Libia A García-Flores

Sonia Medina

Roberto Cejuela

José Miguel Martínez-Sanz

Camille Oger

Jean-Marie Galano

Thierry Durand

Teresa Casas-Pina

Pedro Martínez- Hernández

Federico Ferreres

Ángel Gil-Izquierdo

Angel Gil-Izquierdo
email: angelgil@cebas.csic.es

Assessment oxidative stress biomarkers -neuroprostanes and dihomo-isoprostanes-in elite triathletes urine after two weeks of moderate altitude training

Keywords: Neuroprostanes, Dihomo-isoprostanes, Oxidative stress, Altitude, Physical exercise, UHPLC-QqQ-MS/MS

This randomized and controlled trial investigated whether the increase in elite training at different altitudes altered the oxidative stress biomarkers of the nervous system. This is the first study to investigate four F 4 -neuroprostanes and four F 2 -dihomo-isoprostanes quantified in 24-hour urine. The quantification was carried out by Ultra High Pressure Liquid Chromatography-triple Quadrupole-Tandem Mass Spectrometry (UHPLC-QqQ-MS/MS). Sixteen elite triathletes agreed to participate in the project. They were randomized in two groups, a group submitted to Altitude Training (n=8) and a group submitted to Sea Level Training (n=8), with a Control group of non-athletes (n=8). After experimental period, the Altitude Training group triathletes gave significant data: 17-epi-17-F 2t -dihomo-IsoP (from 5.2 ± 1.4 µg/mL 24 h -1 to 6.6 ± 0.6 µg/mL 24 h -1 ) , ent-7(RS)-7-F 2t -dihomo-IsoP (from 6.6 ± 1.7 µg/mL 24 h -1 to 8.6 ± 0.9 µg /mL 24 h -1 ), and ent-7-epi-7-F 2t -dihomo-IsoP (from 8.4 ± 2.2 µg/mL 24 h -1 to 11.3 ± 1.8 µg/mL 24 h -1 ) increased, while, of the neuronal degenerationrelated compounds, only 10-epi-10-F 4t -NeuroP (8.4 ± 1.7 µg/mL 24 h -1 ) and 10-F 4t -NeuroP (5.2 ± 2.9 µg/mL 24 h -1 ) were detected in this group. For the control group and sea level training groups, no significant changes had occurred at the end of the 2-weeks experimental period. Therefore, and as the main conclusion, the training at moderate altitude increased the F 4 -NeuroPs-and F 2 -dihomo-isoPs-related oxidative damage of the central nervous system (CNS) compared to similar training at sea level.

INTRODUCTION

The practice of training at an altitude is well-known among coaches and athletes, particularly elite athletes. At altitude, exposure to hypoxia is known to influence all functional systems of the body, including the central nervous system (CNS), the endocrine, respiratory, and cardiovascular systems, the blood oxygen-carrying capacity, and morphological and functional adaptations in skeletal muscle [START_REF] Rusko | Altitude and endurance training[END_REF]. The nervous system is especially vulnerable to reactive oxygen species (ROS)-mediated injury some reasons are that the high oxygen consumption of the brain for high energy needs, that is, high O 2 consumption, results in excessive ROS produced. In addition, the neuronal membranes are rich in polyunsaturated fatty acids (PUFA), which are particularly vulnerable to free radical attack [START_REF] Joseph | Why Is the Nervous System Vulnerable to Oxidative Stress?[END_REF]. When exercising at altitude the body responds to the fall in the barometric pressure as well as physical exercise, other factor that contribute to increased oxidative stress (OS) according to literature [START_REF] Askew | Work at high altitude and oxidative stress: antioxidant nutrients[END_REF][START_REF] Calbet | Air to muscle O2 delivery during exercise at altitude[END_REF]. The OS seems to be linearly related to the altitude: higher altitude leads to greater oxidative challenge to the body [START_REF] Dosek | High altitude and oxidative stress[END_REF]. The effects of hypoxia in the brain may influence the training intensity and/or the physiological responses during training at altitude [START_REF] Rusko | Altitude and endurance training[END_REF]. In addition, previous research indicated that the exercise-induced OS may alter the capacity of oxidation and anti-oxidation of brain tissue [START_REF] Camiletti-Moirón | Does exercise reduce brain oxidative stress? A systematic review[END_REF][START_REF] Li | Effect of different methods of hypoxic exercise training on free radical oxidation and antioxidant enzyme activity in the rat brain[END_REF]. Nevertheless the OS related consequence of high altitude training is poorly known [START_REF] Bakonyi | High altitude and free radicals[END_REF]. But there are evidence relatively consistent in human and animal studies that reporting that high altitude-associated hypoxia causes oxidative damage to lipids, proteins, and DNA. This damage can be due to the increased level of ROS production and/or decreased level of antioxidant capacity [START_REF] Radak | The effects of high-altitude exposure on reactive oxygen and nitrogen species[END_REF].

Lipid peroxidation generates a variety of end products, which can then be measured in biological fluid as an indirect index of OS [START_REF] Galano | Isoprostanes and neuroprostanes: total Downloaded by [University of Otago] at 01:26 24 November 2015 synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF][START_REF] Hsiu-Chuan | Detection of F2-isoprostanes and F4-neuroprostanes in clinical studies[END_REF][START_REF] Halliwel | Free radical in biology and medicine[END_REF][START_REF] Roberts | Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid[END_REF]. The most representative end-products of fatty acids oxidation of the system nervous are the F 4 -neuroprostanes (F 4 -NeuroPs), from DHA (Docosahexaenoic Acid), and the F 2 -dihomo-isoprostanes (F 2 -dihomo-IsoPs), from AdA (Adrenic Acid) [START_REF] Galano | Isoprostanes and neuroprostanes: total Downloaded by [University of Otago] at 01:26 24 November 2015 synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF]. The PUFAs, DHA and AdA are highly localized in the nervous tissue and represent the main PUFAs in grey and white matter, respectively [START_REF] Galano | Isoprostanes and neuroprostanes: total Downloaded by [University of Otago] at 01:26 24 November 2015 synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF][START_REF] Signorini | Isoprostanes and 4-Hydroxy-2-nonenal: Markers or Mediators of Disease? Focus on Rett Syndrome as a Model of Autism Spectrum Disorder[END_REF], although AdA also has a high presence in the adrenal gland and kidneys [START_REF] Vanrollins | F2-dihomo-isoprostanes arise from free radical attack on adrenic acid[END_REF] and DHA likewise in adipose tissue, rectal epithelium, muscle, liver and spleen, heart and cheek, red blood cells, and sperm [START_REF] Arterburn | Distribution, interconversion, and dose response of n-3 fatty acids in humans[END_REF]. The quantification of F 4 -NeuroPs and F 2 -dihomo-IsoPs provides a highly sensitive index of oxidative neuronal injury, which likely represents a global measure of oxidant status in the CNS [START_REF] Musiek | Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay[END_REF]. In the literature it is mentioned that some metabolites of F 4 -NeuroPs could have biological activities (anti-arrhythmic activities) [START_REF] Galano | Isoprostanes and neuroprostanes: total Downloaded by [University of Otago] at 01:26 24 November 2015 synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF].

Currently, the detection of F 4 -NeuroPs and F 2 -dihomo-IsoPs is mainly performed in brain tissue and/or body fluids. They are used mainly in clinical trials to elucidate the role of OS in the diseases [START_REF] Durand | F(2)-Dihomo-isoprostanes and brain white matter damage in stage 1 Rett syndrome[END_REF][START_REF] Montine | Peripheral F2-isoprostanes and F4-neuroprostanes are not increased in Alzheimer's disease[END_REF]. Nowadays, no attention has been paid yet concerning to the investigation of these CNS degradation markers and physical exercise and the influence of training at altitude or to sea level to analyze their OS generation. As noted earlier, the exposure to high altitude increased the level of ROS production or decreased level of antioxidant capacity, and then, can lead to oxidative damage to macromolecules [START_REF] Radak | The effects of high-altitude exposure on reactive oxygen and nitrogen species[END_REF]. Therefore, this randomized controlled-trial investigated whether training at different altitudes (~2300 m and 400 m) can alter the OS linked to the nervous system in elite triathletes by analyzing the variations in the values of F 4 -neuroPs/ F 2 -dihomo-IsoPs excreted before and after of the experimental period at different altitudes. To the best of our knowledge, it is the first study concerning the assessment of these non-invasive biomarkers-NeuroPs and dihomo-IsoPs-in elite triathletes subjected to AT (altitude training) or SLT (sea level training).

MATERIALS AND METHODS

Physical characteristics of participants and dietary intake

Sixteen elite triathletes (12 male and 4 female) from the University of Alicante (Spain) agreed to participate in the project. They were randomized in two groups, a group subjected to Altitude Training (AT, n=8) and a group subjected to Sea Level Training (SLT, n=8). The Control group (Cg, n=8) were non-athletes, with similar anthropometric characteristics and the same age range and healthy lifestyle as the triathletes, they have remained at sea level in all study. In the three groups were included 6 men y 2 women for to avoid variations in the analysis. All the volunteers were sea level residents, non-smokers, had stable food habits, and did not receive any medication during the experimental procedure (prescription or over-the counter medication). None had made a trip to high altitude in the three months before the intervention program. The study was approved by the Bioethics Committee of the University Hospital of Murcia and all participants provided written, informed consent to a protocol approved by the institution.

