio of equations ( 1) and (2)

gives:

] [ ] [ . ] [ ] [ / 3 / 3 phenol PC k k phenol d PC d phenol O CP O  (3)
By integrating equation (3), the following equation is obtained:

) ] [ ] [ ln( . ) ] [ ] [ ln( 0 / 3 / 3 0    phenol phenol k k PC PC phenol O PC O (4)
Where [PC]0 and [phenol]0 are respectively phenolic compound and phenol initial concentration (mol/L) and [PC] ∞ and [phenol] ∞ are respectively phenolic compound and phenol concentration at the end of the reaction, while dissolved ozone was consumed (mol/L). In order to determine phenol and phenolic compound concentration at the end of the reaction, ozone has to be added in substoechiometric amount.

In this study three phenolic compounds were studied: gallic acid, p-coumaric acid and p-hydroxybenzoic acid. The ozonation kinetics of dissociating organic compounds strongly depends on pH [START_REF] Hoigné | Rate constants of reactions of ozone with organic and inorganic compounds in water-II: Dissociating organic compounds[END_REF][START_REF] Benitez | Kinetics of phydroxybenzoic acid photodecomposition and ozonation in a batch reactor[END_REF]. For the experiments, initial pH was therefore fixed at 5, which is the average pH value of olive mill wastewaters, by adding H2SO4.

Experiments were carried out at 20°C. In each stirred reactor 200 mL of deionised water, 0.32 mg of phenol and phenolic compound and 10 mg of tBuOH were introduced. In these reactors, different volumes (5 to 30 mL) of ozone saturated water, were introduced. Last one was prepared in a bubble column by introducing ozone in distilled water. Ozone concentration in water was 0.75 mg/L  0,01 mg/L (measured by carmin indigo method, see 2.3.2). After 10 min, phenol and phenolic compound concentrations were determined by HPLC. Different experiments were carried out by changing the volume of ozone saturated water (5 to 30 mL). According to equation ( 4),

ratio phénol O CP O k k / 3 / 3 is the slope of ) ] [ ] [ ln( ) ] [ ] [ ln( 0 0   phénol phénol vs PC PC .

Ozone mass transfer

Efficiency of treatment process depends on the ozone mass transfer. So, it is very important to choose a suitable gas/liquid contactor. The Hatta number is a very important criterion giving information on the competition between the kinetics of the reaction and the diffusion inside the liquid film (table 1). It indicates where the chemical reaction takes place (in the bulk liquid or in the film) and allows to choose the appropriate gas/liquid contactor [START_REF] Roustan | Transferts gaz liquide dans les procédés de traitement des eaux et des effluents gazeux[END_REF]. In the case of a second order reaction the Hatta number is given by [START_REF] Deborde | Kinetics of Aqueous Ozone-Induced Oxidation of Some Endocrine Disruptors[END_REF]:

L O k D A k Ha 3 ]. .[  (5)
where k is the rate constant of the reaction between ozone and compound A (L/mol s),

[A] the concentration of compound A in the bulk liquid (mol/L), DO3 the diffusivity coefficient of ozone into water (m 2 /s) and kL the mass transfer coefficient (m/s). If ozone reacts with two or more compounds in liquid phase, the Hatta number is expressed as:

L O i i k D i k Ha 3 ]. .[   (6)
Where ki is the rate constant of the reaction between ozone and compound i (L/mol s) and [i] is the concentration of compound i in the bulk liquid (mol/L).

Ha

Ozonation pilot

The ozonation pilot (figure 1) uses an ejector as gas/liquid contactor. In this contactor, water passes through a convergent, generating depressurization in the mixing chamber into which the ozone is aspired. This kind of gas/liquid contactor is recommended for a diffusion controlled regime [START_REF] Hoigné | Rate constants of reactions of ozone with organic and inorganic compounds in water-II: Dissociating organic compounds[END_REF]. Initially, the polluted liquid contained in a 40 L tank (figure 1) is first introduced into the heat exchanger, then to the ejector (figure 2). The temperature T of the liquid can be held at between 20°C and 35°C. The liquid flow rate L can be controlled between 0.5 and 1 m 3 /h. The two phases form an emulsion which is then fed into the mixture tube (height: 1.04 m; diameter: 20 mm), allowing a return of the liquid in the tank. The latter is surmounted by a cyclone used to separate the droplets of water from the gas. This gas is fed to the ozone analyzer and to the thermal destructor.

Ozone was produced from pure oxygen in an LABO LOX ozoniser provided by the Trailigaz company. This type of apparatus guarantees an average ozone production of The hydrodynamic properties of this contactor, presented on table 2, were determined in a previous study [START_REF] Chedeville | [END_REF]. It was shown that this ejector presents a high gas hold up and volumic mass transfer coefficient.

Analytical methods

HPLC

Phenolic compound concentration was analysed by means of high performance liquid chromatography with a UV detector (210 nm) and a hypersil C18 column (250 mm long, 4.6 mm internal diameter). Mobile phase was a mixture H2SO4 (4mM) : acetonitril / 80 : 20 at a rate of 1 mL/min.

Dissolved ozone

Dissolved ozone concentration was determined by using the carmin indigo method [START_REF] Bader | Determination of ozone in water by the indigo method[END_REF].

the attack of trisulfonate carmin indigo by O3 in an acid medium leads to discoloration of the solution which was monitored by spectrophotometry at 600 nm.

(spectrophotometer DR2100, HACH).

Total phenolic compounds concentration

Total phenolic compounds concentration was determined by using the Folin Ciocalteu method [START_REF] Singleton | Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents[END_REF]. 1 mL of sample, 500 µL of Folin Ciocalteu reagent, 5 mL of distilled water and 2 mL of 10 % sodium carbonate solution are introduced into a test tube. The mixture is shaken and allowed to stand for two hours in the dark at room temperature.

Absorbance was then measured by spectrophotometry at 760 nm (spectrophotometer DR2100, HACH).

Chemical oxygen demand

The chemical oxygen demand (COD) was measured by method 8000 of HACH with the spectrophotometer DR/2000. High rate constants were obtained at pH 5 (3.8x10 4 < kO3 / PC (L/mol s) < 2.9x10 5 ) showing that these phenolic compounds react strongly with ozone. These results are close to those obtained with other method. Beltran et al estimated the rate constant of ozonation of gallic acid between 0,97x10 5 L/mol s at pH 3 and 5,1x10 5 L/mol s at pH 7 [START_REF] Beltran | kinetics of competitive ozonation of some phenolic compounds present in wastewaters from food processing industries[END_REF]. These high rate constants associated with a high concentration of phenolic compound in margines induce a diffusion controlled regime [START_REF] Amat | Ozonisation coupled with biological degradation for treatment of phenolic pollutants: a mechanistically based study[END_REF]. Subsequently, an ejector, which presents adapted characteristics for this regime (high gas hold up and volumic mass transfer coefficient), should be used to transfer ozone into water during treatment of olive mill wastewater by ozonation.

Results and Discussion

Determination of ozonation rate constants

Ozonation of synthetic effluent

Operating conditions

Process efficiency was evaluated by performing treatment of synthetic effluent prepared by introducing 1,6 g of gallic, p-coumaric and p-hydroxybenzoic acid into 40 L of water. These concentrations were chosen in order to obtain a pollution load compatible with the ozoniser capacities and a diffusion controlled regime for the ozone mass transfer (Ha > 3). Indeed, in these experimental conditions, by using rate constant obtained, Ha number was estimated at 9.9 by using equation ( 6) (with kL = 4x10 -5 m/s and DO3 = 1,73x10 -9 m 2 /s).

This experiment was carried out at 20°C and the effluent was continuously recirculated at a flow rate of 0.9 m 3 /h. Initial pH was fixed at 5 by adding H2SO4. The gas flow rate was 0.3 Nm 3 /h and the ozone concentration in the inlet gas was CO3,i = 23 g/Nm 3 .

Samples were collected at suitable intervals of time. Phenolic compound concentrations were monitored by HPLC, dissolved ozone concentration was measured by the carmin indigo method and ozone mass transfer was determined by monitoring ozone concentration in the inlet and the outlet gas. The efficiency of the mass transfer is given by the ozone mass transfer ratio:

100 . ) ( , 3 , 3 , 3 3 i O o O i O O C C C r   (5)
Where CO3,I is the ozone concentration in the inlet gas (g/Nm 3 ) and CO3,o the ozone concentration in the outlet gas (g/Nm 3 ).

Results and discussion

Evolution of the phenolic compounds concentration and ozone mass transfer ratio are presented on figure 4. It appeared that gallic acid and p-coumaric acid were completely removed in 15 min, and p-hydroxybenzoic acid was removed in 20 minutes. During this step, ozone mass transfer was very high : it was up to 95 % in the first 10 minutes and decrease slowly to be up to 90 % after 20 minutes of treatment. Moreover, no dissolved ozone was detected. So, during this treatment, more than 90 % of produced ozone was transferred into water and consumed to remove phenolic compounds. These results show that ejector is a very efficient gas/liquid contactor in these conditions. High ozonation kinetics constants and phenolic compound concentrations induce a diffusion controlled regime (Ha > 3), and the ejector permit to obtain a very efficient ozone mass transfer which lead to fast an complete removal of pollutants. After 20 minutes of treatment, it appeared that ozone mass transfer is less efficient. It corresponds to ozonation of phenolic compounds byproducts which are less reactive with ozone and do not induce a diffusion controlled regime.

