Norinda Chhim

Elsi Haddad

Thibaut Neveux

Céline Bouteleux

Sébastien Teychené

Béatrice Biscans
email: beatrice.biscans@toulouse-inp.fr

Performance of green antiscalants and their mixtures in controlled calcium carbonate precipitation conditions reproducing industrial cooling circuits

Cooling circuits in many industrial sectors are faced with daily issues of scaling. One preventive treatment consists in injecting a polymer additive in the circuit to inhibit precipitation of calcium carbonate. Among the used additives, very few are "green" and the efficiency of new candidates are difficult to test directly in industrial conditions. The present study compared performance between two "green" polymer additives, polyaspartic acid (PASP) and polyepoxysuccinic acid (PESA), versus a traditional gold-standard, homopolymer of acrylic acid (HA) in a laboratory scale set-up designed to be representative of an industrial circuit. Results showed that HA and PASP are both inhibitors of calcium carbonate crystal growth. This inhibition resulted from adsorption of polymer additive molecules on the crystal surface, as confirmed by adsorption measurement. Under the same conditions, PESA additive, showed a high rate of calcium ion complexation and a very low inhibition rate. But, PESA was shown to be a nucleation delayer. Mixing PESA and PASP can gave nucleation retardation of about 19 h, which approximates the 24 h water residence time in industrial cooling circuits, as well as almost 90% calcium carbonate crystal growth inhibition. This synergy offers promising prospects for preventive scaling treatment.

Introduction

Industrial cooling circuits are subject to clogging and deposit of mineral particles such as calcium carbonate (CaCO 3 ) associated to the suspended matter naturally present in cooling waters. Deposits are localized inside the condenser tubes and on the PVC packing in cooling towers. They can lead to layers of hard scale, increasing heat transfer resistance and thus impairing heat exchange. This is a serious problem in industry, especially in high energy consumption equipment such as thermal and nuclear power plant cooling systems [START_REF] Neveux | Pilot plant experiments and modeling of CaCO 3 growth inhibition by the use of antiscalant polymers in recirculating cooling circuits[END_REF][START_REF] Rahmani | Evaluation of inhibitors and biocides on the corrosion, scaling and biofouling control of carbon steel and copper-nickel alloys in a power plant cooling water system[END_REF], reverse osmosis membrane desalination [START_REF] Al-Hamzah | A comparative study of novel scale inhibitors with commercial scale inhibitors used in seawater desalination[END_REF][START_REF] Phuntsho | Membrane scaling and flux decline during fertiliser-drawn forward osmosis desalination of brackish[END_REF], distillation [START_REF] Al-Rawajfeh | Simultaneous desorption-crystallization of CO 2 -CaCO 3 in multistage flash (MSF) distillers[END_REF][START_REF] Ghani | Impacts of different antiscalant dosing rates and their thermal performance in Multi Stage Flash (MSF) distiller in Kuwait[END_REF] and petroleum production [START_REF] Heming | Synthesis and performance of a polymeric scale inhibitor for oilfield application[END_REF]. In electric power plant cooling systems, CaCO 3 precipitation and/or deposition depends on various factors such as circuit operating conditions (temperature range, concentration factor, water residence time) and circulating water quality (notably, concentrations of calcium and bicarbonate and of suspended matter) [START_REF] Chhim | Inhibition of calcium carbonate crystal growth by organic additives using the constant composition method in conditions of recirculating cooling circuits[END_REF]. Varying hydrodynamic regimes (turbulent in condenser tubes and laminar on the PVC packing of cooling towers) and construction materials (concrete housing, condenser material, PVC packing) also contribute to scale deposition. Technically, deposits impair heat exchange, partially or totally clog tubing and reduce equipment life-expectancy, and financially lead to loss of productivity and increased operating and preventive and curative maintenance costs.

Polymer additives have been widely studied, for their effect on dispersion of suspended matter to reduce clogging and on inhibition of scaling [START_REF] Hasson | State of the art of friendly "Green" scale control inhibitors: a review article[END_REF][START_REF] Bu | Inhibition of calcium carbonate and sulfate scales by a non-phosphorus terpolymer AA-APEY-AMPS. Desalin[END_REF].