The physical parameters (Table 1) and dietary habits (Table 2) of the triathletes were controlled before the onset of the assay and after the experimental period-AT or SLTaccording to their biological and physiological characteristics. Regarding to Cg, their physical parameters and dietary habits also were evaluated at the same time that the triathletes. The anthropometric measurements were made according to the International Society for the Advancement of Kinanthropometry (ISAK) and were performed by the same, internationally certified anthropometrist (level 2 ISAK) in order to decrease technical errors. The body composition was determined by GREC Kinanthropometry consensus [START_REF] Alvero Ramón | Protocolo de valoración de la composición corporal para el reconocimiento médicodeportivo. Documento de Consenso del Grupo Español[END_REF], using a model consisting of: total fat by Withers´s formula [START_REF] Withers | Relative body fat and anthropometric prediction of body density of male athletes[END_REF]; lean weight by the procedure described by Leet et al. [START_REF] Lee | Total-body skeletal muscle mass: development and cross-validation of anthropometric prediction models[END_REF]; and residual mass by the difference in the weight (Table 1).

. The triathletes consumed a constant, equal diet (Table 2) from two weeks before the onset of the study until its conclusion, to avoid any interference with urinary analyses. The calculation of the dietary parameters and calorific intake was accurately designed and overviewed during the experimental intervention by nutritionists, using specific software for the calculation (data calculated by the software available on the website:http://www.easydiet.es),with the additional assistance of the Spanish and USDA databases (http://www.bedca.net/) and http://www.nal.usda.gov/fnic/foodcomp/search/). All the food for the study was prepared and weighed to achieve the desired and constant calorific and nutrient intake for each triathlete.

Training load

The training load quantification was performed using the Objective Load Scale (ECOs) developed by Cejuela Anta and Esteve-Lanao [START_REF] Saugy | Comparison of "Live High-Train Low" in Normobaric versus Hypobaric Hypoxia[END_REF]. Variations in ECOs were recorded as training loads, which were measured and slightly modified daily and weekly to ensure the homogeneity of the training program, taking into account the variable physical characteristics of each athlete during the study (Table 1). The method used allowed the quantification of the training loads in triathlon (swim, bike, run, and transitions). The training loads developed by elite triathletes in the present work were similar to those found in other studies [START_REF] Lucia | Physiological characteristics of the best Eritrean runners-exceptional running economy[END_REF][START_REF] Medina | Assessment of oxidative stress markers and prostaglandins after chronic training of triathletes[END_REF][START_REF] Medina | Physical activity increases the bioavailability of flavanones after dietary aronia-citrus juice intake in triathletes[END_REF].

Experimental design

The study was a randomized controlled trial (Fig. 1) where the athletes were randomly divided into two groups, a group subjected to AT (n=8) and a group undergoing SLT (n=8), during an experimental period of 2-weeks, supervised their training. The hypoxia exposure was carried out in the "Centro de Alto Rendimiento de Sierra Nevada (CAR)" (2320 m altitude; Sierra Nevada, Alpujarra and Valle de Lecrín, Spain). Before the onset of the experimental period, the training load and diet of the two groups were kept similar for two weeks. The experimental training period for the athletes started with an increase of the effort loads, keeping the ECOs during the two weeks. The Cg maintained their lifestyle at sea level (400 m) throughout the assay. The first urine sample was collected 24-hour at the beginning the experimental period y the second urine sample was collected 24-hour at the end of the experimental period. The samples of the Cg were collected at the same time than of the triathletes. Urine samples were aliquoted immediately of its collection and were stored at -80 ºC until further analysis.

Chemicals and Standards

Six F 4 -neuroprostanes (F 4 -NeuroPs) were studied, 4(RS)-4-F 4t -NeuroP, 4-F 4t -NeuroP, 4-epi-4F 3t -NeuroP, 4-F 3t -NeuroP, 10-epi-10-F 4t -NeuroP, and 10-F 4t -NeuroP as well as four F 2-dihomo-isoprostanes (F 2-dihomo-IsoPs): 17-epi-17-F 2t -dihomo-IsoP, 17-F 2t -dihomo-IsoP, ent-7(RS)-7-F 2t -dihomo-IsoP, and ent-7-epi-7-F 2t -dihomo-IsoP. Three deuterated internal standards (d 4 -4(RS)-F 4t -NeuroP, d 4 -10-epi-10-F 4t -NeuroP, and d 4 -10-F 4t -NeuroP) were used too (Fig. 2). All standards were synthesized by Durand's team at the Institut des Biomolecules Max Mosseron (IBMM) (Montpellier, France). The β-glucuronidase, type H2 from Helix pomatia, and bis-(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). All LC-MS grade solvents were obtained from J.T. Baker (Phillipsburg, New Jersey, USA). Strata SPE cartridges (Strata X-AW, 100 mg 3 mL -1 ) were purchased from Phenomenex (Torrance, California, USA).

Sample collection and preparation

A complete clinical analysis -consisting of hematology, chemistry, and urine chemistry analysis -was performed at the onset of and after the experimental period. All samples (blood and urine) were collected by a nurse from the subjects early in the morning, under fasting conditions. Blood samples at rest were obtained by venipuncture, at the beginning and at the end experimental period and were placed in different tubes according to the analytical procedures. Samples were processed within 1 h of collection and stored at -80ºC for the analytical determinations. The hematological parameters were recorded using an automated hematological analyzer (CellDyn 3700 and 4000, Abbott, IL, USA) at the Clinical Analysis Service of the Hospital Virgen de la Arrixaca (Murcia, Spain).

Twenty-four-hour urine samples were collected before and after the 2-weeks training period. They were collected in sterile, dark polystyrene tubes with screw caps. The urine analyses were also performed in a modular analyzer (Roche Diagnostic, Mannheim, Germany). The 24-h -1 urine was used for the absolute calculation of the amounts of F 4 -NeuroPs and F 2 -dihomo-IsoPs excreted. The urinary F 4 -NeuroPs and F 2 -dihomo-IsoPs were analyzed using our previously-described method [START_REF] Medina | Dihomo-isoprostanes-nonenzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography-triple quadrupole-tandem mass spectrometry method[END_REF] .

UHPLC-QqQ-MS/MS analyses

The separation of the F 4 -NeuroPs and F 2 -dihomo-IsoPs in the urine was performed using a UHPLC coupled with a 6460 QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany), using the set-up described previously Medina, et al. [START_REF] Medina | Dihomo-isoprostanes-nonenzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography-triple quadrupole-tandem mass spectrometry method[END_REF]. Data acquisition and processing was performed using MassHunter software version B.04.00 (Agilent Technologies, Walbronn, Germany). The qualitative and quantitative analysis of F 4 -NeuroPs and F 2 -dihomo-IsoPs was performed using the authentic markers synthesized by Durand's team. Three deuterated anal ytes were used as internal standards (Fig. 2).

Statistical analyses

All the statistical analyses were performed using the SPSS 17.0 software package (LEAD Technologies Inc., Chicago, USA). Quantitative data are presented as mean ± SD (standard deviation). The amounts excreted of F 4 -NeuroPs and F 2 -dihomo-IsoPs were calculated as µg/mL per 24-h -1 urine. The normality was analyzed by the Shapiro-Wilk test and the test of equal variances by Levene's test. Specific differences between the hematological, serum, and urinary parameters of SLT and AT triathletes before and after training period were determined by t-test. The different amounts excreted of F 4 -NeuroPs and F 2 -dihomo-IsoPs within the same group, AT or SLT, as a result of the training at different altitudes were examined by paired t-test (before /after). Results were considered to be statistically significant when P< 0.05.

RESULTS

Anthropometric variables and training performance

The kinanthropometric measurements, performed following the ISAK, did not yield representative differences between the experimental groups. The training loads of the triathletes (SLT and AT) were at the onset of the training period ranged from 458 to 727 ECOs and after the experimental period from 766 to 1021 ECOs (Table 1).