Ozonation of olive mill wastewater

Operating conditions

Olive mill wastewaters were obtained from Sfax (Tunisia). The main characteristics of this effluent were: pH = 5, COD = 6.4 g/L, insoluble material = 3 g/L. It is to be noted that this pollution load was lower than those usually reported in literature. In order to have a pollution load compatible with the ozoniser capacities, 6.5 L of this wastewater was mixed with 33.5 L of tap water before introduction in the ozonation pilot. The experiment was carried out at 20°C, with a liquid flow rate L = 0.9 L/h and a gas flow rate G = 0.3 Nm 3 /h. Ozone concentration in the inlet gas was CO3,i = 23 g/Nm 3 . Samples were collected at suitable intervals of time. Total phenolic compound concentration was analysed with the Folin Ciocalteu method. Ozone mass transfer efficiency (rO3) was determined by monitoring the ozone concentration in inlet and outlet gas. The Chemical Oxygen Demand (COD) of each sample was measured.

Results and discussion

The evolution of the total concentration of phenolic compounds (figure 5) shows that these compounds were eliminated at the beginning of the treatment. After 15 min, more than 65 % of the phenolic compounds were removed. Then, removal of these compounds became slower. After 40 min, an abatement of about 80 % was obtained.

Subsequently, no evolution in the total concentration of phenolic compounds was observed.

Evolution of the COD (figure 5) was similar: in the first 40 min, a significant removal of COD was obtained (decrease of about 48 % in COD after 40 min). After this initial period, the COD value remained constant. Other organic compounds present in margines and phenolic compounds byproducts could be responsive of this COD. Evolution of the ozone mass transfer ratio is presented figure 5. It can be seen that in the first 10 min, all the ozone introduced was transferred in into the liquid (rO3 = 100 %).

The lack of dissolved ozone in the liquid (determined by the carmin indigo method) means that all the ozone produced was used to oxidise the pollutants present in the effluent. From 10 to 40 min, rO3 decreased slowly. After 40 min, the ozone transfer ratio was between 50 % and 75 %. After this period, ozone transfer was less efficient, and after 90 min, rO3 was about 50 %, meaning that half of the ozone produced was not used for treatment.

Results show that ozonation is quite an efficient method to remove phenolic compounds from olive mill wastewaters. In spite of the presence of many other organic and inorganic compounds, phenolic compounds were significantly removed at the beginning of the treatment. In this study, abatement up to 80 % was obtained. Some phenolic compounds seem to be recalcitrant to oxidation, no further evolution being observed after 40 min. To remove them, it could be interesting to use an advanced oxidation process (O3/H2O2, O3/UV). However, advanced oxidation processes are less selective than ozonation, and many organic compounds, including non-toxic ones, would be removed, raising the amount of ozone needed to treat these wastewaters.

Using the ejector as gas / liquid contactor resulted in a very efficient transfer of ozone into water during the first 15 minutes (ozone mass transfer was up to 95 %). This phenomenon is due to the presence of phenolic compounds, which react strongly with ozone and induce a diffusion controlled regime. Subsequently, the decrease in ozone mass transfer efficiency is due to the removal of phenolic compounds. Results therefore show that in a very short contact time (mean residence time is about 1 second), this process can obtain very interesting mass transfer efficiencies since target compounds are present in solution. By modifying the ozoniser capacities, it should be possible to use this process in an open circuit configuration. Results show that in this configuration, the ejector could transfer all the produced ozone in water and hence remove a high rate of phenolic compounds in a very short time.

Conclusions

Phenolic compounds present in olive mill wastewaters react strongly with ozone. In a first step, the ozonation kinetics constant of gallic acid, hydroxybenzoic acid and coumaric acid were determined by using competition kinetic model. The high values of these constants (from 3.8x10 4 L/mol s to 2.9x10 5 L/mol s) and the high concentration of phenolic compounds induce a diffusion controlled regime. In a second time, ozonation of a synthetic effluent containing the phenolic compounds was performed by using an ejector as gas/liquid contactor. It appeared that in a diffusion controlled regime (Ha >3), the process was very efficient: more than 90 % of ozone produced was transferred into water and used to remove pollutants. So, phenolic compounds removal was fast and complete. Finally an ozonation process using an ejector as gas / liquid contactor was used to perform the treatment of olive mill wastewaters coming from Sfax (Tunisia).

Positive results were obtained:

-phenolic compounds present in wastewaters were removed at the beginning of the treatment, showing that other products present in this industrial matrix do not interfere with treatment; -abatement of phenolic compounds reached 80 %; -ozone mass transfer was very efficient since the removal of phenolic compounds: rO3 was up to 95 % during the first 15 min. This process could be an efficient pre-treatment enabling the effluent then to be fed to a classical wastewater treatment plant.

Figure 1

 1 Figure 1. ozonation pilot

is plotted on figure 3 .Figure 3 .

 33 Figure 3. Ozonation kinetics of phenolic compounds

Figure 4 .

 4 Figure 4. Evolution of phenolic compounds abatement and ozone mass transfer ratio (◊) gallic acid; (+) p-coumaric acid; (Δ) p-hydroxybenzoic acid; (○) rO3 T = 20°C ; G = 0,3 Nm 3 /h ; L = 0,9 m 3 /h ; CO3,i = 23 g/Nm 3

Figure 5 .

 5 Figure 5. Evolution of COD, total phenolic compounds removal and ozone mass transfer ratio -(Δ) [PC]; (+) COD; (○) rO3 T = 20°C ; G = 0,3 Nm 3 /h ; L = 0,9 m 3 /h ; CO3,i = 23 g/Nm 3

Table 2

 2

	. Hydrodynamic properties [16]
	0.8 < L (m 3 /h) < 1 ; 0.2 < G (Nm 3 /h) < 0.5
	Volumic mass transfer coefficient (m s -1 )	kL	4x10 -5
	Gas hold up	εG	0.21 < εG < 0.32
	Main residence time (s)	τ	0.75 < τ < 1.15

Table 3 .

 3 Ozonation rate constant of phenolic compounds -pH 5

	Phenolic compound	r 2	slope	kO3	/ PC (L/mol s)
	(PC)
	gallic acid	0.99	5.82		2.9 10 5
	p-hydroxybenzoic acid	0.99	0.76		3.8 10 4
	p-coumaric acid	0.99	0.96		4.8 10 4

The authors wish to thank Caroline Wisniewski (Sanofi Aventis), Marielle Coste, Florencio Martin (Anjou Recherche), Naouel Chetouane and ANVAR for supporting this investigation.


Professor Damian Miles Bailey
email: damian.bailey@southwales.ac.uk

Marcel Culcasi

Teresa Filipponi

Julien V Brugniaux

Benjamin Stacey

Christopher J Marley

Rodrigo Soria

Stefano F Rimoldi

David Cerny

Emrush Rexhaj

Lorenza Pratali

Carlos Salinas Salmòn

Carla Murillo Jáuregui

Mercedes Villena

Francisco Villafuerte

Antal Rockenbauer

Sylvia Pietri

Urs Scherrer

Claudio Sartori

PhD FPVRI FRSC Facsm Ftps

EPR spectroscopic evidence of iron-catalysed free radical formation in chronic mountain sickness: Dietary causes and vascular consequences

Keywords: chronic mountain sickness, free radicals, oxidative-nitrosative stress, systemic vascular function, oxidative catalysis Redox-regulation of systemic vascular function in hypoxia

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Chronic mountain sickness (CMS) is a high-altitude (HA) maladaptive syndrome that affects between 5-10% of the world's estimated 140 million highlanders who permanently reside >2,500 meters above sea-level [1]. It is characterised by excessive erythrocytosis (EE) and severe hypoxemia, frequently associated with moderate to severe pulmonary hypertension, that in advanced cases may evolve to cor pulmonale, congestive heart failure and stroke [START_REF] Penaloza | The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic mountain sickness[END_REF]. Currently, there are no effective long-term treatments given its complex pathogenesis.

It is well recognized that CMS associates with pulmonary vascular dysfunction [START_REF] Penaloza | The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic mountain sickness[END_REF], with emerging evidence indicating that the systemic circulation may be equally compromised, further compounding disease risk and premature mortality. In support, more marked impairments in flow-mediated dilation (FMD) and elevations in vascular stiffness and carotid intima-media thickness (cIMT) have been observed in comparatively more hypoxemic CMS patients (CMS+) compared to healthy welladapted highlander (CMS-) and lowlander (sea-level) controls [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF][START_REF] Rexhaj | Sleep-Disordered Breathing and Vascular Function in Patients With Chronic Mountain Sickness and Healthy High-Altitude Dwellers[END_REF][START_REF] Rimoldi | Systemic vascular dysfunction in patients with chronic mountain sickness[END_REF][START_REF] Tremblay | Global REACH 2018: High blood viscosity and hemoglobin concentration contribute to reduced flow-mediated dilation in high-altitude excessive erythrocytosis[END_REF].