Additives commonly used for scale inhibition are polymers containing carboxylic acid groups such as polyacrylic acid [START_REF] Moulaya | Low molecular weight poly(acrylic acid) as a Salt scaling inhibitor in oilfield operations[END_REF][START_REF] Al-Hamzah | Inhibition of homogenous formation of calcium carbonate by poly(acrylic acid). The effect of molar mass and end-group functionality[END_REF][START_REF] Amjad | Calcium sulfate dihydrate (gypsum) scale inhibition by PAA, PAPEMP, and PAA/PAPEMP blend[END_REF], polymaleic acid [START_REF] Shen | The effect of synthesized hydrolyzed polymaleic anhydride (HPMA) on the crystal of calcium carbonate[END_REF][START_REF] Amjad | Calcium sulfate dihydrate (gypsum) scale inhibition by PAA, PAPEMP, and PAA/PAPEMP blend[END_REF] and polyaspartic acid [START_REF] Quan | Experimental study on scale inhibition performance of a green scale inhibitor polyaspartic acid[END_REF][START_REF] Pramanik | Antiscaling effect of polyaspartic acid and its derivative for RO membranes used for saline wastewater and brackish water desalination[END_REF], which is a well-known green inhibitor in terms of biodegradability, non-toxicity and non-bioaccumulation [START_REF] Martinod | Electrodeposition of a calcareous layer: effects of green inhibitors[END_REF][START_REF] Hasson | State of the art of friendly "Green" scale control inhibitors: a review article[END_REF]. These polymers have important properties, including low concentration effectiveness and high temperature endurance.

Those last years, a lot of effort s have been done to generate green inhibitors. In their review, [START_REF] Chaussemier | State of art of natural inhibitors of calcium. A review article[END_REF] focused on some green antiscalants, obtained either by using "natural" organic molecules or extracted from plants. The authors described some positive performance in laboratory tests but underlined that trials of these molecules in pilot plant are limited [START_REF] Schweinsberg | New stable biodegradable scale inhibitor formulations for cooling water: development and field tests[END_REF] . However, a study on the inhibition performances of polyaspartic acid on a cooling-water pilot installation by Laborelec company (Belgium) can be found in [START_REF] Girasa | Polyaspartate, an new alternative for the conditionning of cooling water[END_REF] .

Several works in the literature were focused on polyepoxysuccinic acid (PESA) and polyaspartic acid (PASP) because they are green scale inhibitors given their non-nitrogenous, nonphosphorus, and biodegradable features. Experimental results of [START_REF] Zhou | Inhibition and dispersion of polyepoxysuccinate as a scale inhibitor[END_REF] obtained by static beaker test on inhibition to CaCO3, demonstrated that PESA functioned excellent nucleation inhibition to CaCO3. The scale inhibition ratio was calculated from the change in cation concentration. Tests were performed at temperature 80 °C, solution pH 9.0, and reaction time 10 hr. Above 99.0% of the scale inhibition ratio was obtained when PESA dosage is 30.2 mg/L with 500 and 720 mg/L of Ca2 + concentrations.

In another work, [START_REF] Sun | Study on polyepoxysuccinic acid reverse osmosis scale inhibitor[END_REF] used the reverse osmosis scale inhibitor for static and dynamic study of PESA inhibition at 30 °C and pH 7.4. PESA (5, 10, 15 mg/L) was added to the testing water samples. Their results suggest that PESA should be an effective nucleation scale inhibitor that is applicable to reverse osmosis treatment of waters with a wide range of ion compositions. The scale inhibition ratio of PESA is above 90% and at the same water quality is directly proportional to the dosage of PESA, and scale inhibition ratio at the same PESA dosage decreased with the increase of calcium concentration. [START_REF] Liu | Comparative performance of polyepoxysuccinic acid and polyaspartic acid on scaling inhibition by static and rapid controlled precipitation methods[END_REF] compared the anti-scaling performance of PESA and PASP using the static and rapid controlled precipitation methods (Ca2 + : 500 mgL-1, pH: 9, T:80 °C, t: 8 h). The scale inhibition efficiency, was calculated from the change in cation concentration. During the experiments, the variation in pH and resistivity with time of the water samples was measured. The maximum in the pH-time curves corresponds to the precipitation threshold in the water. In these conditions PESA showed better performance than PASP on nucleation inhibition. Their tests showed that PESA could be very efficient at very low concentration.

More recently [START_REF] Peronno | Study of the inhibition effect of two polymers on calcium carbonate formation by fast controlled precipitation method and quartz crystal microbalance[END_REF] investigated the inhibition effect of poly(acrylic acid-co-maleic acid) (PAMA) and polyaspartic acid (PASP) on the nucleation/growth process of CaCO3 in a bulk solution and on a metallic surface using fast controlled precipitation(FCP) method and electrochemical quartz crystal microbalance (EQCM). They showed that these inhibitors were very efficient at low concentration (4 mg •L -1 ) and that they modified the morphology of calcium carbonate crystals.