Hematology, chemistry, and urine chemistry modifications

Regarding the results of the tests and profiles, significant variation in the hematology, chemistry, and urine chemistry was detected between the AT and SLT triathletes, after the training program. In this study, we only mentioned the parameters that showed significant changes according to the different tests and profiles of the clinical analyses and that could be relevant -in biochemical terms -to the F 4 -NeuroPs and F 2 -dihomo-IsoPs, according to previous investigations [START_REF] Ciccoli | Morphological changes and oxidative damage in Rett Syndrome erythrocytes[END_REF][START_REF] Gureeva | Pancreatic lipase activation, inhibition, and relationship to peroxide oxidation of lipids[END_REF][START_REF] Kurtul | Leukocyte lipid peroxidation, superoxide dismutase and catalase activities of type 2 diabetic patients with retinopathy[END_REF][START_REF] Ohmori | The relationship between body mass index Downloaded by [University of Otago] at 01:26 24 November 2015 and a plasma lipid peroxidation biomarker in an older, healthy Asian community[END_REF][START_REF] Signorini | Free iron, total F-isoprostanes and total F-neuroprostanes in a model of neonatal hypoxic-ischemic encephalopathy: neuroprotective effect of melatonin[END_REF] (Table 3). Regarding the clinical urinary parameters (urinary density (g mL -1 ), pH, proteins (mg dL -1 ), calcium (mg dL -1 ), phosphorus (mg dL -1 ), uric acid (mg dL -1 ), urea (mg dL -1 ), creatinine (mg dL -1 ), and potassium (mEq L -1 )), no significant differences between the AT and SLT groups of triathletes before and after the experimental period were observed.

Qualitative analysis of F 4 -neuroprostanes and F 2 -dihomo-isoprostanes

Ten biomarkers were screened in the urine of volunteers. Their identification was confirmed according to their molecular masses, the characteristic MS/MS fragmentation product ions, and the retention time relative to the corresponding standard. The mass spectral information on the F 4 -NeuroPs and F 2 -dihomo-IsoPs is summarized in our previously reported [START_REF] Medina | Dihomo-isoprostanes-nonenzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography-triple quadrupole-tandem mass spectrometry method[END_REF].

The NeuroPs deriving from DHA were not detected in the samples of the triathletes in the SLT and Cg groups, under any condition-perhaps because they were present at very low levels, below the limit of detection and/or quantification (LOD / LOQ). Only two NeuroPs were detected in the urine of the AT group after training (10-epi-10-F 4t -NeuroP, 10-F 4t -NeuroP) (Table 4). The analytes deriving from AdA were detected in triathletes and nontriathletes at the beginning and end of the experimental period. In the present study, F 3 -NeuroPs (4-epi-4-F 3t -NeuroP and 4-F 3t -NeuroP) formed by the oxidation of docosapentaenoic acid were analyzed, but were below the LOD/ LOQ. Therefore, these data are not shown.

Quantification of F 4 -neuroprostanes and F 2 -dihomo-isoprostanes

A total of six biomarkers were quantified in the triathlete's urine, as described in Table 4. All of the urinary biomarkers were normalized to the total 24-h excretion volume. The values are presented as the mean (± SD) total urinary excretion at the onset of and after the experimental period for all groups (µg/mL 24 h -1) . In the AT group, only the analytes 10 epi-10F 4t -NeuroP and 10-F 4t -NeuroP (8.4 ± 2.1 and 5.2 ± 1.2 µg/mL 24 h -1 , respectively) were detected, after physical training during exposure to altitude.

On the other hand, the markers of lipid peroxidation derived from AdA were quantified in all groups, and showed statistically-significant variation in the AT group. The 2weeks exposure to moderate altitude produced significant increases in the urinary levels of 17-epi-17-F 2t -dihomo-IsoP, ent-7(RS)-7-F 2t -dihomo-IsoP, and ent-7-epi-7-F 2t -dihomo-IsoP, compared to their corresponding concentrations at the start of the training periodaccording to the paired t-test (Table 4). The urinary excretion of F 2 -dihomo-IsoPs in the Cg and SLT group had not changed significantly after two weeks of the experimental period at sea level.

DISCUSSION

When comparing the urinary excretion of F 4 -NeuroPs and F 2 -dihomo-IsoPs at the onset of and after the experimental period in all three groups, four points emerged primarily:

1) The markers of lipid peroxidation derived from DHA were analyzed but were detected under the LOD and LOQ in the urine samples of Cg and SLT triathletes. The LOD are as follows: 4(RS)-4F 4 -NeuroP: 5.90 ng/mL, 4-F 4 -NeuroP: 5.90 ng/mL, 10-epi-10-F 4 -NeuroP: 0.15 ng/mL, and 10-F 4 -NeuroP: 0.10 ng/mL). The LOQ are as follows: 4(RS)-4F 4 -NeuroP: 11.81 ng/mL, 4-F 4 -NeuroP: 11.81 ng/mL, 10-epi-10-F 4 -NeuroP: 0.34 ng/mL, and 10-F 4 -NeuroP: 0.15 ng /mL. Both the LOD and LOQ were based on the method by [START_REF] Medina | Dihomo-isoprostanes-nonenzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography-triple quadrupole-tandem mass spectrometry method[END_REF]. This fact suggested that our healthy people, as well as athletes in physical training at sea level, did not show changes in their F 4 -NeuroPs values.

2) In our study, after two weeks of AT, the analytes 10-epi-10-F 4t -NeuroP (8.4 ± 2.1 µg/mL 24 h -1 urine) and 10-F 4t -NeuroP (5.2 ± 1.2 µg/mL 24 h -1 urine) -derived from DHAwere detected only in this group. This suggests that 10-epi-10-F 4t -NeuroP and 10-F 4t -NeuroP may be potential biomarkers of lipid peroxidation physical exercise under hypoxia (low levels of oxygen) at moderate altitude (2320 m altitude). The effects of hypoxia in the brain may influence the training intensity and/or the physiological responses during training at altitude [START_REF] Rusko | Altitude and endurance training[END_REF]. In cerebral cortex of newborn pigs, an increase in the levels of F 4 -NeuroP and other OS markers, after hypoxia and resuscitation with supplementary oxygen (reoxygenation), was detected [START_REF] Solberg | Resuscitation with supplementary oxygen induces oxidative injury in the cerebral cortex[END_REF]. Other study in cerebral tissue, observed that DHA seems to be more damaged by ischemia (restriction in blood supply to tissues) compared with hypoxia, suggesting that the increase of F 4 -NeuroPs could represent a specific marker for ischemia damage mainly [START_REF] Signorini | Free iron, total F-isoprostanes and total F-neuroprostanes in a model of neonatal hypoxic-ischemic encephalopathy: neuroprotective effect of melatonin[END_REF]. In human research was indicated that acute hypoxia on Rett syndrome patients, increases plasma levels of F 4 -NeuroPs by two orders of magnitude, as compared to those of healthy controls [START_REF] De Felice | Systemic oxidative stress in classic Rett syndrome[END_REF]. These results supported to previous studies in vivo, about possible hypoxic state links and an increased NeuroPs values.

According to the literature, F 4 -NeuroPs not only might be biomarkers of lipid peroxidation, but also could have anti-arrhythmic effects [START_REF] Galano | Isoprostanes and neuroprostanes: total Downloaded by [University of Otago] at 01:26 24 November 2015 synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF]. Therefore, the detection of 10epi-10F 4t -NeuroP and 10-F 4t -NeuroP, besides indicating an increase in lipid peroxidation in athletes submitted to altitude, also suggests a role for lipid metabolism against arrhythmias induced by altitude. Since arrhythmia starts at an altitude of about 2000 m, an immediate increase in ventilation, mediated by peripheral chemoreceptors, is observed according to the literature [START_REF] Burtscher | Short-term exposure to hypoxia for work and leisure activities in health and disease: which level of hypoxia is safe?[END_REF]. In addition, arrhythmia is one of the physiological responses to hypoxia exposure, caused by ventilator and circulatory responses that are accompanied by an increase in the sympathetic activity and local vasoregulatory effects. Therefore, these are undoubtedly key mechanisms improving oxygen delivery to tissues [START_REF] Rusko | Altitude and endurance training[END_REF][START_REF] Burtscher | Short-term exposure to hypoxia for work and leisure activities in health and disease: which level of hypoxia is safe?[END_REF]. The increase in the amount of NeuroPs in urine is also evidence that the hard work involved in elite training results in OS in tissues where DHA is present -like muscle, as well as adipose tissue, rectal epithelium, liver and spleen, heart and cheek, red blood cells, and sperm. Further research is required to elucidate the biological role of the NeuroPs in triathletes.