While preliminary evidence supports a contributory role for an exaggerated and sustained elevation in basal systemic oxidative-nitrosative stress (OXNOS) subsequent to enhanced free radical-mediated scavenging of vascular nitric oxide (NO) bioavailability [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF][START_REF] Bailey | Exaggerated systemic oxidativeinflammatory-nitrosative stress in chronic mountain sickness is associated with cognitive decline and depression[END_REF], the contributing species, underlying mechanisms and functional significance remain obscure. Cellular hypoxia has consistently been shown to promote mitochondrial superoxide ( -) formation primarily from complex III in vitro [START_REF] Smith | Redox signaling during hypoxia in mammalian cells[END_REF], due to a Na + -mediated reduction in mitochondrial inner membrane fluidity resulting in the entrapment and accumulation of ubisemiquinone at the Qo site [START_REF] Hernansanz-Agustin | Na(+) controls hypoxic signalling by the mitochondrial respiratory chain[END_REF]. Subsequent formation of hydrogen peroxide (H 2 O 2 ) can diffuse rapidly into the cytoplasm, facilitated by a reduction in mitochondrial matrix and intermembrane space peroxidase activities [START_REF] Murphy | How mitochondria produce reactive oxygen species[END_REF] to initiate extracellular 'blood-borne' lipid peroxidation, that in the presence of 'free' ferrous (Fe 2+ ) iron (see below) can give rise to secondary lipid-derived alkoxyl (LO • ) and alkyl (LC • ) radicals [START_REF] Bailey | Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness?[END_REF]. Intravascular can also be generated from extramitochondrial sources including nicotinamide adenine dinucleotide phosphate-and xanthine oxidases, cytochrome P-450 enzymes, lipoxygenases, phagocytes and hydrogen ion formation subsequent to hypoxia-induced metabolic acidosis [START_REF] Bailey | Hypoxia compounds exercise-induced free radical formation in humans; partitioning contributions from the cerebral and femoral circulation[END_REF][START_REF] Munzel | Vascular consequences of endothelial nitric oxide synthase uncoupling for the activity and expression of the soluble guanylyl cyclase and the cGMP-dependent protein kinase[END_REF][START_REF] Hernansanz-Agustin | Acute hypoxia produces a superoxide burst in cells[END_REF].

These radical species are all thermodynamically capable of initiating additional chain reactions (L • LC • + L-H → L • ) [START_REF] Buettner | The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate[END_REF] and reacting directly with intravascular NO at diffusion-controlled rates (k = ~10 9 M/s) to yield peroxynitrite ( -L • LC • -) [START_REF] Nauser | The rate constant of the reaction of superoxide with nitrogen monoxide: approaching the diffusion limit[END_REF] that in turn can mediate lipid peroxidation/protein tyrosine nitrosylation and uncouple endothelial NO synthase [START_REF] Munzel | Vascular consequences of endothelial nitric oxide synthase uncoupling for the activity and expression of the soluble guanylyl cyclase and the cGMP-dependent protein kinase[END_REF].

However, to what extent -formation proceeds directly in vivo including the potential for reductive formation of Fe 2+ (Eq. 1) capable of catalysing Fenton (Eq. 2) and Haber-Weiss (Eq. 3)-induced generation of hydroxyl radicals (HO•) has not previously been investigated. Overall, these biological pathways predominantly reflect a transition metal catalysed reaction stemming from Fridovich's original -theory of O 2 toxicity, given that the Haber-Weiss reaction (Eq. 3) has a negligible rate constant [START_REF] Fridovich | The biology of oxygen radicals[END_REF].

Fe 3+ + -→ Fe 2+ (1)

Fe 2+ + H 2 O 2 → H • H -+ Fe 3+ (2) -+ H 2 O 2 → O 2 + H • H - (3)
Furthermore, to what extent dietary deficiency of antioxidant micronutrients due to inadequate consumption of fruit and vegetables (F+V) further compounds systemic OXNOS is equally unknown, despite preliminary evidence for lower circulating concentrations of ascorbate and α ß-carotene in CMS+ [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF].

To address these limitations, the present study combined electron paramagnetic resonance (EPR) spectroscopy with ozone-based chemiluminescence (OBC) for the direct detection and molecular characterisation of OXNOS biomarkers, to test the following hypotheses. First, Me-β-cyclodextrin (MeβCD)-assisted spin trapping with the novel stereoselective phosphorylated nitrone, 2-methyl-2-(5,5-dimethyl-2-oxo-1,3,2dioxaphosphinan-2-yl)-3,4-dihydro-2H-pyrrole 1-oxide (Me 2 CyDEPMPO), would demonstrate more marked elevations in systemic -and H • formation in CMS+ relative to healthy lowlander and CMS-controls specifically related to increased catalytic ('free') iron bioavailabilty. Second, this would be related to inadequate intake of antioxidant micronutrients as a consequence of lower F+V consumption. Third, exaggerated OXNOS would be associated with more marked impairments in systemic vascular function and structure extending prior observations [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF][START_REF] Bailey | Exaggerated systemic oxidativeinflammatory-nitrosative stress in chronic mountain sickness is associated with cognitive decline and depression[END_REF] given its more informed mechanistic basis.

METHODS

Ethics

The experimental protocol was approved by the Institutional Review Boards for Human Identifier: NCT01182792). All procedures adhered to the guidelines set forth in the Declaration of Helsinki and participants provided written informed consent following explanation of the purposes of clinical experimentation and risks involved.

Design

We conducted a cross-sectional, population-based, observational study in accordance with the STROBE statement [START_REF] Elm | The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: guidelines for reporting observational studies[END_REF]. The Neurovascular Research Laboratory in the UK (~80 m) and Instituto Boliviano de Biologia de Altura in La Paz, Bolivia (~3,600 m) were the primary sites of investigation. Select aspects of the cardiopulmonary data have previously been published as part of a wider investigation focused exclusively on the mechanisms underlying cerebrovascular dysfunction in CMS+ [START_REF] Bailey | Exaggerated systemic oxidativeinflammatory-nitrosative stress in chronic mountain sickness is associated with cognitive decline and depression[END_REF]. Thus, although the present study adopted an identical experimental design, it constitutes an entirely separate question with additional experimental measures of free radicals, catalytic iron, dietary intake and systemic vascular function and structure. Fig. 1 provides a schematic summary of the experimental design.

Participants

For all participants, inclusion criteria specified that they were born and had lived permanently at their resident altitude and were sedentary, defined as no formal recreational activity outside of everyday living [START_REF] Bailey | Elevated aerobic fitness sustained throughout the adult lifespan is associated with improved cerebral hemodynamics[END_REF]. Exclusion criteria included those with significant developmental delay or learning difficulties, diagnosis of any central neurological disease such as aneurysm, stroke, transient ischemic attack, epilepsy, multiple sclerosis and psychiatric disorders including any history of traumatic brain injury and hypertension. None of the participants were taking nutritional supplements including over-the-counter antioxidant or anti-inflammatory medications. We specifically chose to focus on males given their higher prevalence of CMS notwithstanding our inability to control for differences in circulating estrogen known to impact redox-regulation of systemic vascular function in females. Prior to inclusion, all participants were subject to an extensive clinical examination that consisted of a thorough medical history, chest auscultation and 12-lead ECG. Participants were subsequently familiarised with the equipment and all experimental procedures.

Highlanders

We recruited 10 male patients with primary CMS (CMS+) and 10 healthy age-and education-matched controls without CMS (CMS-) native to La Paz, Bolivia (Table 1).

Symptoms of CMS were scored and clinical diagnosis confirmed by an excessive erythrocytosis including hemoglobin (Hb) > 20 g/dL in the presence of normal pulmonary function and no history of working in the mining industry [1]. All participants identified themselves as Aymaras and were from similar socio-economic backgrounds having been born and bred in La Paz (Cerro de Pasco for secondary study) with Spanish spoken as their first language.

Lowlanders

We recruited 10 age and education matched healthy Caucasian males in the UK (Table 1) who were all born and bred close to sea-level with no prior history of ascending above 1,500 m.

Socioeconomic status (SES)

In addition to educational attainment (completed Secondary School/University) for the purposes of group matching, we also assessed occupational prestige using established methods that employed the 1989 modification of scores taken from the original 1980 US Census Occupational Category [START_REF] Fujishiro | What does "occupation" represent as an indicator of socioeconomic status?: Exploring occupational prestige and health[END_REF]. In brief, published scores (highest reflecting most prestigious occupation) were assigned to individual occupational titles classified under the following (nine) general categories: managerial and administrative, professional specialty, technical, sales, administrative support, service, farming/ forestry/fishing, precision production/craft/repair, and laborer.

Cardiopulmonary assessments

Metabolic assessments

Participants were asked to refrain from physical activity, caffeine and alcohol and maintain their normal dietary behaviors before and during formal experimentation. They were 12 h overnight fasted when they attended the laboratory at 8 am. We obtained blood samples without stasis following 20 min of seated rest to control for the confounds associated with plasma volume shifts.

Chemicals

All chemicals including dimethylsulfoxide (DMS ), MeβCD (CAS 128446-36-6), diethylenetriaminepentaacetic acid (DTPA), desferrioxamine mesylate (DFX), ferric chloride (FeCl 3 ), ferrous sulfate (FeSO 4 ), H 2 O 2 and the spin trap N-tert-butyl-αphenylnitrone (PBN) were of the highest available purity from Sigma-Aldrich. The spin trap Me 2 CyDEPMPO, a cyclic analogue of 5-diethoxyphosphoryl-5-methyl-1-pyrroline 1oxide (DEPMPO; see Fig. 1 inset), was synthesised according to [START_REF] Gosset | CyDEPMPOs: a class of stable cyclic DEPMPO derivatives with improved properties as mechanistic markers of stereoselective hydroxyl radical adduct formation in biological systems[END_REF].

Blood sampling

Blood was obtained from an indwelling cannula located in a cephalic vein into Vacutainers® (Becton, Dickinson and Company, Oxford, UK). Centrifugation was performed at 600g (4° C) for 10 min to yield hemolysis-free supernatant [START_REF] Bailey | Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness?[END_REF]. Plasma samples were decanted directly into cryogenic vials (Nalgene® Labware, Thermo Fisher Scientific Inc, Waltham, MA, USA) and immediately snap-frozen in liquid nitrogen (N 2 )

and shipped/stored under N 2 gas (Cryopak, Taylor-Wharton, Theodore, AL, USA) prior to analysis in the UK. Samples were left to defrost at 37 °C in the dark for 5 min prior to batch analysis.