Moreover, little is known of polymer additive action on scaling under conditions representing river-water quality (pH, supersaturation, suspended matter, etc.) and industrial cooling circuit operation (temperature, residence time, etc.).

Different tests conditions were applied in the previous works cited above for evaluating the inhibition effects of PASP and PESA. It is important to notice that PESA was demonstrated to be a nucleation inhibitor at low concentration, in water initially free of calcium carbonate particles. But considering the previous experimental set-ups, it is difficult to withdraw nucleation and growth kinetics laws separately and to include in these laws process parameters such as supersaturation. Indeed, one of the main issues is of these previous studies is to extrapolate the results to industrial pilot plants.

In our work, seeds of CaCO3 are introduced in the reactor in order to mimic suspended matter in river water and favor crystal growth in controlled conditions. Inhibition rate is then defined in this work by comparing R0 and Ra which are respectively calcium carbonate growth rate, in mol •m -2 •min -1 , without and with additive. This enable to use very low quantity of polymers.

In a previous study [START_REF] Chhim | Inhibition of calcium carbonate crystal growth by organic additives using the constant composition method in conditions of recirculating cooling circuits[END_REF], the constant composition method, was adapted for calcium carbonate precipitation, in order to replicate experimentally the operating conditions of recirculating cooling water systems, especially with respect to the supersaturation range, presence of suspended matter (such as illite, silica, calcite) and the temperature range encountered. Determination of the thermodynamic driving force (supersaturation) was based on the relevant chemical equilibria, total alkalinity and calculation of the activity coefficients, using PHREEQC software.

In this study, this constant composition method precipitation (constant supersaturation, pH and ionic force) was applied to investigate the influence of polyepoxysuccinic acid (PESA), polyaspartic acid (PASP) and their mixture and to compare the inhibition results to homopolymer of acrylic acid (HA), a classical polymeric additive. So, scaling inhibition performance was investigated through dynamic constant composition precipitation experiments to obtain CaCO 3 growth kinetics in conditions representative of an industrial circuit. The calcium carbonate scale samples were characterized on scanning electron microscopy (SEM) and X-ray diffraction (XRD), and possible mechanisms of inhibition are proposed. These results provide a basis for using new green additives in industrial circulating water systems by synergic treatment of chemicals.

Material and methods

Materials

Commercial calcite powder (99% pure, Merck, reference 1.020 6 6) was used as seeds in all experiments. Measurement of calcite particle size distribution in solution was performed by laser light scattering granulometry showing a mean size of 34.7 μm and a specific area of 0.266 m 2 •g -1 . Calcium chloride dihydrate (purity 99-105 % ref. 141232.1210) was purchased from Panreac. Anhydrous sodium carbonate (99.95 to 100.05 % purity, ref. 223484) and sodium chloride (ACS reagent, purity ≥99.0%) were purchased from Sigma-Aldrich.

All the reagents benzethonium chloride, trisodium citrate dehydrate, ethylenediaminetetraacetic acid (with purity ≥ 99%) were purchased from Sigma Aldrich.

Green polymer scale inhibitors have been chosen according to the their biodegrability [START_REF] Hasson | State of the art of friendly "Green" scale control inhibitors: a review article[END_REF][START_REF] Harris | Biodegradation and testing of scale inhibitors[END_REF][START_REF] Wilson | Development of a 'Green' hydrothermally stable scale inhibitor for geothermal wells and pipelines[END_REF], their lethal concentration and their bioaccumulation properties defined by [START_REF] Hasson | State of the art of friendly "Green" scale control inhibitors: a review article[END_REF] . They were used as received without any further modification. Table 1 shows the polymers used in this study and their characteristics and suppliers.

Constant composition method

Fig. 1 shows the main elements of the constant composition set-up. Precipitation in presence of seeds particles took place in a 1,0 0 0 mL agitated double-jacketed reactor. A PTFE cover was designed to minimize the gaseous head space above the solution to prevent ambient CO 2 exchange liable to reduce solution pH.