In ours AT athletes, 17-epi-17-F 2t -dihomo-IsoP, ent-7(RS)-7-F 2t -dihomo-IsoP, and ent-7-epi-7-F 2t -dihomo-IsoP increased compared with the amount before the short-term training at moderate altitude (Table 4). F 2 -dihomo-IsoPs reflect the oxidative status of brain white matter, but also could reflect the OS of the other organs where they are present [START_REF] Durand | F(2)-Dihomo-isoprostanes and brain white matter damage in stage 1 Rett syndrome[END_REF]. Altitude training stimulates the adrenergic nervous system responsible for the acute cardiovascular response to hypoxia, playing a crucial role in the adaptation to acute hypoxia during exercise [START_REF] Favret | Exercise and hypoxia: the role of the autonomic nervous system[END_REF]. Dosek, et al. [START_REF] Dosek | High altitude and oxidative stress[END_REF], they mentioned that physical exercise at high altitude could further increase the altitude-induced oxidative stress-as can be seen in this study-and the associated oxidative damage although the OS seems to be linearly related to the altitude: higher altitude leads to greater oxidative challenge to the body. On the other hand, the excretion of F 2 -dihomo-IsoPs by Cg and SLT triathletes had not changed significantly after two weeks. This result indicates that an acute increase in training at sea level for elite athletes using ECOs did not influence the urinary excretion of F 2 -dihomo-IsoPs. An increase of the OS biomarkers followed to aerobic and anaerobic acute physical exercise has been shown in numerous investigations, but also is mentioned that in well-training athletes, this is not always fulfilled [START_REF] Fisher-Wellman | Acute exercise and oxidative stress: a 30 year history[END_REF]. In studies developed, in vivo , it was found that chronic exercise could increase the resistance against OS, providing enhanced protection [START_REF] Lin | Chronic treadmill running protects hippocampal neurons from hypobaric hypoxia-induced apoptosis in rats[END_REF][START_REF] Liu | Chronically and acutely exercised rats: biomarkers of oxidative stress and endogenous antioxidants[END_REF][START_REF] Nikolaidis | F(2)-isoprostane formation, measurement and interpretation: the role of exercise[END_REF] for this reasons is mentioned that some athletes do not show changes in its levels of biomarkers of OS, since is associated with an adaptive process [START_REF] Dosek | High altitude and oxidative stress[END_REF][START_REF] Nikolaidis | F(2)-isoprostane formation, measurement and interpretation: the role of exercise[END_REF].

4) The last point concerns anthropometric, biochemical, and hematological parameters and their connection with F 4 -NeuroPs and F 2 -dihomo-IsoPs, since some of them have been associated with an increase or decrease in lipid peroxidation. As regards the anthropometric parameters and their relation to an increase in lipid peroxidation, a study carried out by Ohmori and co-workers reported that body mass index (BMI) in humans is related to an increase of lipid peroxidation [START_REF] Ohmori | The relationship between body mass index Downloaded by [University of Otago] at 01:26 24 November 2015 and a plasma lipid peroxidation biomarker in an older, healthy Asian community[END_REF], but in this sense, in our volunteers (triathletes and Cg) no changes in BMI were found during the study and there were no statistical differences between the two groups. On the other hand, the activities of the pancreatic enzymes (amylase and lipase) were increased after two weeks at altitude. The pancreatic lipases are involved in the mobilization of fatty acids from fat deposits -for example, during stress -and play a role in lipolysis, which, together with the biogenic monoamines, could influence in the process peroxide oxidation of natural lipid [START_REF] Gureeva | Pancreatic lipase activation, inhibition, and relationship to peroxide oxidation of lipids[END_REF]. Another biochemical parameter in plasma that showed significant changes was ferritin, the concentration of which decreased. In our study the plasma iron remained constant whilst ferritin levels declined. Previous study demonstrated, in cerebral cortex from rats with hypoxia-ischemia, an increase in desferoxamine-chelatable free iron that could have induced cerebral OS [START_REF] Signorini | Free iron, total F-isoprostanes and total F-neuroprostanes in a model of neonatal hypoxic-ischemic encephalopathy: neuroprotective effect of melatonin[END_REF]. These authors reported increases in F 2 -IsoPs and F 4 -NeuroPs concomitant with that of iron, suggesting a dual interaction in relation to this oxidative damage.

Normal physiological increases in the red blood cells count occur at high altitudes or after strenuous physical training [START_REF] Bailey | Physiological implications of altitude training for endurance performance at sea level: a review[END_REF]. According to literature 2-weeks at moderate altitude exposition the hemoglobin concentrations and hematocrit increased their values in elite athlete [START_REF] Heinicke | A three-week traditional altitude training increases hemoglobin mass and red cell volume in elite biathlon athletes[END_REF][START_REF] Wehrlin | Linear decrease in .VO2max and performance with increasing altitude in endurance athletes[END_REF]. Although the AT group showed higher hemoglobin concentration values after training, these have not been significant in the statistical tests due to the variability of the results (Table 3). But AT group showed significant increases in ACV (averaged corpuscular volume) and ACH (averaged corpuscular hemoglobin), compared to the baseline determinations. Hence, these data reflect a general activation of erythropoiesis in response to presumed renal tissue hypoxia for the AT athletes. Red blood cell morphology is an important biosensor for OS imbalance and chronic hypoxemia (low oxygen in the blood), in neurodegenerative diseases [START_REF] Signorini | Isoprostanes and 4-Hydroxy-2-nonenal: Markers or Mediators of Disease? Focus on Rett Syndrome as a Model of Autism Spectrum Disorder[END_REF][START_REF] Ciccoli | Morphological changes and oxidative damage in Rett Syndrome erythrocytes[END_REF]. Moreover, the leukocyte concentrations in peripheral blood after the AT effort also exhibited significant modifications. An earlier study mentioned that leukocyte antioxidants, in patients with type 2 diabetes, are related to lipid peroxidation [START_REF] Kurtul | Leukocyte lipid peroxidation, superoxide dismutase and catalase activities of type 2 diabetic patients with retinopathy[END_REF]. Whether due to physical exercise in altitude and/or exposure altitude, influenced significantly in to alter metabolic process and lipid peroxidation of the AT group, however it still not clear whether they are causative or associative, is necessary further investigation to clarify these results in elite athletes.

In conclusion, our study F 2 -dihomo-IsoPs and F 4 -NeuroPs have been detected for the first time in the urine of elite triathletes subjected to two weeks of training at altitude or sea level. The F 4 -NeuroPs were only detected in the group training at moderate altitude, suggesting that the altitude factor could be related to their production from DHA, through lipid peroxidation. The F 2 -dihomo-IsoPs also showed increases in their urinary excretion in athletes subjected to AT, versus their baseline amounts. Therefore, and as the main conclusion, the training at moderate altitude increased the F 4 -NeuroPs-and F 2 -dihomo-isoPsrelated oxidative damage of the CNS compared to similar training at sea level.

FIGURE CAPTIONS

Figure 1

 1 Figure 1 Study design. The triathletes were randomized in two groups, a group subjected to Altitude Training (AT, n=8) and a group subjected to Sea Level Training (SLT, n=8). The Control group (Cg, n=8) were non-athletes. For two weeks, the training loads and diet were kept similar. Urine samples were collected before and after the training period (SLT or AT) and at the same times for the Cg.

Figure 2

 2 Figure 2 Chemical structures of F 4t -neuroprostanes, F 2t -dihomo-isoprostanes, and deuterated internal standards. A: F 4t -Neuroprostanes, B: F 2t -dihomo-isoprostanes.

Figure 3 F 4 -

 34 Figure 3 F 4 -neuroprostanes and F 2 -dihomo-isoprostanes (µg/mL 24 h -1 ) determined in the urine of the three groups before and after the experimental period. The values shown are mean ± standard deviation. A) Cg: Control group (n=8), B) SLT: Elite triathletes training at sea level (n=8), and C) AT: Elite triathletes training at altitude (n=8).The average volume of urine excreted by volunteers was 1560.62 ± 627.32 mL per 24 h in the two periods. The level of statistical significance was set at P <0.05 (*) and P <0.001 ( †), by the paired t-test. DHA: docosahexaenoic acid and AdA: adrenic acid.

TABLE 1

 1 Physical parameters and metabolic characteristics at the beginning and at the end of the experimental period both of control group (n=8) and of the triathletes according to their altitude level training (n=16, divided in two, at sea level n=8 and at altitude level n=8) y with their objective load scale (ECOs)

					Triathletes
		Control Group	Sea level	Altitude
	Variable	(<400 m)	(<400 m)	(~2320 m)
		1	2	1	2	1	2
	Age Year (yr)	21.9 (6.2)	21.9 (6.2)	20.8 (2.0) 20.8 (2.0) 20.3 (1.8)	20.3 (1.8)
	Weight (kg)	64.4 (8.2)	64.7 (8.1)	68.4 (13.1)	65.4 (11.5)	68.1 (7.1)	67.2 (7.1)
	Height (m)	1.7 (0.1)	1.7 (0.07)	1.7 (0.1)	1.7 (0.1)	1.8 (0.1)	1.8 (0.1)
	BMI (kg m -2 )	22.0 (0.9)	22.2 (0.5)	21.4 (2.5) 21.8 (0.1) 21.3 (0.6)	21.2 (0.7)
	Total fat (kg)	9.3 (1.6)	9.5 (1.7)	8.4 (1.3)	8.7 (0.8)	6.2 (1.2)	6.0 (1.2)
	Lean weight (kg)	26.7 (6.3)	26.3 (5.9)	29.1 (7.2) 27.3 (6.7) 32.2 (7.3)	30.1 (4.2)
	Subscapular skinfold (mm)	11.5 (1.5)	11.8 (1.1)	10.6 (2.0) 11.0 (1.0) 7.8 (1.6)	7.7 (1.7)
	Tricipital skinfold (mm)	12.9 (5.2)	12.7 (5.0)	12.3 (6.8) 12.8 (5.2) 7.4 (3.4)	8.2 (3.7)
	Bicippital skinfold (mm)	4.6 (1.3)	5.3 (0.6)	4.7 (1.4)	5.0 (1.1)	4.6 (1.7)	3.7 (1.0)
	Ileocrestal skinfold (mm)	14.1 (0.8)	14.4 (0.09)	13.9 (4.7) 15.9 (3.4) 10.4 (3.4)	8.9 (2.3)
	Supraespinal skinfold (mm)	10.8 (0.6)	10.8 (0.7)	10.5 (3.6) 11.2 (2.8) 7.3 (1.9)	6.7 (1.9)
	Abdominal skinfold (mm)	18.1 (3.7)	18.3 (3.7)	17.7 (7.1) 19.3 (4.9) 10.5 (4.6)	9.5 (3.8)
	Thigh skinfold (mm)	17.8 (5.8)	17.7 (3.3)	16.1 (9.3) 17.9 (9.2) 10.4 (4.3)	10.5 (4.3)
	Calf skinfold (mm)	12.3 (3.5)	12.6 (5.3)	10.2 (3.8) 10.6 (2.6) 7.1 (2.9)	7.4 (3.2)
	Fat mass (%)	14.0 (10.2) 14.2 (10.5)	13.7 (3.4) 13.6 (5.7)	10.25 ( 2.2)	10.4 (1.8)
	Training loads (ECOs)			553(95)	870(104)	933(88)	651(76)
	Values are Mean (Standard Deviation).1= data corresponding to before experimental period; 2= data
	corresponding to after experimental period. BMI: Body Mass Index