Free radicals

X-band EPR spectra were recorded at room temperature using a Bruker (Karlsruhe, Germany) EMX spectrometer, except for studies with Me 2 CyDEPMPO where a Bruker ESP 300 spectrometer was used. Throughout, 100 kHz modulation frequency, 10 or 20 mW microwave power (MP) and a standard TM 110 cavity were used.

Preliminary in vitro EPR controls

In order to assist the assignments of Me 2 CyDEPMPO adducts detected ex vivo in the blood of participants (see Results section), in vitro tests were performed in the presence of an excess of MeβCD. This cyclodextrin (Fig. S1, inset; Supplementary data) was included in the spin-trapping milieu on the basis of the reported formation of cyclodextrin-guest inclusion complexes with DEPMPO spin adducts that yield increased stability toward bioreductive EPR silencing mechanisms in buffers [START_REF] Karoui | Spin trapping of superoxide in the presence of beta-cyclodextrins[END_REF] and cells [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF]. In addition, these preliminary experiments, using a Fenton driven

Ex-vivo EPR studies in blood

Three experiments were performed for the molecular detection and characterisation of free radicals in blood.

[i] MeβCD-assisted spin-trapping on Me 2 CyDEPMPO: Heparinised whole blood samples (1 mL) were obtained from all participants and placed into 2 mL cryovial tubes pre-filled with a mixture of (final concentration) 0.2 mM DTPA, 80 mM MeβCD, 40 mM PBS and 0.5 mL of aqueous Me 2 CyDEPMPO (0.1 M), and immediately frozen in liquid N 2 . EPR acquisition in glass capillaries was initiated 1 min after complete thawing with the same parameters as for preliminary tests above, except that 2 cumulative scans were averaged due to low signal-to-noise ratio. In both in vitro and ex vivo experiments using Me 2 CyDEPMPO, relative spin adduct concentrations were derived from the simulated spectra [START_REF] Rockenbauer | Automatic computer simulation of ESR spectra[END_REF] and the unequivocal assignment of trans/cis-Me 2 CyDEPMPO-OH and trans-Me 2 CyDEPMPO-Me adducts (see Fig. 2A, with R = Me) was based on the close similarity (< 5%) of their calculated EPR hyperfine couplings (hfcs) with those reported earlier using the same free radical generators in buffer [START_REF] Gosset | CyDEPMPOs: a class of stable cyclic DEPMPO derivatives with improved properties as mechanistic markers of stereoselective hydroxyl radical adduct formation in biological systems[END_REF].

PBN spin trapping and catalytic iron experiments were conducted in a random subsample of 5 CMS+ patients to provide additional 'proof-of-concept' insight into the mechanisms underlying Me 2 CyDEPMPO-OH adduct(s) formation (see Limitations).

[ii] Spin-trapping using PBN: Whole blood (4. [iii] Catalytic iron: Additional EPR measurements were performed on the same blood samples as in (ii). The basic principle of the assay, which relies on relative changes in the EPR signal intensity of the persistent ascorbate radical A •-(yielding an EPR doublet with a H ~1.76 G; Fig. 3) during air auto-oxidation of ascorbate, has been used for probing catalytic iron ex vivo [START_REF] Buettner | The catalytic activity of iron in synovial fluid as monitored by the ascorbate free radical[END_REF]. Briefly, separate vials of serum (200 µL) were treated with 500 µM (final concentration) of DFX (reduces iron-catalysed oxidation) and 10 µM of FeCl 3 -EDTA (stimulates iron catalysed oxidation). Instrument settings were: MP, 20 mW; R, 4096 points; MA, 2.0 G; RG, 1.0  10 5 ; TC, 41 ms; SW, 10 G, for 15 cumulative scans. Spectra were filtered identically using Bruker WinEPR version 2.11 software and simulated using Bruker SimFonia or SimEPR32 (Bruker Biospin) [START_REF] Adamski | Application of EPR spectroscopy for elucidation of vanadium speciation in VOx/ZrO 2 catalysts subject to redox treatment[END_REF]. Relative free radical concentrations were calculated by double integration of simulations using Origin 8 software (OriginLab Corp., Northampton, MA, USA). We also chose to 'normalise' A •- formation for differences in ambient ascorbate concentrations. Following deproteination with 10% metaphosphoric acid, ascorbate was assayed via fluorimetry based on the condensation of dehydroascorbic acid with 1,2-phenylenediamine [START_REF] Vuilleumier | Fluorimetric assay of vitamin C in biological materials using a centrifugal analyser with fluorescence attachment[END_REF].

Protein carbonyls (PC)

Plasma was diluted with 4 vol of PBS 0.1X prior to analysis and 100 μL placed in 96well microplates and left to react for 15 min at 21ºC with an aliquot (100 μL) of the NBDH assay solution (Carbofax®, Yelen Analytics, Marseille, France) [START_REF] Stocker | A fluorescent homogeneous assay for myeloperoxidase measurement in biological samples. A positive correlation between myeloperoxidase-generated HOCl level and oxidative status in STZ-diabetic rats[END_REF].

Concentration of NBDH-reacted PC was determined by fluorescence (emission determined at 560 nm using an excitation wavelength at 480 nm) with reduced/oxidized bovine serum albumin employed as calibrants. The concentration of PC was calculated relative to total protein content, the latter determined using the Pierce BCA protein assay kit (Fisher Scientific, France). Intra-and inter-assay CVs were <10%.

NO metabolites

Plasma NO metabolites were measured via reductive ozone-based chemiluminescence (Sievers NOA 280i, Analytix Ltd, Durham, UK) [START_REF] Bailey | Nitrite and S-Nitrosohemoglobin exchange across the human cerebral and femoral circulation: relationship to basal and exercise blood flow responses to hypoxia[END_REF]. Plasma (200 µL) was injected into tri-iodide reagent for the combined measurement of nitrite and S-Nitrosothiols ( -+ RSNO). A separate sample (400 µL) was mixed with 5% acidified sulphanilamide (9:1)

and left to incubate in the dark at 21 ºC for 15 min to remove NO Analysis package was used to calculate the area under the curve and subsequently converted to a concentration using standard curves of sodium . Intra-and interassay CVs for all metabolites were <10%.

Iron metabolites

Iron metabolism was assessed in a separate group of age-matched lowlanders/highlanders given ethical constraints imposed during blood volume sampling in the primary study. Serum iron and transferrin were measured using a colorimetric and immunoturbidimetric assay respectively via the C501 module on a Cobas 6000 analyzer (Roche) [START_REF] Gibson | Principles of nutritional assessment[END_REF]. Serum ferritin was determined using an electrochemiluminescence immunoassay using a Cobas e601 module (Roche). Assuming that 1 mol of transferrin (molecular mass 79,570 Da) has the capacity to bind two atoms of iron (atomic mass 56), transferrin saturation (TSAT) was calculated as:

TSAT (%) = × 70.9 [START_REF] Kasvosve | Total iron binding capacity and transferrin concentration in the assessment of iron status[END_REF] Total iron binding capacity (TIBC) was calculated as: TIBC = (μg dL) = Transferrin (mg/dL) × 1.41 [START_REF] Kasvosve | Total iron binding capacity and transferrin concentration in the assessment of iron status[END_REF]

Dietary analysis

Nutritional intake was assessed using the 24 h dietary recall (24-HDR) method conducted on two separate days [START_REF] Gibson | Principles of nutritional assessment[END_REF]. The 24-HDR is considered the least biased structured self-report instrument that is the most accurate for low income populations [START_REF] Gibson | Measurement Errors in Dietary Assessment Using Self-Reported 24-Hour Recalls in Low-Income Countries and Strategies for Their Prevention[END_REF][START_REF] Thompson | Dietary Assessment Methodology[END_REF]. Participants were asked to provide detailed information about the food and beverages consumed including preparation methods, ingredients used, amount consumed, leftover, brand name of commercial products, time of the day and type of meal. Standard household measuring tools, food models, photos and other visual aids were also employed to help judge and estimate portion size in order to improve accuracy. Dietary data were analyzed using NetWISP dietary analysis software (Version 4.0, Tinuviel Software; Anglesey, UK).

Vascular assessments

All resting measurements were performed following 10 min of supine rest, breathing room air at each respective location at the prevailing barometric pressures (normoxic normocapnia for lowlanders, hypoxic hypocapnia for highlanders).

Systemic arterial function

Systemic conduit artery endothelial function was assessed by determining the increase of the brachial artery (BA) diameter evoked during reactive hyperemia using highresolution ultrasound and automatic wall tracking software [START_REF] Jayet | Pulmonary and systemic vascular dysfunction in young offspring of mothers with preeclampsia[END_REF] according to

International Guidelines [START_REF] Deanfield | Endothelial function and dysfunction. Part I: Methodological issues for assessment in the different vascular beds: a statement by the Working Group on Endothelin and Endothelial Factors of the European Society of Hypertension[END_REF]. Briefly, the BA was identified approximately 5 cm above the antecubital fossa with high-resolution duplex ultrasound (Acuson Sequoia C512, Acuson Siemens, Mountain View, CA, USA or Esaote MyLab30 Gold, Genova, Italy) and a high frequency (7-10 MHz) linear array probe. The ultrasound probe was fixed in a stereotactic clamp (AMC Vascular Imaging, Amsterdam) and flow recorded continuously throughout. After 1 min of baseline measurements, a pressure cuff placed around the forearm was inflated to 250 mmHg for 5 min. Following cuff-deflation, the hyperemia-induced changes in BA diameter and flow were continuously monitored for 3 min. B-mode ultrasound images were analysed with a validated system for automatic real-time measurement of BA diameter (FMD Studio, Computer Vision Group, Pisa, Italy) [START_REF] Gemignani | A system for real-time measurement of the brachial artery diameter in B-mode ultrasound images[END_REF]. Flow-mediated dilation (FMD) was expressed as the maximal percentage change in BA diameter from baseline and allometrically scaled (adjusted FMD) to account for differences in baseline BA diameter [START_REF] Atkinson | The percentage flow-mediated dilation index: a largesample investigation of its appropriateness, potential for bias and causal nexus in vascular medicine[END_REF]. Endothelium-independent dilation was assessed by measuring the percentage increase in BA diameter to an acute oral dose ( 50 μg) of glyceryl trinitrate (GT ; UCB-Pharma SA). The intra-observer CV was <5%.