In each experiment, solution temperature, supersaturation and pH were held constant. A temperature regulator kept working temperatures within the industrial cooling circuit operational range of 25-45 °C. A conductivity electrode monitored variation of the ionic force in solution [START_REF] Chhim | Inhibition of calcium carbonate crystal growth by organic additives using the constant composition method in conditions of recirculating cooling circuits[END_REF]. The constant composition method relies on the use of two automated burettes, controlled by Tiamo TM 2.5 software (Metrohm), filled with solutions of calcium chloride (reagent 1) and sodium carbonate (reagent 2) corresponding to concentrations of 7.08 × 10 -2 mol •L -1 of calcium and carbonate ions (calculated by PHREEC software), respectively and a sensor able to follow reagent consumption due to crystal growth. In the case of calcium carbonate, according to Eq. (1) , calcium carbonate precipitation leads to consumption of Ca 2 + and HCO 3 -ions accompanied by formation of H + protons, reducing pH below the threshold value defined initially.

Ca 2 + + HCO 3

- CaCO 3 + H + (1)
Consequently, a high-precision pH electrode (Unitrode Easyclean, Metrohm) was used to detect pH variations during CaCO 3 precipitation and to trig reagent injection in order to maintain a constant pH during the precipitation process.

To compensate the reactant consumption due to crystal growth and thus to restore the pH at its initial value, the burettes injected equal volumes of reagents 1 and 2 with a precision of 0.001pH. Injected reagent volume variation over time showed a dV dt slope on the basis of which the growth rate (R) of calcium carbonate crystals introduced in the reactor, expressed as mol •m -2 •min -1 , is given by Eq. ( 2

) : R = dV dt C R S T (2)
where dV dt is the reagent compensation slope in L •min -1 , C R and S T are respectively the reagent concentration in mol •L -1 and the total surface area of the seeds initially introduced in the precipitation reactor (in m 2 ).

The typical curves obtained during constant composition crystallization experiments are schematically presented in Fig. 2 .

In order to study the effect of additives on calcium carbonate precipitation, in conditions that mimic the operating conditions of recirculating cooling water systems, for all the experiments, a set of operating conditions were kept constant and are given in Table 2 .

Calcite crystals characterization

Each synthesized sample was characterized by powder X-ray diffraction (XRD), and scanning electron microscopy (SEM). The powder X-ray diffraction data were collected with a Bruker D8-2 diffractometer equipped with a Bragg -Brentano θ -θ geometry and a copper anode ( λ (Cu K α1) = 1.54060 Å and λ (Cu K α2) = 1.54439 Å). The X-ray diffraction patterns were obtained for a range of 2 θ angles from 5 to 50 °with a step of 0.2 °. Scanning electron microscopy (SEM) micrographs were obtained using a SEM-FEG JEOL JSM 7100F microscope. The samples were goldplated before observation.

Free Ca 2 + ion volumetric assay protocol

Free Ca 2 + ion concentration was assessed by volumetric assay in titrated EDTA (ethylene-diamine tetra-acetic acid) solution at 4.10 -3 mol •L -1 in presence of 30 μL of colored Patton-Reeder indicator at 1 g •L -1 and 1 mL NaOH solution at 2 mol •L -1 . To limit solution volume variation in the precipitation reactor due to successive sampling, sample volume was limited to 1 mL and sample number to 3 or 4.

Measurement of polymer adsorption isotherms

Adsorption experiments were performed in a series of 8 identical hermetically capped bottles. Each bottle was filled with 200mL of supersaturated solution of calcium carbonate, 20mg of calcite seed, and polymer with a concentration ranging from 0.1 to 1.2 mg •L -1 . The bottles were immersed in a water thermostatic bath and the temperature was kept constant at 35 °C. After 6 days, the solutions were filtered at 0.2 μm, collecting filtrate for assay of residual additive and of calcite seeds for SEM characterization.

Residual additive concentration (C e ) in the solution at 7.6 supersaturation at end of each test was assayed on UV-Visible spectrophotometry using 2 reagents: benzethonium chloride and trisodium citrate dihydrate. The assay principle is based on proportionality between turbidity and additive concentration. Turbidity is

•L -1 ) Calcite seed concentration (mg •L -1 ) 100
The seeds concentration corresponds to values measured in the river during moderate flooding [START_REF] Idlafkih | Comportements des contaminants en trace dissous et particulaires (As, Cd, Cu, Hg, Pb, Zn) dans la Seine[END_REF] caused by the reaction between benzethonium chloride and the functional groups of the polymer additive. Trisodium citrate dihydrate is needed to complex the free calcium ions in the sample in order to reduce interference resulting from complexation by polymer additive molecules. All assays were performed on a doublebeam Shimadzu 1800 spectrophotometer with 10 cm quartz optical trajectory tanks, with working wavelength set at 306 nm. This tank length reduces the limit of quantification to 0.1 mg •L -1 , enabling assay of low polymer additive concentrations after the adsorption tests (more details on the procedure are given in Appendix).