TABLE 2 .

 2 Dietary parameters and calorific intake

		Control group	Triathletes
		Male	Female	Male	Female
		(n=6)	(n=2)	(n=12)	(n=4)
	Energy intake (kcal d -1	2913.1 (601.6)	2289.7 (270.7)	3483.5 (673.6)	2585.0 (376.4)
	Carbohydrate (g d -1 )	309.4 (62.4)	257.0 (7.6)	409.1 (79.1)	324.3 (31.8)
	Dietary fiber (g d -1 )	29.5 (9.3)	25.2 (11.9)	26.6 (4.9)	26.4 (8.6)
	Sugar (g d -1 )	125.7 (37.7)	147.8 (16.0)	169.8 (68.0)	147.6 (11.97)
	Proteins (g d -1 )	130.8 (32.0)	97.6 (28.4)	145.7 (33.3)	109.9 (34.7)
	Total lipids (g d -1 )	127.9 (27.3)	96.8 (14.0)	140.4 (25.69)	94.1 (12.2)
	SFA a (g d -1 )	38.1 (11.0)	29.36 (5.8)	36.5 (10.5)	28.5 (6.7)
	MUFA b (g d -1 )	64.5 (15.4)	47.3 (4.0)	73.8 (14.0)	46.6 (2.4)
	PUFA c (g d -1 )	17.2 (3.9)	10.8 (0.4)	19.2 (1.7)	10.4 (1.0)
	Vitamin C (mg d -1 )	160.9 (101.2)	42.8 (12.5)	254.4 (69.6)	153.6 (63.1)
	Vitamin E (mg d -1 )	24.0 (10.8)	9.65 (0.3)	29.5 (8.42)	14.9 (1.0)
	Vitamin D (mg d -1 )	6.3 (4.1)	2.5 (3.4)	5.7 (3.2)	3.6 (2.6)
	Iron (mg d -1 )	24.7 (9.6)	17.0 (7.14)	26.9 (5.1)	24.6 (1.3)
	Selenium (mg d -1 )	100.5 (68.5)	133.3 (8.2)	235.1 (91.2)	168.4 (73.6)

a Saturated fatty acids

TABLE 3 .

 3 Hematology, chemistry, and urine chemistry parameters of the elite triathletes before and after training at different altitude (n = 8 each group) Values are means (SD). Abbreviation: ACV, averaged corpuscular volume; ACH, averaged corpuscular hemoglobin; PDW: Platelet distribution width (it analyzed in blood) a These parameters were analyzed in serum. *Significant differences at p<0.05 measured at different altitudes at matched time points. †Significant differences at p<0.05 between measurements before and after training periods at the same altitude.

			Before training		After training
	Variable	SLT (<400 m)	AT (~2320 m)	Sign	SLT (<400 m)	AT (~2320 m)	Sign
	Pancreatic amilase (U L -1 ) a	22.33 (5.01) 25.25 (7.65)		22.50 (4.46)	30.13 (7.83) †
	Lactate dehydrogenase (U L -1 ) a	179.5 (38.87)	213.63 (36.25)		157.83 (32.98) † 203.50 (25.00) *
	Lipase (U L -1 ) a	25.33 (6.22) 26.88 (8.34)		22.67 (4.97)	32.63 (10.17) † *
	C-Reactive Protein mg/dL a	0.10 (0.09) 0.11 (0.21)		0.12 (0.10)	0.03 (0.0)
	Ferritin (µg L -1 ) a	48.33 (10.44)	55.88 (17.16)		58.00 (17.44)	38.50 (14.76) † †	*
	Iron (µg L -1 ) a	79.50 (28.65)	100.75 (20.81)		112.00 (62.03) 109.63 (72.99)
	Hemoglobin (g dL -1 )	15.00 (1.31) 14.83 (1.13)		15.08 (0.86)	19.01 (10.78)
	Hematocrit (%)	45.60 (4.50) 44.81 (3.82)		44.83 (2.4)	45.79 (4.24)
	PDW (%)	16.57 (1.05) 17.29 (1.65)		16.38 (0.44)	15.85 (0.37) †	*
	ACV (fL)	82.22 (2.86) 91.64 (2.22)		89.12 (2.81)	93.84 (2.21) †
	ACH (pg cell -1 )	29.40 (1.51) 30.39 (0.94)		30.23 (1.16)	31.30 (1.04) †

Downloaded by [University of Otago] at 01:26 24 November 2015

b Monounsaturated fatty acids c Polyunsaturated fatty acids Values are Mean (SD) Downloaded by [University of Otago] at 01:26 24 November 2015

ACKNOWLEDGEMENTS

This study was supported by the project AGL2011-23690 (CICYT) (Spanish Ministry of Economy and Competitiveness). LAGF was granted with a pre-doctoral FPI fellowship BES2012-060185 by the Spanish government. The authors are also grateful to the University of Alicante for its collaboration. Sonia Medina was appointed under a research contract from the project AGL2011-23690 (CICYT). We are grateful to Dr. David Walker for the review of the English grammar and style of the current report.

The authors declare that they have no conflict of interest.


Adrien Gans
email: adrien.gans@univ-amu.fr

Olivier Pouliquen
email: olivier.pouliquen@univ-amu.fr

Maxime Nicolas
email: maxime.nicolas@univ-amu.fr

A cohesion-controlled granular material

We present a simple method to prepare a granular material with a controlled cohesion between particles. The granular material is made of spherical glass beads coated with a polyborosiloxane polymer. This material is proved to be stable in time and non-sensitive to temperature and humidity. The inter-particle force is measured and related to the size of the grain and the polymer coating thickness. Classical measurements (packing fraction, repose angle, macroscopic cohesion), are performed with this cohesion-controlled granular material. This model material opens many perspectives to study in a controlled manner the flow of cohesive grains.

I. INTRODUCTION

Handling powders and cohesive materials is an everyday challenge in many industrial processes. Beyond the difficulty of handling a very large number of particles, the cohesion between grains is a serious source of difficulty. Cohesion often comes into play when manipulating small particles, for which humidity [START_REF] Bocquet | Moisture-induced ageing ingranular media and the kinetics of capillary condensation[END_REF][START_REF] Mitaraiand | Wet granular materials[END_REF] or attractive forces like Van der Walls [START_REF] Castellanos | The relationship between attractive interparticle forces and bulk behaviour in dry and uncharged fine powders[END_REF] or electrostatic forces [START_REF] Konopkaand | Discrete element modeling of electrostatic charging of polyethylene powder particles[END_REF], become important. The adhesion between the grains decreases the ability of powders to flow easily, and the dynamics then differs from dry granular materials made of large particles interacting solely trough frictional dry contact interactions. Whereas our knowledge of granular flows has improved over the last 20 years, the behavior of powders and cohesive granular media still represents a real challenge.

From an industrial point of view, different techniques have been developed to characterize and to quantify the "flowability" of powders [START_REF] Antequera | Evaluation of an adequate method of estimating flowability according to powder characteristics[END_REF][START_REF] Geldart | Characterization of powder flowability using measurement of angle of repose[END_REF]. This concept based on a compilation of different measurements (compaction, repose angles,...), although very useful when checking the quality of a product or when comparing different materials, lacks clear physical bases.