Systemic arterial stiffness

In accordance with International Guidelines [START_REF] Laurent | Expert consensus document on arterial stiffness: methodological issues and clinical applications[END_REF], large artery stiffness was assessed non-invasively via the carotid-femoral pulse wave velocity (PWV) using the Complior® device (Artech Medical, Pantin, France) [START_REF] Laurent | Expert consensus document on arterial stiffness: methodological issues and clinical applications[END_REF][START_REF] Asmar | Assessment of arterial distensibility by automatic pulse wave velocity measurement. Validation and clinical application studies[END_REF]. Briefly, common carotid and femoral arterial pressure waveforms were recorded simultaneously with mechano-transducers.

The mean wave transit time for 10 heart beats was calculated by the system software using the foot-to-foot method. To determine aortic PWV (aPWV), the surface distance between the recording sites was determined with a flexible tape measure. Central pulse wave analysis was performed non-invasively using the SphygmoCor ® system (AtCor Medical Pty Ltd, Sydney, Australia). The radial artery pressure waveform was recorded at the wrist using applanation tonometry with a high-fidelity micromanometer (SPT-301, Millar Instruments, Houston, TX, USA) and the central (ascending aortic) pressure waveform derived using a validated transfer function [START_REF] O'rourke | Arterial stiffness, systolic blood pressure, and logical treatment of arterial hypertension[END_REF]. AIx was calculated as the difference between the 1 st and 2 nd peaks of the central arterial waveform, expressed as a percentage of the pulse pressure (PP) and normalised to 75 b/min heart rate (AIx-75) [START_REF] Wilkinson | The influence of heart rate on augmentation index and central arterial pressure in humans[END_REF]. The intra-observer CV for all metrics was <5%.

Systemic arterial structure

Duplex ultrasound scanning of the right and left common carotid arteries (CCA), carotid bifurcations and the origin of the internal carotid arteries was performed in conjunction with simultaneous ECG acquisition in participants placed supine with slight hyperextension of the neck at 45º. The right and left carotid arteries were imaged in the anterolateral, posterolateral and mediolateral planes, 1-2 cm proximal to the carotid bulb to identify the optimal angle of incidence [START_REF] Stein | Use of carotid ultrasound to identify subclinical vascular disease and evaluate cardiovascular disease risk: a consensus statement from the American Society of Echocardiography Carotid Intima-Media Thickness Task Force. Endorsed by the Society for Vascular Medicine[END_REF][START_REF] Touboul | Mannheim carotid intima-media thickness consensus[END_REF]. Radiofrequency signals with a 21 µm resolution (RF QIMT, Esaote, Genova, Italy) were employed to measure cIMT. After scanning the vessel, each radiofrequency line was automatically analysed forward and backward in real time by the echo-device at a site free of any discrete plaque. The mean of 3 measures from the right and 3 from the left carotid artery was documented.

The intra-observer CV was <5%.

Statistical analysis

Power calculation

Prospective power calculations were performed using G* Power 3.1 software. Given the lack of pilot data in Me 2 CyDEPMPO-adducts in CMS+/CMS-, we focused on baseline differences in (absolute) FMD and aPWV based on our prior research. Assuming comparable differences and corresponding effect sizes (η = 0.51 for FMD and 0.55 for aPWV), we required a total sample size ranging from 12-15 participants (4-5 per group) to achieve a power of 0.80 at P < 0.05. We chose to further inflate to a total sample size of 30 (10 per group) to accommodate potential data loss (prospective power of >0.95).

Inferential statistics

Data were analysed using using the Statistics Package for Social Scientists (IBM SPSS Statistics V 28.0). Shapiro-Wilk W tests (P > 0.05) confirmed that all data sets were normally distributed. Demographic (Table 1), dietary (Table 2), and metabolic (Table 3)

data were analysed using one-way analyses of variance (ANOVA) and post-hoc Bonferonni-adjusted independent samples t-tests assuming a main effect was observed.

Relationships between variables were assessed using Pearson Product Moment Correlations. Significance was established at P < 0.05 for all two-tailed tests (with individual P values for all comparisons shown) and data expressed as mean ± standard deviation (SD).

RESULTS

Demographics

By design, groups were of a similar age and educational attainment, whereas highlanders, in particular CMS+, were more hypoxemic, hypocapnic and erythrocytotic (Table 1). Despite comparable body mass, CMS+ were generally shorter with a corresponding higher BMI and waist-to-hip ratio (Table 1). Resting HR, SV and were generally elevated and PET O2 , PET CO2 , DBP and TPR were lower in the highlanders (Table 1). No between differences were observed in any of the SES metrics including occupational prestige scores (Table 1).

Metabolic assessments

In vitro studies (quality control)

MeβCD does not affect Me 2 CyDEPMPO spin adduct formation in vitro

Trapping of Fenton-derived HO• on Me 2 CyDEPMPO (30 mM) in plain PBS yielded a primary symmetrical 14-lined EPR spectrum with calculated hfcs consistent with a mixture of trans-Me S1; Supplementary data). This implies a general lack of coordination between MeβCD and these spin adducts that would typically yield distorted EPR signals characteristic of the superimposition of free and complexed radicals, as reported for the DEPMPO/superoxide (-OOH) adduct [START_REF] Bailey | Elevated aerobic fitness sustained throughout the adult lifespan is associated with improved cerebral hemodynamics[END_REF]. However, a near two-fold decrease in the total EPR signal was observed when MeβCD was present before vs. after Fentoninduced Me 2 CyDEPMPO-OH formation, reflecting competitive 'side attack' of HO• on the MeβCD backbone forming non-spin-trapped radicals (Fig. S1; Supplementary data).

Accordingly, pre-inclusion of MeβCD in a [Fenton + DMSO] system also resulted in a loss of intensity for the expected trans-Me 2 CyDEPMPO-Me signal [START_REF] Fridovich | The biology of oxygen radicals[END_REF], with formation of a strong unassigned alkyl adduct that may have originated from the MeβCD structure (Fig. S1D; Supplementary data).

Stability of Me 2 CyDEPMPO adducts and trans-OH/cis-OH ratios

All spin adduct concentrations decreased progressively with time (up to [START_REF] Buettner | The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate[END_REF] Fenton reagent [START_REF] Gosset | CyDEPMPOs: a class of stable cyclic DEPMPO derivatives with improved properties as mechanistic markers of stereoselective hydroxyl radical adduct formation in biological systems[END_REF], except when CD was added before the Fenton reaction, where the ratios were considerably higher (Fig. S2C; Supplementary data).

Ex vivo studies (clinical experimentation)

Me 2 CyDEPMPO spin-trapping in the presence of MeβCD

Unlike lowlanders (no adducts detected), highlander blood was paramagnetic and consistently exhibited either a single component consistent with a Me 2 CyDEPMPO-alkyl adduct (i.e., having a H ~20 G; all CMS-, all CMS+) or combination of cis/trans-Me 2 CyDEPMPO-OH and -alkyl adducts (one CMS-, all CMS+). In combination with the in vitro controls (above), all EPR spectra in blood were satisfactorily simulated assuming that no penetration of spin adducts in the inner cavity of CD occurred. Representative EPR spectra obtained from a (single) lowlander, CMS-participant and CMS+ patient with simulated composite adducts and relative intensities are illustrated in Fig. 2B. The group signal intensities of total and composite Me 2 CyDEPMPO-OH adducts (no difference in -alkyl adducts) were consistently higher in CMS+ (Fig. 2C) representing 8 ± 25% (CMS-) vs. 45 ± 18% (CMS+, P = 0.001) for Me 2 CyDEPMPO-OH and 92 ± 25% (CMS-) vs. 55 ± 18% (CMS+, P = 0.001) Me 2 CyDEPMPO-alkyl adducts respectively, of the total signal intensities recorded. The trans:cis ratios for CMS-was 1.427 AU (n = 1) and 1.741 ± 0.266 (1.151-2.125) AU for CMS+.

PC

Baseline PC were higher in CMS+ compared to CMS-(Fig. 2D).

Catalytic iron

Compared to control (untreated) samples, DFX decreased A •-by 30 ± 12% (P = 0.029) whereas Fe(III)-EDTA increased A •-by 125 ± 17% (P = 0.002). Differences persisted following correction for ambient ascorbate (Fig. 3 A,B).

PBN spin-trapping

We observed EPR sextets from toluene-extracted blood samples with a primary signal that was consistent with an alkoxyl adduct (PBN-OR: a N = 13.59 G; a H β = 1.85 G, Fig. 3 C, D).

NO metabolites

Total bioactive NO was consistently suppressed in highlanders due to lower whereas no differences were observed in RSNO (Fig. 4A). Both -and RSNO were comparable between CMS+ and CMS-(Fig. 4A).

Iron metabolism (sub-study)

No between group differences were observed in total iron, ferritin, transferrin, TSAT or TIBC (Table S2; Supplementary data).