Results

Crystal growth experiments without additives

To assess the inhibitory effect of the selected additives, reference experiments were performed using the constant composition method for three different tem peratures (25 °C, 35 °C and 45 °C). The chosen temperatures correspond to the operational temperature range of an industrial cooling circuit. Each experiment was repeated at least three times. The evolution of the volume added during the experiment for each experiment are presented in Supplementary Material (Fig. SM1) and calcium carbonate crystal growth rate without additive (R 0 ), calculated from Eq. ( 2) are given in Table 3 .

Calcium carbonate crystal growth rate (R 0 ) without additive, based on Eq. ( 2) , increased with temperature ( Table 3 ) in the oper-ational range of an industrial circuit, suggesting that kinetics under our experimental conditions followed Arrhenius's law relating calcium carbonate growth (R 0 ) to activation energy (E a ) by Eq. ( 3) :

R 0 = k 0 e -E a R g T (3)
where R 0 is the growth rate without additive (mol •m -2 •min -1 ), k 0 is a pre-exponential constant, E a is the activation energy (kJ •mol -1 ), R g the universal constant for perfect gas (under normal conditions, R = 8.314 J •mol -1 •K -1 ) and T the absolute temperature (Kelvin).

The activation energy of calcium carbonate growth thus obtained is 53.4 kJ •mol -1 suggesting that the calcium carbonate precipitation could be surface-controlled or diffusion controlled. This value is in good agreement with the literature involving CaCO 3 precipitation on crystal seeds, where it ranges between 22 and 155 kJ •mol -1 . For example, [START_REF] Parsiegla | Calcite growth inhibition by copper (II) I. Effect of supersaturation[END_REF] At 25 °C and 35 °C, Powder X-Ray Diffraction (PXRD) measurements (not shown here) have shown that only calcite crystals were obtained during these experiments. In addition, SEM pictures show well defined calcite crystals with their typical rhombohedral shape, and smooth surface (see Fig. SM2 of Supplementary Material). At 45 °C, Powder-XRD characterization have shown that calcite and aragonite were obtained at the end of the experiment. The SEM image presented in Fig. SM2 (supplementary material) shows a typical image of the powder obtained at 45 °C. The obtained powder is a mixture of rhombohedral shape crystals as well as needle shape crystals characteristic of the aragonite phase. These results agree with those of Rodriguez [START_REF] Rodriguez Blanco | The kinetics and mechanisms of amorphous calcium carbonate (ACC) crystallization to calcite, via vatérite[END_REF] .

Effect of additive nature on calcium carbonate crystallization

As it was already mentioned in literature [START_REF] Sun | Study on polyepoxysuccinic acid reverse osmosis scale inhibitor[END_REF] the scale inhibition ratio of tested additives at the same water quality is directly proportional to their dosage. In the conditions of our experiments, we found that 0.5mgL-1 was the threshold value above which, the inhibition efficiency does not evolve. This additive concentration was then chosen for testing their effect. A first series of experiments was performed at 35 °C to understand the effect of the nature of the additives on the crystallization of calcium carbonate. The results of the constant composition crystal growth experiments obtained for the three tested polymeric additives (HA, PASP and PESA) at a concentration of 0.5mgL -1 , are shown in Fig. 3 . This figure clearly shows that for PASP and HA, crystal growth starts few minutes after the introduction of seeds and the volume addition rate is much lower than the one obtained without additives. The corresponding slopes and the calculated growth rate are given in Table 4 . On the contrary, when PESA is added to the solution, the addition of the reagent starts few hours after the introduction of the seeds in the medium. In addition, when crystallization starts, the reagent addition rate is of the same order of magnitude or higher than the one obtained without additives. Another significant phenomenon, which has been observed only in the experiments performed with PESA, is the increase of pH at the beginning of the experiments, as shown in Fig. 3 . To explain this unexpected evolution of pH during the experiment, the amount of free calcium ions in solution was titrated using EDTA. The results show that quickly after the beginning of the experiments, the amount of free Ca 2 + ions drops from 2.7 × 10 -3 mol •L -1 to 0.65 × 10 -3 mol •L -1 . The decrease of free Ca 2 + induces excess negative charge due to excess HCO 3 -ions in the solution. To ensure solution electroneutrality, CO 2 degassing occurs, accompanied by OH -ion formation [START_REF] Pesonen | Density functional complexation study of metal ions with poly(carboxylic acid) ligands. Part 2. Poly(acrylic acidco-maleic acid), poly(methyl vinyl ether-co-maleic acid), and poly(epoxy succinic acid)[END_REF]. OH -ion formation in the solution during CO 2 degassing accounts for the rise in pH seen in Fig. 3 .