From a fundamental point of view, one difficulty in studying cohesive material is the control of the cohesion forces between the particles. Whereas in numerical studies, simplified interaction laws might be introduced in DEM approaches to simulated cohesive grains [START_REF] Luding | Cohesive, frictional powders: contact models for tension[END_REF], experimentally, the control of the cohesion is more challenging. Working with actual powders faces many difficulties. Powder particles are often very polydispersed with a great variety of shape and surface roughness. A humidity and temperature control of the ambient air is required to ensure a good reproducibility of the experiments [START_REF] Howell | Dynamics of electrostatically-driven granular media. effects of humidity[END_REF][START_REF] Jones | Inter-particle forces in cohesive powders studied by afm: effects of relative humidity, particle size and wall adhesion[END_REF]. While some comparative tests between benchmark powders (such as lactose) are useful [START_REF] Alexander | Avalanching flow of cohesive powders[END_REF], it is still very difficult to link the particle properties to the bulk flow behavior of the powder [START_REF] Castellanos | The relationship between attractive interparticle forces and bulk behaviour in dry and uncharged fine powders[END_REF], mainly because of a lack of a controlled particle interaction. Most of the recent experimental works on cohesive granular materials have focused on capillary cohesion, studying the properties of an assembly of beads mixed with a small amount of a viscous liquid. The influence of the amount of liquid on the packing fraction [START_REF] Vandewalle | How relative humidity affects random packing experiments[END_REF][START_REF] Xu | Analysis of the packing structure of wet spheres by voronoidelaunay tessellation[END_REF], on the repose or avalanche angle [START_REF] Hornbaker | What keeps sandcastles standing?[END_REF][START_REF] Albert | Maximum angle of stability in wet and dry spherical granular media[END_REF][START_REF] Halseyand | How sandcastles fall[END_REF][START_REF] Nowak | Maximum angle of stability of a wet granular pile[END_REF][START_REF] Richefeu | Shear strength properties of wet granular materials[END_REF], on the stability of a column [START_REF] Pakpour | How to construct the perfect sandcastle[END_REF] has been studied. The strength of a wet granular assembly has been trialed through the measurement of the shear modulus [START_REF] Bonn | The shear modulus of wet granular matter[END_REF] and more recently, rheology experiments have been also conducted [START_REF] Badetti | Rheology of wet granular materials in shear flow: experiments and discrete simulations[END_REF][START_REF] Badetti | Rheology and microstructure of unsaturated granular materials: Experiments and simulations[END_REF] in a pressure imposed rheometer for glass beads coated by viscous silicon oil. Despite a good knowledge of the cohesion force arising from capillary bridges [START_REF] Willett | Capillary bridges between two spherical bodies[END_REF] the bulk behavior and the rheological measurements suggest a complex dynamics due to the evolution of the capillary bridges during shear, which may migrate and coalesce [START_REF] Mani | Liquid migration in sheared unsaturated granular media[END_REF][START_REF] Gagneuxand | An analytical framework for evaluating the cohesion effects of coalescence between capillary bridges[END_REF]. Recently [START_REF] Jarray | Cohesion-driven mixing and segregation of dry granular media[END_REF], silanization has been used to modify the surface properties of glass particles and build a cohesive force of 0.1 mN order of magnitude. Experimental research on cohesive materials would strongly benefit from a model system making it possible to tune at will, and through a simple process, the cohesion between the grains.

In this paper we present a new method to prepare a controlled-cohesion granular material (CCGM) made of polyborosiloxane (PBS) coated glass particles, which suits many of the requirements to achieve experiments with a controlled cohesion. The main point is that the cohesion force between particles can be easily tuned through the PBS coating. The coating of the particles is easy and does not require heavy chemical equipment. Furthermore, the CCGM is very stable on a long time scale, is insensitive to humidity of the ambient air, and is also insensitive to room temperature.

The preparation method is first presented in Sec. II. A detailed studied of the inter-particle cohesive force induced by the presence of the PBS coating is presented in Sec. III. In Sec. IV the CCGM is tested in different classical configurations used for characterizing granular media : measurements of the bulk density, pile angle, inclined plane experiments. Concluding remarks and perspectives are given in Sec. V.

II. DESIGN OF A COHESION CONTROLLED GRANULAR MATERIAL

The particle coating material is a polyborosiloxane (PBS) made from a -OH terminated Polydimethylsiloxane (PDMS) cross-linked with boric acid (H 3 BO 3 ) [START_REF] Thuresson | Material composition and lmethod for its manufacturing[END_REF][START_REF] Li | Synthesis of polyborosiloxane and its reversible physical crosslinks[END_REF]. Each batch of CCGM is prepared with a mass m g of spherical glass beads (diameter d and density ρ G = 2600 kg•m -3 ) with a small polydispersity, a mass m P of PDMS (density ρ P = 970 kg•m -3 , viscosity 750 mPa•s and gyration radius r g = 17 Å), and a mass m A of boric acid. We kept a constant mass ratio of boric acid over PDMS m A /m P = 0.14. The boric acid is first dissolved in a small volume (50 ml) of purified water heated at 60 • C. The particles, the PDMS and the H 3 BO 3 solution are then mixed together in a heating mixer (Kenwood Cooking Chef) at 110 • C during 90 min to ensure the evaporation of the water and a homogeneous PBS coating of the spherical particles. After cooling, the batch is ready to use for experiments. The PBS has been characterized in a rheometer using a plane-plane geometry and the real and imaginary part of the shear modulus are G = 35 kPa and G = 7.5 kPa with a relaxation time constant of 3.8 s.

The range of particle size was d = [0.8 -10] mm in experiments of Sec. III concerning the measurement of the inter-particle force, and d = [0.2 -1.4] mm in the macroscopic experiments of Sec. IV. From atomic force microscopy imaging, the average roughness of 0.8 mm particles was 30 ± 2 nm. The effect of the PBS coating is qualitatively illustrated in Fig. 1 where a sample of 3 mm diameter coated glass beads reveals the cohesive nature of the material. The parameter controlling the cohesion in our study is the averaged thickness b. Assuming an homogeneous coating of a Ω P = m P /ρ P volume of PBS over perfectly spherical particles of volume Ω G = m G /ρ G gives an average thickness

b = d 2 1 + Ω P Ω G 1/3 -1 (1)
In the limit of a small coating averaged thickness (b d), the PBS volume ratio Ω P /Ω G ≈ 6b/d.

An attempt to visualize the PBS layer is presented in Fig. 2. On the pictures, the PBS appears as a grey and foamy fluid, which is optically different from the clean glass surface. The pictures of the clean surfaces have been obtained after a careful removal of the PBS with a spray of heptane without touching or moving the particle. The iridescence seen in Fig. 2(b) indicates that the PBS layer is not perfectly uniform and may suffer from thickness variation, and that some "holes" in the coating layer may also exist (Fig. 2a). However, a statistical analysis of the inter-particle force discussed later in the paper (see Sec. III) shows that the presence of defects weakly affects the cohesion between two particles. Since the coating layer is very thin, no capillary structure has been observed in optical microscopy when putting two

beads in contact.

A last important remark is that the strong Si-OH link between the polymer and the glass bead surface helps the PBS to stick permanently on the particles. No drainage of the fluid was observed even for very long time (on the timescale of a year), making the material very stable in time, as will be discussed in section Sec. IV.

III. INTER-PARTICLE COHESION FORCE MEASUREMENTS

The cohesion force between two particles is a key parameter to control the macroscopic cohesion of a granular assembly. In this section we present the results of different experiments designed to measure the contact force between two particles due to the PBS coating. As presented in the previous section, the control parameters are: the size of the beads (diameter d), the PBS averaged layer thickness b, and the duration of the contact. We designed two methods to accurately measure the cohesion force. The first method uses the torque-meter of a commercial rheometer (MCR501 Anton Paar). It provides accurate measurements but performing a statistical analysis is tedious to. The second method is a home-designed force measurement device allowing simultaneous measurements on 10 pairs of particles.

A. Role of the pre-compression load

The cohesion force between two CCGM particles has been first measured using the rotating head of a Anton-Parr MCR501 rheometer. A sketch of the experimental set-up is given in Fig. 3a. A coated particle is attached to a fixed rigid structure through a linear spring and a similar coated particle from the same batch is glued at the end of an arm attached to the rheometer head. The two particles were put in contact and a pre-compression torque T pc corresponding to a pre-compression force F pc was applied before slowing reversing the applied torque up to the point when the two particles suddenly detach. The critical torque when detachment occurs provides the measurement of the cohesion force F c . (Fig. 3a).

We measured the cohesion force using d = 10 mm particles and with a coating b = 5 µm, and the precompression force F pc was varied from 0.08 N to 2 N. Fig. 4(a) shows that the cohesion force F c does not depend on the pre-compression force F pc , and that the order of magnitude of the cohesion force is F c ≈ 5 mN (dashed line). This independence of the cohesion with the compression force has been also observed in a different system by Kobayashi et al. [START_REF] Kobayashi | Dem-cfd analysis of fluidization behavior of geldart group a particles using a dynamic adhesion force model[END_REF]. With this setup, we also studied if the cohesion force was affected by the number of successive contacts. One can wonder if the polymer layer can be altered after the first sticking contact. Fig. 4(b) shows for three different pre-compression forces that the cohesion force is independent of the number of successive contacts. We therefore conclude that the PBS layer is strongly attached to the glass bead surface and that the stick-pull process occurring for a binary contact is reversible. A last important remark is that variation in the mean cohesion force is observed in Fig. 4(b): the mean cohesion is F c = 4.3 mN for the triangle symbols and F c = 3.7 mN mean force for the star symbols. This is an indication that the cohesion force may vary from one pair of particle to another and that a statistical analysis is necessary. This has motivated us to develop a second experimental set-up to measure in parallel the cohesion force for 10 pairs of particles.