Dietary intake

Caloric and macronutrient (protein, carbohydrate, fat and fiber) intake were comparable across groups and generally consistent with current recommendations (Table 2). In contrast, F+V consumption was markedly lower in highlanders resulting in a reduced intake of vitamins C and E, carotene, magnesium and retinol compared to lowlanders (Table 2). The lower vegetable and vitamin C intake was more pronounced in CMS+ compared to CMS-(Table 2).

Vascular assessments

Function

Baseline BA diameters were similar across groups (Table 3). Absolute and adjusted FMD were consistently lower in highlanders, although no differences were observed between CMS+ and CMS-(Table 3). In contrast, GTN-induced dilation was lower in CMS+ compared to CMS-(Table 3), although there was a tendency for baseline BA diameter to be higher (4.69 ± 0.55 mm vs. 4.27 ± 0.32 mm, P = 0.086).

Stiffness

Central SBP was consistenly higher and central DBP lower in highlanders, whereas no differences were observed between CMS+ and CMS-(Table 3). In contrast, aPWV was elevated in highlanders and greater in CMS+ compared to CMS-(Table 3). No between group differences were observed in AIx-75 (Table 3).

Structure

Elevated cIMT was observed in CMS+ compared to CMS-(Table 3).

Correlational analyses

Me 2 CyDEPMPO adducts, NO

NO  2 and vascular function

In all participants (pooled Lowlanders, CMS-and CMS+), a positive relationship was observed between -and absolute FMD (Fig. 5 D) with inverse relationships between -and aPWV (Fig. 5 E) and AIx-75 (Fig. 5 F).

DISCUSSION

The present study has identified three important findings that extend our understanding of redox-regulated mechanisms underlying lifelong mal/adaptation to HA. First, basal systemic OXNOS was permanently elevated in highlanders and further exaggerated in CMS+. This was reflected by elevated global protein carbonylation implying increased oxidation and H • formation subsequent to liberation of 'free' catalytic iron consistent with a Fenton and/or NA mechanism(s), coupled with lower -. Second, this was accompanied by inadequate intake of select antioxidant micronutrients due to lower F+V consumption. Third, we confirm that systemic vascular function and structure were collectively more impaired in CMS+ in direct proportion to the systemic OXNOS response. Collectively, these findings are the first to suggest that lifelong exposure to iron-catalysed systemic OXNOS, compounded by a dietary deficiency of antioxidant micronutrients, likely contributes to the systemic vascular complications and increased morbidity/mortality in CMS+.

Systemic OXNOS and iron catalysis

We specifically employed EPR spin-trapping with Me 2 CyDEPMPO to optimise detection of blood-borne -and H • given short half-lives and established roles in regulating vascular endothelial function either directly or through secondary formation of reactive oxygen/nitrogen species [START_REF] Cobley | 13 reasons why the brain is susceptible to oxidative stress[END_REF]. We also chose to include an excess of MeβCD in the trapping milieu to further improve adduct stability by forming inclusion complexes that protect against ascorbate, α-tocopherol and glutathione-induced bioreduction to EPR 'silent' hydroxylamines [START_REF] Karoui | Spin trapping of superoxide in the presence of beta-cyclodextrins[END_REF][START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF] (Forrester-Hepburn mechanism) that can be released during systemic OXNOS and subsequently reduced to Fe 2+ by -(Eq. 1) [START_REF] Gosset | CyDEPMPOs: a class of stable cyclic DEPMPO derivatives with improved properties as mechanistic markers of stereoselective hydroxyl radical adduct formation in biological systems[END_REF][START_REF] Fréjaville | 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals[END_REF][START_REF] Culcasi | The line asymmetry of electron spin resonance spectra as a tool to determine the cis:trans ratio for spintrapping adducts of chiral pyrrolines N-oxides: the mechanism of formation of hydroxyl radical adducts of EMPO, DEPMPO, and DIPPMPO in the ischemic-reperfused rat liver[END_REF].

The trans:cis ratios obtained in CMS+ patients were more consistent with a Fenton and/or NA mechanism (typical ratios of ~1.4 AU observed in vitro), since appreciably higher ratios (~3.0 AU) arise during GSH + GPx-induced reduction of primary OOH [START_REF] Gosset | CyDEPMPOs: a class of stable cyclic DEPMPO derivatives with improved properties as mechanistic markers of stereoselective hydroxyl radical adduct formation in biological systems[END_REF].

While this may argue against -formation, it is conceivable that OOH-adducts may have initially formed, yet were rapidly reduced to OH-adducts before entry into the MeβCD inner cavity requisite for EPR detection. This is plausible given that the oxidative stress cascade is typically initiated by -with HO• formed as a secondary species.

Detection of A •-, albeit in a random (sub) sample of CMS+ patients, suggests that 'free' iron was bioavailable with a catalytic efficiency comparable to Fe(III)-EDTA, lending further support to the proposed Fenton and/or NA mechanisms. This was complimented by detection of PBN-adducts assigned to secondary •OR formed downstream of Fe 2+catalysed reductive decomposition of lipid hydroperoxides (LOOH) since equivalent hfcs have been observed in vitro following (Fe 2+ -catalysed) auto-oxidation of cumene-OOH [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF]. That the minor signal ascribed to Me 2 CyDEPMPO-alkyl adducts were likely artefacts originating from the MeβCD structure potentially explains why further elevation was not observed in CMS+. In contrast, carbonylation was selectively elevated indicating that proteins are more vulnerable to OXNOS-mediated oxidative damage to amino acid side chains and protein backbone [START_REF] Stadtman | Free radical-mediated oxidation of free amino acids and amino acid residues in proteins[END_REF].

The complimentary examination of iron metabolites indicated that free iron in CMS+ did not arise as a result of iron exceeding transferrin binding capacity since TSAT, as indeed all associated metrics, were normal and comparable to healthy lowlander/CMSparticipants. Intravascular hemolysis caused by mechanical stress associated with increased intrinsic blood viscosity [START_REF] Tremblay | Global REACH 2018: High blood viscosity and hemoglobin concentration contribute to reduced flow-mediated dilation in high-altitude excessive erythrocytosis[END_REF] and/or oxidative damage to the RBC membrane [START_REF] Luneva | Deoxygenation Affects Composition of Membrane-Bound Proteins in Human Erythrocytes[END_REF] is a plausible alternative. In support, a recent study documented an inverse relationship between free plasma haptoglobin and hematocrit in CMS+, suggesting that these patients are potentially exposed to lifelong hemolysis [START_REF] Macarlupu | Sub-maximal aerobic exercise training reduces haematocrit and ameliorates symptoms in Andean highlanders with chronic mountain sickness[END_REF].

Dietary antioxidants

Only one study has previously estimated dietary intake in CMS+, albeit using a nonvalidated questionnaire limited to vitamins A, C and E. Despite indirect evidence for elevated lipid peroxidation in (Andean) CMS+, vitamin intake was comparable to healthy lowlander and CMS-controls [START_REF] Jefferson | Increased oxidative stress following acute and chronic high altitude exposure[END_REF]. In contrast, our validated, more comprehensive approach identified that intake of select antioxidant micronutrients including vitamins C and E, carotene, magnesium and retinol was generally lower in highlanders due to reduced F+V consumption.

Although the traditional Bolivian diet is predominantly based on foods of plant origin including potatoes and cereals, F+V have been subject to frequent price increases [START_REF] Pérez-Cueto | Monitoring food and nutrient availability in a nationally representative sample of Bolivian households[END_REF] that may have contributed to the lower consumption observed. However, why intake was further supressed in CMS+ remains unclear, especially in light of comparable SES.

Indeed, vegetable and vitamin C intake were especially inadequate, falling below the minimum amount advised by UK [START_REF] Health | Dietary reference values for food and nutrients for the United Kingdom. Report of the Panel on Dietary Reference Values of the Committee on Medical Aspects of Food Policy[END_REF] and Bolivian [START_REF] Bolivia | Ministerio de Salud. Bases técnicas de las guías alimentarias para la población Boliviana[END_REF] Governments.

ur findings disagree with the latest Bolivian households' survey data [START_REF] Pérez-Cueto | Monitoring food and nutrient availability in a nationally representative sample of Bolivian households[END_REF] and first comprehensive review of dietary intake in Andean countries, indicating that vitamin C intake is generally sufficient in this population [START_REF] Berti | A systematic review of the nutritional adequacy of the diet in the Central Andes[END_REF]. Inadequate intake may thus contribute, at least in part, to the lower systemic concentration of ascorbate (in combination with α-and β-carotene) previously documented in CMS+ [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF]. This may further enhance vulnerability to OXNOS-mediated impairments in systemic vascular endothelial function and structure (see 4.3) given that ascorbate is the primary watersoluble chain-breaking antioxidant required for targeted 'repair' of -, HO•, alkyl, peroxyl, • R and tocopheroxyl radicals during chain propagation, the latter synergising regeneration of α-tocopherol, the primary fat-soluble chain-breaking antioxidant [START_REF] Sharma | Interaction of vitamin C and vitamin E during free radical stress in plasma: an ESR study[END_REF].