During the pH decrease, formation of CaCO 3 can occur: Ca 2 + + HCO 3 -⇔ CaCO 3 + H + , forming H + ions which neutralize OH -ions. It is why the delay time before crystal growth is long. After pH reached the setpoint (pH = 8.5) and as the quantity of PESA introduced is very low (0.5mgL -1 ) the crystal growth rate obtained is comparable to the one obtained without additive: all the PESA was consumed by complexation. So, PESA turns out to be a nucleation inhibitor with an efficiency of 80%, and hardly inhibits growth (1%).

Table 4 presents results for these three additives at 35 °C, 7.6 supersaturation and calcite seeds concentration of 100 mg •L -1 , for additive concentration at 0.5 mg •L -1 .

Powder characterization shows that when crystallization experiments were performed in presence of HA and PESA, only calcite crystal were obtained as for the crystallization experiments performed without additives.

SEM images performed on crystals obtained in presence of HA ( Fig. 4 (A)) show that the crystals have the typical rhombohedral shape of calcite. However, compared to the initial seeds, the surface of the crystals seems to be rougher.

In the case of PESA ( Fig. 4 (C)), the roughness of the crystal surfaces is more pronounced and these surfaces present some vacancies, and the crystals seem to be formed with an assembly of sheets.

When crystallization experiments were performed in presence of PASP, XRPD patterns show that vaterite is preferentially crystallized (see Fig. SM3 in Supplementary Material). In addition, SEM images ( Fig. 4 (B)) show that a wide range of crystal shapes are obtained: smooth rhombohedral calcite (probably initial seed crystals), porous spherical particles (with a core-shell structure) and spherulitic crystals (wheat sheaf crystals or type 2 spherulites).

Effect of temperature on scale inhibition

With 0.5mg L -1 polymer additives in the original solution, under the same conditions as without additive, calcium carbonate growth inhibition decreases with increasing temperature ( Fig. 5 ) within the operating range for an industrial cooling circuit (25-45 °C). The inhibition rate is given by Eq. ( 4) :

Inhibition rate ( % ) = 100 R 0 -R a R 0 (4)
where R 0 and R a are respectively the growth rate, in mol •m -2 •min -1 , without and with additive. HA showed the best inhibition performance (94.8% at 25 °C, compared to 87.9% for PASP or 3% for PESA at the same temperature), and likewise at other temperatures.

Activation energy was calculated for the three additives. Activation energy with PASP (84.3 kJ •mol -1 ) and HA (76.8 kJ •mol -1 ) were similar, and much higher than the one obtained without additives, confirming the inhibitory effect of both polymers on crystal growth. Activation energy with PESA (54.3 kJ •mol -1 ) was more or less the same as that found previously for calcite seeds without additive (53.4 kJ •mol -1 ), suggesting absence of inhibitory action, in contrast to HA and PASP. However, with PESA, apparent delay time (before reagent burette triggering as start of growth) is longer (around 3 hours). These results suggest that, PESA polymer plays a different role in crystal growth inhibition compared to the two other polymers.

Effect of polymer additive concentration on crystal growth

When the concentration of PESA in the solution increases from 0.1 mg •L -1 to 1 mg •L -1 , the addition rate of reagent is almost not affected and is of the same order of magnitude or higher than the one obtained for the experiments performed without additives. However, as shown in Fig. 6 , the apparent delay time of crystallization is increased from 63min to 438 min for C PESA = 0.1 mg •L -1 to C PESA = 1 mg •L -1 . It is worth to note that an increase of pH is observed at the beginning of the experiments for all the concentrations tested.

In the case of HA and PASP, the opposite is observed. The delay time of crystallization is almost not affected by the increase of additive concentration (from 0.1 to 0.5 mg •L -1 ) and it ranges from 1 to 10 min. However, when crystallization starts the reagent addition rate, and thus the crystal growth rate, decreases when the additive concentration increases.

By taking the assumption that the crystal growth rate inhibition is due to the polymer adsorption at the surface of the grow-ing crystal, the polymer additive molecule adsorption characteristics can be derived from these experiments.