B. Role of the contact waiting time

The second home-made force measurement device is sketched in Fig. 3b and consists in a set of 10 independent parallel pendulums. Each pendulum had one particle (B) attached at the bottom of the arm (Fig. 3b), which came into contact with a fixed particle (A). A third particle (C) was also glued on the other side of the pendulum arm and played the role of a counter-weight. The setup was mounted on a table that can be inclined. Starting from a nearly horizontal position (step 1 in Fig. 3, the table was slowly inclined with a rate 10 • •min -1 (step 2) until all the pairs of particles detached (step 3). The whole measurement process was recorded with a camera, and each time a pair of particles was detaching, the angle α c was recorded and the cohesion force F c was computed from the torque balance.

With this device, the influence of the contact waiting time between two coated particles has been investigated from 5 s to 2 hours. We also investigated a 24 hours waiting time but the results were not significantly different from the 2 hours result. Fig. 5 shows that the cohesion force varies with contact time t c for t c 600 s) but eventually saturates for long contact times t c > 1000 s. This confirms the qualitative observations made when 0.00 0.25 0.50 0.75 1.00 1. [START_REF] Jarray | Cohesion-driven mixing and segregation of dry granular media[END_REF] handling the CCGM out of storage. A CCGM stored during a long time looks more cohesive, although a vigorous shaking of the packing which renews all the contacts seems to diminish the cohesive nature of the sample. In the following, we now refer to "short" waiting time experiments when t c = 10 s, and "long" waiting time experiments when t c = 10 min.

The cohesion force distribution has been measured for a hundred pairs of particles out of the same batch (d = 5 The averaged cohesion force is 0.56±0.1 mN and 1.14±0.3 mN for short and long contact time respectively. For t c = 10 s the cohesion force distribution is narrower than for t c = 10 min. We have not investigated in more details the influence of the contact time and the origin of the force distribution, which are certainly related to the coating property of the particle and to the entanglement dynamics of the polymer chains.

C. Scaling of the cohesion force

To understand the physical origin of the cohesion force, we have systematically studied how it varies with the particle diameter d and the averaged PBS coating thickness b. In Fig. 7, the cohesion force is plotted as a function of the particle diameter d for a constant layer thickness b = 2 µm, and for the short and long waiting contact times. In the range 0.8 < d < 7 mm the cohesion force increases with the particle diameter. The cohesion force varies linearly with the diameter for short contact time but exhibits a more rapid increase for long contact time. The linear variation can be well described by a capillary model at contact [START_REF] Johnson | Surface energy and the contact of elastic solids[END_REF]

F c = 3 2 πγd, (2)
where the surface tension γ ≈ 24 mN•m -1 , a relevant order of magnitude for PDMS. For long contact times, other molecular phenomena may occur, such as a slow polymer entanglement between PDMS polymers and we did not investigate further the long time correlation between the cohesion force and the particle radius.

The influence of the coating PBS thickness b on F c is studied in Fig. 8 for different particle diameters d. The cohesion force normalized by the expression ( 2) is plotted as a function of b. We first observe that all the data obtained for different particle diameters collapse on a single curve. The normalized cohesion force starts from zero when there is no coating, increases and reaches a plateau equal to 1 when the averaged thickness of PBS is larger than 1µm. An ad-hoc expression for the cohesion force can be proposed:

F c = 3 2 πγd 1 -e -b/B , (3)
where B ≈ 230 nm is a characteristic thickness, which is a few times the particle roughness value (30 ± 2 nm). This behavior is reminiscent of what is observed with liquid capillary bridges. In this latter case the cohesion force increases when increasing the amount of liquid, up to the point where the liquid screens the surface roughness and that a single bridge exists, giving rise to the saturated force given by Eq. 2 [START_REF] Halseyand | How sandcastles fall[END_REF].

From this expression, we can write the expression for a Bond number, i.e. the ratio of the weight of the particle over the cohesion force, a dimensionless number that will be useful in the following :

Bo = 1 9 ρ G gd 2 γ 1 -e -b/B . (4)
The threshold value Bo = 1 gives a critical particle size for which the weight is balanced by the cohesion force. A typical example of Bo ≈ 1 is given by a coating thickness b = 2 µm with particles d = 3 mm, as illustrated in figure 1.

IV. BULK BEHAVIOR OF THE CCGM

The previous sections were dedicated to the measure of the cohesion force between pairs of particles. In this section we discuss the collective behavior of the coated particles in several classical configurations used in the literature for characterizing granular media. The goal is to show that the ability to precisely control the cohesion force between the grains open new perspectives to understand the influence of the inter-particle force in the dynamics of cohesive granular media. This section presents experimental results for the bulk density, for the angle of repose of static piles, and for the onset of the flow of a layer of CCGM resting on an inclined plane.

A. Packing fraction

In many industrial processes, the bulk density of a granular assembly is a qualitative indicator of the cohesive property of the medium [START_REF] Standish | An experimental study of the packing of a coal heap[END_REF][START_REF]Powder Flow: Theory, Characterisation and Application[END_REF]. The Haussner ratio (H = ρ T /ρ B ) or the Carr index (defined as 100(1-1/H)) are often used both implying the measurement of the bulk density in two different compaction states: the aerated density ρ B (similar to the random loose packing state) and the tapped density ρ T / (similar to the random close packing). In the following we investigated how the loose packing fraction changes for CCGM batches when varying the PBS coating. The random loose packing fraction φ rlp was evaluated through mass and volume measurements in a 250 cm 3 graduated cylindrical test tube. The results are presented in Fig. 9 for d = 680 mm particles and for a coating layer thickness varying from 72 to 624 nm. The best collapse of experimental data is obtained with a plot of the packing fraction versus the volume ratio Ω P /Ω G which is proportional to b/d.

For very low values of the coating (Ω P /Ω G ≈ 10 -4 ), the packing fraction is equal to the packing fraction of clean and dry glass beads. The packing fraction decreases for an increasing PBS content, and a very low packing fraction φ rlp ≈ 0.45 may be reached for a typical Ω P /Ω G ≈ 10 -2 value. This can be explained by the existence of large-scale voids and arches in the bulk sustained by strong cohesive links between particles.

B. Angle of repose

The measure of the slope angle of a granular heap is also a way to emphasize the role of the cohesion in a powder or in a granular material [START_REF] Albert | Maximum angle of stability in wet and dry spherical granular media[END_REF][START_REF] Miles | The relation between the moisture content and the test weight of corn[END_REF][START_REF] Wolfand | Experimental study of the flow of coal in chutes[END_REF][START_REF] Tegzes | Liquid-induced transitions in granular media[END_REF][START_REF] Samadaniand | Angle of repose and segregation in cohesive granular matter[END_REF][START_REF] De Ryck | Shape of a cohesive granular heap[END_REF]. With our CCGM, static piles were made from a chute flow from a hopper on a 5 cm diameter rough disc. A side-view camera captured the image of a pile and the angle of repose α r is obtained from image analysis. Examples of images of piles are given in Fig. 10. The value of the repose angle is averaged over 20 measurements. The repose angle is observed to increase when increasing the PBS thickness. Without coating (Fig. 10a), the heap presents a smooth surface with a constant angle. Adding some cohesion gives rise to steeper slopes, and also to abrupt local variations of the local angle as seen illustrated in Fig. 10(b-d).

Fig. 11 shows that the angle θ r increases with the coating thickness b, with a sharp increase for b ≈ 40 nm and seems to saturate for coatings larger than 50 nm. We compare our results with the repose angle obtained with the crater method using vacuum pump oil coated particles [START_REF] Albert | Maximum angle of stability in wet and dry spherical granular media[END_REF][START_REF] Tegzes | Liquid-induced transitions in granular media[END_REF] in figure 11. While the heap formation methods are different, the CCGM presents a similar trend. Our experimental setup is limited to low-cohesion values because the materials prepared with a large cohesion (b > 100 nm) experience difficulties to flow through the hopper under gravity only.

The heap angle experiment is also a benchmark test to assess the stability and the durability of the CCGM. The first test concerns the stability with temperature since the cohesion is based on a cross-linked polymer. Piles have been prepared with a CCGM stored in controlledtemperature devices. As shown in Fig. 12 perature.

We have also investigated the stability of the CCGM with time by measuring the heap repose angle for the same batch of particles at different ages. Fig. 12(b) shows the evolution of the repose angle α r for three different materials at different ages from preparation. This plot shows that for thin coatings (b = 16 or b = 31 nm), the heap angle remains identical even for sample prepared one year ago. For a thicker coating (b = 62 nm), a slow decrease of the repose angle has been observed. Nevertheless, the PBS-coated CCGM seems to be stable for months and large batches can then be prepared before performing large-scale experiments. This CCGM also seems to be unaffected by the moisture content of the ambient air since our experiments were performed without humidity control across different temperature and humidity conditions over a year.