Equally, lower dietary intake of nitrate, primarily sourced through green, leafy vegetables with bioactivity (likely) further reduced due to overcooking [START_REF] Ahluwalia | Dietary Nitrate and the Epidemiology of Cardiovascular Disease: Report From a National Heart, Lung, and Blood Institute Workshop[END_REF] when added to traditional Bolivian 'sopas' (soups), may have also contributed to the lower plasma -observed (4.2.). Clinically, inadequate F+V compounds OXNOS and increases corresponding risk of cardiovascular disease (CVD) and stroke [START_REF] Wang | Fruit and Vegetable Intake and Mortality: Results From 2 Prospective Cohort Studies of US Men and Women and a Meta-Analysis of 26 Cohort Studies[END_REF], the primary causes of mortality in CMS+ patients who exhibit >3.6 fold higher CVD risk relative to healthy CMS- [START_REF] Corante | Excessive Erythrocytosis and Cardiovascular Risk in Andean Highlanders[END_REF]. It has recently been identified that ≈5 daily servings daily of F+V confers the lowest risk of total and cause-specific mortality (albeit in lowlanders), with 2 (daily) servings for fruit and 3 for vegetables identified as the minimum required for risk reduction [START_REF] Wang | Fruit and Vegetable Intake and Mortality: Results From 2 Prospective Cohort Studies of US Men and Women and a Meta-Analysis of 26 Cohort Studies[END_REF].

Clearly, the dietary behavior of CMS+ patients in the present study places them at higher CVD risk yet provides an opportunity for therapeutic intervention (pharmacological and/or dietary) in a bid to bolster endogenous antoxidant defences.

Our findings provide a mechanistic basis justifying implementation of a prospective randomised trial to assess the impact of dietary manipulation on CMS+ given that OXNOS and corresponding hemodynamic risk factors may be at least, partially reversible. This may take the form of a randomised, controlled parallel-group community trial with CMS+ patients randomly assigned to either an interventional (encouraged to consume 5 daily servings of F+V consistent with aforementioned guidelines [START_REF] Wang | Fruit and Vegetable Intake and Mortality: Results From 2 Prospective Cohort Studies of US Men and Women and a Meta-Analysis of 26 Cohort Studies[END_REF], also incorporating transtheoretically-determined readiness for change combined with educational activities and problematising-dialogic pedagogy [START_REF] Menezes | Promoting fruit and vegetable consumption: Methodological protocol of a randomized controlled community trial[END_REF]) or control (maintenance of habitual dietary intake) group. We recommend a trial duration of 6 months given that this approach has been shown to augment circulating water and lipidphase antioxidants (α-carotene, β-carotene, lutein, β-cryptoxanthin and ascorbic acid)

and improve corresponding vascular funtion (reduction in systolic/diastolic blood pressure) [START_REF] John | Vegetable Study, Effects of fruit and vegetable consumption on plasma antioxidant concentrations and blood pressure: a randomised controlled trial[END_REF].

Systemic vascular function and structure

In addition to direct scavenging of endothelial NO via diffusion-limited reactions with -, RO • and RC • [START_REF] Nauser | The rate constant of the reaction of superoxide with nitrogen monoxide: approaching the diffusion limit[END_REF], hemolysis-induced extravasation of cell-free Hb into plasma can further compound elimination via additional Fenton, NO dioxygenation [Hb-Fe 2+ (O 2 ) + NO → Hb-Fe 3+ OONO • / -) and iron nitrosylation of deoxy-Hb [Hb-Fe 2+ → Hb(NO)] reactions [START_REF] Kato | Intravascular hemolysis and the pathophysiology of sickle cell disease[END_REF]. These outcompete reaction with intra-erythrocytic Hb by ~1000fold [START_REF] Reiter | Cell-free hemoglobin limits nitric oxide bioavailability in sicklecell disease[END_REF], reducing rates of S-nitrosothiol formation and/or deoxyHb-mediated reduction of -to NO, stable (i.e. RBC-encapsulated) metabolites that conserve/transfer bioactivity within the hypoxic microcirculation. Indeed, hemolysis-induced NO scavenging and corresponding impairments in vascular endothelial function are common to (other) diseases characterised by increased RBC mass, including polycythemia vera and Chuvash polycythemia [START_REF] Gladwin | The role of red blood cells and hemoglobin-nitric oxide interactions on blood flow[END_REF].

In the present study, plasma -was generally lower in highlanders' blood and consistently associated with reduced systemic vascular endothelial function (FMD), elevated arterial stiffness (aPWV/AIx-75) and morphological impairment (elevated cIMT) confirming our prior observations [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF][START_REF] Rimoldi | Systemic vascular dysfunction in patients with chronic mountain sickness[END_REF]. However, that plasma -was not further reduced in CMS+ despite more pronounced impairments in systemic vascular function and structure, suggests that vasculopathic complications cannot solely be ascribed to disordered NO metabolism (notwithstanding interpretive challenges associated with a small sample size, see 4.4. Experimental limitations). In support and contrary to prior observations [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF][START_REF] Rimoldi | Systemic vascular dysfunction in patients with chronic mountain sickness[END_REF], GTN-induced dilation was also selectively attenuated in CMS+ albeit complicated by a tendency towards an elevated baseline diameter (arguably constraining further vasodilation), tentatively implying additional impairments in smooth muscle function and/or structure previously ascribed to direct vasculotoxic/constrictive effects of hypoxia-induced -formation [START_REF] Lewis | Conduit artery structure and function in lowlanders and native highlanders: relationships with oxidative stress and role of sympathoexcitation[END_REF].

Intriguingly, aPWV widely considered the gold standard metric of aortic stiffness and independent predictor of coronary heart disease and stroke [START_REF] Mitchell | Arterial stiffness and cardiovascular events: the Framingham Heart Study[END_REF], provided the most robust differences and associations distinguishing vascular impairment in CMS+. While we cannot discount (aforementioned) statistical idiosyncrasies associated with a small sample size combined with differences in biomarker sensitivity/specificity, our findings tentatively suggest that elevated OXNOS may have selectively targeted (impaired) aortic stiffness over vascular endothelial function. Although speculative, this may be due to the fact that aortic stiffness is largely governed by structural degeneration of the scaffolding proteins, collagen and elastin, that are especially (if not perhaps more) vulnerable to mitochondrial ROS [START_REF] Zhou | Mitochondrial oxidative stress in aortic stiffening with age: the role of smooth muscle cell function[END_REF]. Figure 6 provides an overall schematic summarising the proposed molecular pathways underlying systemic OXINOS and corresponding link(s) to vasculopathic complications of CMS (including novel contributions in the present study), extending our previously published findings [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF][START_REF] Bailey | Exaggerated systemic oxidativeinflammatory-nitrosative stress in chronic mountain sickness is associated with cognitive decline and depression[END_REF].

Experimental limitations

Several limitations warrant careful consideration to provide a more balanced perspective to the present findings. All findings were based exclusively on crosssectional comparisons and correlational analyses in the absence of mediation analysis, warranting future interventional approaches to better define a causal role for OXNOS as mediators of vascular dysfunction and/or CMS disease process (see 4.5 Conclusions).

We also acknowledge the interpretive limitations associated with a relatively small sample size and associated loss of statistical power; including caveats associated with

Type II and M errors [START_REF] Gelman | Beyond Power Calculations: Assessing Type S (Sign) and Type M (Magnitude) Errors[END_REF], that for select variables, in particular FMD, AIx-75, -and vitamin E, limited our ability to detect post-hoc differences between CMS+ and CMS-. In terms of (absolute %) FMD values, we (retrospectively) observed an effect size of 1.067 and corresponding power of 0.998 (α = 0.05), having adhered strictly to established guidelines that have been shown to reduce variability [START_REF] Greyling | Adherence to guidelines strongly improves reproducibility of brachial artery flow-mediated dilation[END_REF], indicating that our study was well powered to detect a treatment effect. However, our conservative approach (Bonferroni adjustments to control for false positives), combined with recent evidence documenting CVs of 16.6% and 14.2% and intra-class correlation coefficients of 0.528 and 0.714 for intra-and inter-session brachial FMD test-retests respectively [START_REF] Kimble | Methodological Considerations for a Vascular Function Test Battery[END_REF] suggests that this technique is at best, only 'moderately' reliable, that warrants consideration during future research.

We also recognise the need to exercise caution when attempting to unequivocally assign spin-adducts to specific free radical species/mechanisms; while these can be better approached in vitro using 'stopped generators', this was not possible ex vivo given that continuous free radical formation persists over the course of blood sampling, notwithstanding the potential for artefactual -HO• generation during the course of incubation and sample processing. Indeed, this is a universal challenge when attempting to detect 'any' reactive metabolite ex vivo in the human circulation (i.e. not just free radicals) that are formed clearly downstream of the primary reaction pathway that we assume reflects dynamic events in vivo. Recently, we pretreated PBN trapped blood with ethylgallimidate in an attempt to inhibit (neutrophil) NADPH oxidase activation as originally recommended [START_REF] Bailey | Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness?[END_REF], although initial findings indicated this had little to no measurable effect in terms of PBN-OR formation (DM Bailey, unpublished observations). Follow-up quality control approaches are warranted given the reactivity of free radicals and complexity of blood as a biological matrix. Future studies aimed at elucidating the targets of the generated -and HO• are also warranted, including 'proteomics' to explore the thiol redox proteome [START_REF] Yang | The Expanding Landscape of the Thiol Redox Proteome[END_REF] and 'immuno-spin trapping' that combines the specificity of spin trapping with improved sensitivity of immunological techniques with nitrone adducts that can be detected by mass spectrometry, NMR or immunochemistry using anti-DMPO nitrone antibodies [START_REF] Mason | Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping[END_REF][START_REF] Mason | Immuno-spin trapping of macromolecules free radicals in vitro and in vivo -One stop shopping for free radical detection[END_REF].