The various studies in the literature [START_REF] Klepetsanis | The inhibition of calcium carbonate formation by copolymers containing maleic acid[END_REF][START_REF] Kırboga | The inhibitory effects of carboxymethyl inulin on the seeded growth of calcium carbonate[END_REF][START_REF] Lioliou | Calcium sulfate precipitation in presence of water-soluble polymers[END_REF] agree on a growth inhibition mechanism involving polymer additive adsorption on the growing crystal surface. We therefore represented the concentration effect by Langmuir's isotherm ( Eq. ( 5) ) in the inhibition ratio:

R 0 R 0 -R a = 1 ( 1 -b ) + 1 ( 1 -b ) K a C add ( 5
)
where R 0 and R a are the crystal growth rate respectively without and with polymer additive, in mol •m -2 •min -1 ; (1 -b) is the additive efficacy factor; K a is the adsorption affinity constant for the additive, in L •mol -1 ; and C add is the additive concentration, in mol •L -1 .

The additive efficacy factor (1 -b) and adsorption affinity constant (K a ) can be derived experimentally by

R 0 R 0 -R a = f( 1 C add
) . Fig. 7 shows the experimental representation of the inhibition ratio for HA and PASP.

The adsorption constants obtained are reported in Table 5 .

In order to test this hypothesis, adsorption experiments of HA and PASP additives on the surface of calcite crystals were performed (the details of the experimental procedure and the data treatments are given in Appendix). The linearized plot of the adsorption isotherm (i.e. the evolution of C e Q e vs C e ) are presented in

Table 5

Adsorption characteristics at 35 °C, with 100 mg •L -1 calcite seeds and 7.6 supersaturation ratio.

Adsorption characteristics HA PASP

(1 -b) 0.971 0.900 K a (L •mol -1 ) 5.0 × 10 7 1.0 × 10 8 Supplementary Material Fig. SM4. In the case of experiments performed with HA polymer, the correct fit confirms that the main mechanism of crystal growth inhibition is due to adsorption of the polymer on the surface of calcite crystals.

When PASP is added to the solution, the obtained result is less clear. The hypothesis of adsorption mechanism seems to be valid to a concentration up to 5 mg •L -1 . But, for higher concentrations, a deviation from linearity is observed that could be due to an adsorption mechanism that is not described by the Langmuir model,

m (mol •g -1 seeds) 2.5 × 10 -6 10 -6 K a (L •mol -1 ) 2.3 × 10 7 1.1 × 10 8
or by another phenomenon that consume the polymer in solution.

In these experiments, the adsorption constants are obtained from the linearized Langmuir model and are given in Table 6 . So, the value of the adsorption affinity constant (K a ) obtained by two different types of experiments (constant composition method and adsorption measurement) are of the same order of magnitude.

Effect of mixing PASP and PESA or HA and PESA

It is therefore deemed interesting to study the effect of mixtures of growth and nucleation inhibitor additives, under the same experimental conditions: i.e., Ω = 7.6, temperature 35 °C, calcite seeds at 100 mg •L -1 . Two series of tests were carried out: PASP + PESA and HA + PESA. Growth inhibitor (HA or PASP) concentration was held constant at 0.5 mg. L -1 while nucleation inhibitor concentration was varied between 0.3 and 1.5 mg •L -1 . The performance indicators of scale inhibition were the delay time of crystallization and the inhibition rate.

Fig. 8 A shows the evolution of reagent volume addition during the experiments at constant composition for PESA + PASP mixtures and Table 7 shows the values of the delay time of crystallization and inhibition rate for various concentrations of PESA in the PESA + PASP mixture.

In the case of PESA and PASP mixtures ( Fig. 8 A), the results show that a positive synergetic effect is obtained on delay time when additives are mixed. For instance, for the experiment performed with 0.5 mg •L -1 of PESA in the mixture, a delay time of 6 6 6 min is measured whereas it is of 190 min with the same concentration of PESA used alone. In addition, this experiment was performed with a total additive concentration of 1 mg •L -1 , and can be compared with experiment performed with PESA alone at the same concentration with a lower delay time for crystallization (i.e. 438 min).

Apparent delay time increased with increasing PESA concentration in the mixture. In addition, as PESA concentration in the mixture was increased from 1 mg •L -1 to 1.5 mg •L -1 , apparent delay increased from 1,072.3 minutes to 1,128.6 minutes ( Fig. 8 A), which is more than twice the delay time obtained when PESA is not mixed with PASP. After the delay time of crystallization, crystallization starts with approximatively the same rate as the one obtained with PASP used alone. The inhibition rate is 87% for PASP alone and ranges between 85% to 89% when PASP is used in a mixture with PESA.