C. Onset of flow on an inclined plane

Measuring the onset of flow of a layer of particles lying on a rough inclined bed is another way to investigate the friction and the cohesion of a material. The simplest description of the plasticity of a granular material assumes that the yield stress follows a cohesive Mohr-Coulomb criterion, τ yield = µP + τ c , where µ is the friction coefficient, P the confining pressure and τ c the cohesive stress. Starting from an horizontal plane and increasing progressively the inclination θ, a layer of thickness h will start to flow at a critical angle θ start when the shear stress at the base reaches the yield stress value:

ρ G φgh sin θ start = µρ G φgh cos θ start + τ c ( 5
)
where φ is the volume fraction of the layer. This equation can be simplified as

h sin θ start = µh cos θ start + h c (6)
where h c = τ c /(φρ G g) is a characteristic cohesive length, which represents the maximum thickness of a self-standing vertical layer of granular medium stuck to a rough surface under gravity. Eq. 6 shows that the cohesion length h c and the friction coefficient µ can be identified by systematically measuring the critical starting angle θ start for different thicknesses h. We have conducted such a series of experiments with our model cohesive material. For seek of efficiency, we have not used a uniform layer as initial state, but rather a prismatic deposit, as sketched in Fig. 13(a). With this geometry, it is possible in a single experiment to perform several measurements of (h, θ start ).

A typical experiment is conducted as follows. The CCGM is poured on a 20 × 6 cm 2 rectangular rough plate (the roughness is made with CCGM particles glued on a double sided adhesive tape) with two prismatic side walls. The free surface of the deposit is then leveled following the two side walls. The final prismatic volume has a thickness varying linearly from 15 to 25 mm (see Fig. 13a). The thickness of the granular layer is measured with a laser sheet technique and the angle with a clinometer. Starting from a very low angle of inclination (typically 10 • ), the setup is slowly inclined at a constant rate. A first avalanche occurs at the bottom thick side (Fig. 13b), leaving a thinner and shorter prism. When the angle is further increased, a second avalanche occurs (Fig. 13c), which corresponds to a different h, and so on. In one experiment, one can then extract the critical angle θ start for 4 to 5 different thicknesses. For a single CCGM batch, this experiment is repeated several times. The collected data are then plotted in a (h cos θ start , h sin θ start ) plane. According to Eq. 6, a linear fit of experimental data gives the slope µ and the intercept value h c for a given CCGM. Fig. 13(d) shows the (h cos θ start , h sin θ start ) plot for small glass beads of diameter d = 202 ± 4 µm. Results are given for 4 coatings of increasing thickness b. Despite some experimental noise, the linear expression (5) fits well the experimental data. Increasing the coating thickness b increases the cohesive length h c (the intercept of the linear fit with the vertical axis), but does not significantly affect the friction coefficient (the slope of the lines). From the measure of h c , one can then estimate the cohesive stress τ c .

The stress τ c is a macroscopic measurement of the cohesion, which can be compared to the inter-particle cohesion force measured in section III. From a dimensional analysis, the scaling relating the cohesive shear stress and the cohesion force is τ c d 2 ∝ F c . Following [START_REF] Richefeu | Shear strength properties of wet granular materials[END_REF], a theoretical expression based on a mean field approach relates the macroscopic cohesion τ c to the cohesion force:

τ c = 3µφZF c 2πd 2 , (7)
with µ the friction coefficient, φ the volume fraction and Z the averaged coordinance number (number of contacts per particle). Fig. 14 gathers our data for different particle sizes and different PBS coatings and shows a linear trend between τ c d 2 and F c although it is not perfect especially for small particles. The presence of other cohesive forces like humidity may explain this discrepancy observed for small particles. Taking the experimental averaged value µ = 0.4 along with φ = 0.6 and Z = 6, the theoretical prediction is plotted in Fig. 14 and gives indeed a good estimate of the measured cohesion. The insert of Fig. 14 also shows that the macroscopic friction coefficient µ seems independent of the coating property of the particles.

V. CONCLUDING REMARKS AND PERSPECTIVES

A cohesion-controlled granular material (CCGM) has been designed with glass beads coated by a thin layer of polyborosiloxane polymer. The preparation technique is simple, the resulting CCGM is very robust and can be used several times in various situations to model industrial or natural cohesive granular materials. We have investigated how the inter-particle cohesive contact force varies with the thickness of the coating and with the particle size and have showed that the cohesion force does not depend on the pre-compression force. We also showed that the cohesion force slightly evolves with time and eventually saturates for long time. Different experimental setups were used to put in evidence the influence of the cohesion on various macroscopic quantities. We have illustrated the effect of the cohesion on the packing fraction of loose samples, on the repose angle of heaps, on the onset of flow of a granular deposit on a rough bed. In all cases we have shown that controlling the inter-particle force is a way to controlled the macroscopic properties. This CCGM seems to be very stable in time and weakly affected by the humidity or the temperature of the laboratory. This is a major advantage compared to previous capillary cohesion techniques based on Newtonian fluids where drainage, evaporation, capillary bridges recombination may occur during an experiment.

In this paper we have restricted our observation to static configurations. However, the possibility to control the cohesion open many perspectives to study also the flow properties of cohesive materials. Configurations such as flows down inclined planes, flows in hoppers and silos, flows in rotating drums, mixing or segregation may also be investigated, with the hope that it will help to better understand the physics beyond the concept of "flowability" of cohesive granular materials.

FIG. 1 .

 1 FIG. 1. Example of a cohesion-controlled granular material: a pile of glass beads d = 3 mm with a PBS coating layer of thickness b = 2.2 µm .

FIG. 3 .

 3 FIG. 3. (a)Sketch of the setup to measure the cohesion force for different pre-compression force using the rheometer torque-meter. The two particles are put in contact with a precompression force Fpc = Tpc/L, where L = 3.5 cm is the arm length and the cohesion force Fc = Tc/L is measured when the two particles detaches. (b) Sketch of the pendulum experimental setup. Particle A is attached to a rigid structure, particle B and C are attached to the two sides of a pendulum. Fc is measured by inclining the setup. 10 pendulums were mounted in parallel.

Fpc = 1 . 25 NFIG. 5 .

 1255 FIG. 4. (a) Cohesion force measured for different precompression forces d = 10 mm, b = 5 µm. The dashed line indicates the mean cohesion force. Empty coloured symbols refer to the legend of (b). (b) Cohesion force for successive contacts, and for different pre-compression forces. The contact waiting time was kept constant equal to 10 minutes. If not visible, the error bars are smaller than the symbol size.

FIG. 6 .

 6 FIG. 6. Probability distribution function of the cohesion force measured for approximately 100 pairs of particles, for two different contact times (tc = 10 s and tc = 10 min) and for d = 5 mm, b = 2 µm coated particles.

FIG. 7 .

 7 FIG. 7. The cohesion force Fc as a function of the particle diameter d for short (10 s) (circles) and long (10min) (squares) contact times. The dashed line is the linear expression (2).

FIG. 8 .

 8 FIG. 8. Cohesion force normalized by3 2 πγd as a function of the mean PBS layer b for short contact times and for different particle sizes.

FIG. 9 .

 9 FIG. 9. Random loose packing fraction of various CCGM with different particle sizes and different coating values. The packing fraction decreases with the volume ratio ΩP /ΩG. The legend indicates the beads diameter.

FIG. 10 .

 10 FIG. 10. Images of piles for a CCGM with d = 480 µm with increasing PBS coating: (a) no coating, αr = 27.7 ± 0.8, (b) b = 31 nm, αr = 30.1 ± 0.9, (c) b = 52 nm, αr = 40.3 ± 1.9, (d) b = 62 nm, αr = 42.5 ± 2.2

FIG. 12

 12 FIG. 12. (a) Effect of the temperature on the repose angle of a CCGM (d = 480 µm, b = 62 nm). (b) Heap repose angle αr as a function of the time since the preparation of the CCGM. Experiments were made with d = 480 µm particles.

FIG. 13 .

 13 FIG. 13. (a)-(c): Sketch of the inclined plane setup with the variable granular thickness and a progressive inclination. (d): Inclined plane results for d = 202 µm CCGM particles with increasing coating thickness. Dashed lines are best fits using Eq. (6).

dFIG. 14 .

 14 FIG.[START_REF] Albert | Maximum angle of stability in wet and dry spherical granular media[END_REF]. The macroscopic cohesion force τcd 2 measured from inclined plane experiments as a function of the inter-particle cohesive force Fc. The dashed line is the prediction from Eq. (7).

ACKNOWLEDGEMENTS

We thank Inès Basses, Davide Di Giusto and Amina Abbedou for their help during the experiments and Sandip Mandal for fruitful discussion and improvement of the paper. This work is part of the COPRINT [37] project and was supported by the ANR grant ANR-17-CE08-0017.