Due to logistics constraints, lowlander controls were recruited from the UK and not Bolivia. Expected differences in socio-cultural influences (e.g., access to healthcare, social/family structure) likely complicated our ability to accurately distinguish whether observed differences were due to HA residence or CMS per se. Likewise, future researchers need to consider investigating females, especially those who are postmenopausal, given that this is a risk factor for CMS [START_REF] Villafuerte | glyceryl trinitrate-stimulated FMD (endotheliumindependent), flow-mediated dilatation (endothelium-dependent); aPWV, aortic pulse wave velocity; cIMT, carotid intima-media thickness. Values are mean ± SD; CMS-/CMS+, highlanders without/with chronic mountain sickness (CMS); BMI, body mass index; Hb, hemoglobin[END_REF]. Furthermore, catalytic iron detection and general assessment of iron metabolism were confined to either a select (sub) sample of CMS+ patients or entirely separate group respectively, as 'follow-ups' upon successful detection of Me 2 CyDEPMPO-OH adducts. Complimentary assays of intravascular hemolysis specifically cell-free Hb, hemopexin and haptoglobin are also encouraged in future studies to complement the present findings.

Finally, while select aspects of elevated OXNOS biomarkers and systemic vascular dysfunction have previously been reported in CMS+ [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF][START_REF] Bailey | Exaggerated systemic oxidativeinflammatory-nitrosative stress in chronic mountain sickness is associated with cognitive decline and depression[END_REF], the new data generated by the present study (Figure 6) further extends our mechanistic understanding of the systemic OXNOS pathway.

Conclusions

In conclusion, the present study suggests that elevated systemic OXNOS and associated vasculopathic complications in CMS+ may be the consequence of ironinduced oxidative catalysis that may have a Fenton/NA/hemolytic basis. Future studies need to further differentiate these mechanisms and consider related pharmacological/dietary approaches that selectively target the OXNOS pathway including iron chelation (e.g. desferroxamine) to constrain iron-mediated oxidative catalysis. Equally, prospective randomized trials and/or public health interventions aimed at encouraging more F+V consumption will help address dietary antioxidant deficiency, reduce vulnerability to OXNOS and improve clinical outcome. [START_REF] Health | Dietary reference values for food and nutrients for the United Kingdom. Report of the Panel on Dietary Reference Values of the Committee on Medical Aspects of Food Policy[END_REF] and Bolivian [START_REF] Bolivia | Ministerio de Salud. Bases técnicas de las guías alimentarias para la población Boliviana[END_REF] reference values. *Sum of α β-carotene and cryptoxanthins. *No Reference Nutrient Intake set, recommendation expressed as Safe Intake [START_REF] Health | Dietary reference values for food and nutrients for the United Kingdom. Report of the Panel on Dietary Reference Values of the Committee on Medical Aspects of Food Policy[END_REF]. ND, not determined. Samples preparation and EPR acquisition were as described in the legend of Fig. S1. Time refers to iron addition to the Fenton reagent.

  5 mL) was added to a 6 mL glass vacutainer (SST) primed with 1.5 mL of cold freshly prepared PBN dissolved in physiological saline (50 mM final concentration). The vacutainer was gently mixed then placed in the dark to clot for 10 min. Following centrifugation, 1.5 mL of the serumadduct was removed, flash-frozen and stored in liquid N 2 prior to batch analysis. During the analysis, samples were thawed on ice and 1 mL of the serum-adduct was added to a boro-silicate glass tube containing 1 mL of spectroscopic grade toluene and vortexmixed for 10 s. The sample was centrifuged for a further 10 min and 200 µL of the organic supernatant added to a (N 2 -flushed) precision-bore quartz EPR tube and vacuum-degassed to remove O 2 prior to EPR spectroscopy. All sample handling was performed in a darkened room to minimise photolytic degradation. Instrument settings were: MP, 20 mW; R, 4096 points; MA, 0.5 G; RG, 1.0  10 5 ; TC, 82 ms; SW, 50 G, for 10 cumulative scans.

 2 2 +

 22 prior to injection into tri-iodide reagent for the direct measurement of RSNO. Plasma NO  RSNO) -RSNO. Total NO bioactivity was calculated as the cumulative concentration of all metabolites. Signal output was plotted against time using Origin 8 software and smoothed using a 150-point averaging algorithm. The Peak

2

 2 CyDEPMPO-OH (trans-OH; a N = 13.94 G, a P = 48.89 G, aH β = 12.40 G) and cis-Me 2 CyDEPMPO-OH (cis-OH; a N = 13.93 G, a P = 50.59 G, aH β = 14.08 G) diastereoisomers (Fig.1B)[START_REF] Fridovich | The biology of oxygen radicals[END_REF]. A small contaminant Me 2 CyDEPMPO/alkyl radical adduct (Fig.1B; a N = 14.46 G, a P = 50.79 G, aH β = 20.86 G; ~10% of the total signal), likely a decomposition nitroxide, was also detected. A typical signal recorded 2 min after HO• formation was triggered is illustrated in Fig.S1A(Supplementary data). Strikingly, inclusion of excess MeβCD (50 mM) in the above [Fenton + nitrone] system yielded mixtures of trans-OH, cis-OH and alkyl spin adducts with EPR spectra retaining similar shapes and hfcs within experimental accuracy (Fig.S1B, C and Table

  min after iron added), with no clear influence of MeβCD (Fig. SA, B; Supplementary data). Signal acquisitions started 1 min after thawing of the sample given that we anticipated weak EPR signals in blood [Me 2 CyDEPMP MeβCD]. Here, trans/cis ratios recorded after 2 min of continuous Fenton-mediated HO• formation were in general agreement with the published value of 1.4 (AU) observed in the same conditions for a stopped

 2

 2 and CMS Inverse relationships were observed between Me 2 CyDEPMPO-alkyl and -(Fig. 5 A) and FMD (Fig. 5 B) in CMS+. A positive relationship was observed between (total) Me 2 CyDEPMPO-OH/alkyl adducts and CMS scores (Fig. 5 C) in the highlanders (pooled CMS-and CMS+).

Figure 3 .

 3 Figure 3. EPR studies of catalytic iron and PBN spin adductsA. Changes in A •-concentration following ex vivo addition of DFX (500 µM) and Fe(III)-

Figure 4 . 2 +

 42 Figure 4. Nitric oxide metabolites

Figure 5 .

 5 Figure 5. Relationships observed between free radical and systemic

Figure 6 .

 6 Figure 6.

Fig. S2 .

 S2 Fig. S2. Effect of MeβCD on spin trapping kinetics and diastereoisomeric ratios of Me 2 CyDEPMPO adducts in the non-stopped

  . Importantly, control experiments confirmed that MeβCD did not affect Me 2 CyDEPMPO spin adduct formation in vitro, yet despite our best efforts, we consistently failed to detect Me 2 CyDEPMPO-OOH adducts in participants'

	blood.
	In contrast, highlander blood exhibited paramagnetic spectra consistent with cis/trans-
	Me 2 CyDEPMPO-OH and/or -alkyl adducts with the former selectively elevated in CMS+
	in direct proportion to CMS scores. The average OH-adduct concentration was
	comparable to that induced by maximal exercise in lowlanders following 2-
	hydroxypropyl-β-CD-assisted DEPMPO spin-trapping [46] at identical (final)
	concentrations to those employed herein, placing the magnitude of 'basal' oxidative
	stress to which the CMS+ patient is chronically exposed over their lifetime into clearer
	perspective.
	These include (i) bioreduction of primary Me 2 CyDEPMPO-OOH by endogenous thiols or
	enzymes including reduced glutathione (GSH) and glutathione peroxidase (GPx), (ii)
	direct trapping of biologically relevant HO• by a metal ion-catalysed (e.g. Fe 2+ ) Fenton
	mechanism initiated by dismutation of primary	-to H 2 O 2 (Eq. 2) and (iii) nucleophilic
	addition (NA) of water to the nitronyl function catalysed by metal ions such as Fe 3+

In chiral nitrones such as DEPMPO or Me 2 CyDEPMPO that form EPR distinguishable cis/trans-OH diastereoisomers upon trapping HO•, the trans/cis ratio determined under controlled conditions (e.g. at a given time after direct inhibition of HO-adduct formation) can differentiate between competing mechanisms that give rise to Me 2 CyDEPMPO-OH.

Acknowledgements

We thank Mrs Catherine Romero and staff of the Instituto Boliviano de Biologia de Altura (La Paz, Bolivia) for technical support. ADInstruments provided exceptional

Funding

This work was supported by a Royal Society Wolfson Research Fellowship (#WM170007) and grants from the Higher Education Funding Council for Wales (DMB), Swiss National Science Foundation, Cloëtta Foundation, Eagle Foundation, Leenaards Foundation and Placide Nicod Foundation (US and CS).

technical input/support during the period of data logging. We also thank Professor Martina Muckenthaler (University of Heidelberg, Heidelberg, Germany) for specialist input relating to iron metabolism.

Competing interests

The authors declare that they have no competing interests.

Author contributions

DMB, US and CS obtained funding, conceived and designed the research. DMB, MC, TF, JVB, BS, CJM, RS, SFR, DC, ER, LP, CSS, CMJ, MV, FV, AR, SP, US and CS contributed to data collection and/or analysis. DMB, MC, TF, AR, SP, US and CS interpreted the results of the experiments. DMB drafted the first draft manuscript and revisions thereof with input from MC, US and CS. DMB, MC, TF, JVB, BS, CJM, RS, SFR, DC, ER, LP, CSS, CMJ, MV, FV, AR, SP, US and CS edited and revised the manuscript(s) and approved the final version submitted for publication.

Appendix A. Supplementary material

Supplementary data associated with this article can be found in the online version. reported by the present study that compliment our previously published findings [START_REF] Bailey | Oxidativenitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF][START_REF] Bailey | Exaggerated systemic oxidativeinflammatory-nitrosative stress in chronic mountain sickness is associated with cognitive decline and depression[END_REF].

LEGENDS

Fig.