Fig. 8 B shows the evolution of reagent volume addition during the experiments at constant composition for the PESA + HA mixtures and Table 8 shows the values of the delay time of crystallization, the growth rate and the inhibition rate for various concentrations of PESA in the PESA + HA mixtures. The results obtained show that no synergetic effect is obtained when both additives are mixed. The result obtained is a combination of both effects: PESA addition increases the delay time of crystallization whereas HA addition decreases the crystal growth rate. The experiment performed with 0.5m.L -1 of PESA (corresponding to a total concentration of additives of 1 mg •L -1 ) in the mixture have a much lower delay time for crystallization than the one obtained for PESA alone. In addition, under the same conditions as for the PESA + PASP mixture, apparent delay time was much shorter with PESA + HA ( Table 8 ). The inhibition rate of crystal growth is slightly increased (from 92% to 97%) by the presence of PESA when compared to the experiment performed with HA alone.

Fig. 9 shows SEM images of crystals grown with various PESA and PASP mixtures. The mixture showed predominance of calcite and vaterite crystals, whatever the concentration of PESA.

Discussion

The results obtained in this study, raise three important questions. Why vaterite is mainly formed when PASP is added to the supersaturated solution even in presence of stable calcite? Why such crystallization delay time is obtained in presence of PESA? Why only PESA and PASP mixture shows a significant synergetic effect? When HA is added to the growing medium, no crystallization delay is observed, but crystal growth is strongly slowed down. Based on adsorption experiments and simulations results found in literature [START_REF] Tribello | A molecular dynamics study of the early stages of calcium carbonate growth[END_REF][START_REF] Bulo | Site binding of Ca2+ ions to polyacrylates in water: a molecular dynamics study of coiling and aggregation[END_REF] it is more likely that crystal growth inhibition is mainly due to adsorption of the polymer on the crystal surface rather than binding of the acrylate polymer to Ca 2 + ion / carbonate ions in solution. Moreover, because of the very low concentration of additive and due to the use of the constant composition set-up (presence of calcite seeds), no effect on nucleation was observed at the opposite of the study of [START_REF] Gebauer | The multiple roles of additives in CaCO 3 crystallization: a quantitative case study[END_REF] . In these conditions, HA inhibitor can be referenced as a crystal growth inhibitor by adsorption on crystal faces (type IV of the [START_REF] Gebauer | The multiple roles of additives in CaCO 3 crystallization: a quantitative case study[END_REF] classification: "the additive adsorbs onto the nucleated particles and stabilizes them").

The influence of PASP on calcium carbonate crystallization is subtler to analyze. The results of constant composition crystal growth experiments show that with the addition of PASP, a slight increase of delay time is observed and crystal growth on calcite seeds is also strongly slowed down. Adsorption experiments show that the adsorption of PASP on crystal surfaces is less effective, and a change on the adsorption isotherm is observed beyond a certain concentration. In addition, the introduction of PASP in the growing medium, moves the crystallization of calcium carbonate towards the crystallization of vaterite, even in presence of calcite. As proposed by Grower and Odom (20 0 0) , it is clear that the adsorption of polymer on crystal surface cannot provide a full explanation of the PASP additive action on crystal morphology. As presented in Fig. 4 (B), the crystallization pumpkin particles and the "double-leaf" type of particles suggest that, based on the work of [START_REF] Xu | Fabrication and growth mechanism of pumpkinshaped vaterite hierarchical structures[END_REF] , the main crystallization mechanism proceeds via the formation of an amorphous calcium carbonate (ACC) phase. Compared to the reference experiments (without additives) or experiments performed in presence of HA, the orientation of crystallization of calcium carbonate towards a metastable phase with PASP (i.e. vaterite) suggests that one of the main effects of PASP is to control the local structure of the nucleated secondary phase (type VI of additives in the Gebauer's classification). Consequently, PASP inhibits the formation of calcite. A possible mechanism of crystal inhibition due to the presence of PASP, could be that this additive promotes the formation of a Polymer Induced Liquid Phase (PILP). Indeed, as the concentration of polymer is low, this liquid phase could not be stabilized and was quickly transformed in ACC, as suggested in some conditions by [START_REF] Gebauer | The multiple roles of additives in CaCO 3 crystallization: a quantitative case study[END_REF] and Grower and Odom (20 0 0) . Then, this ACC turns into vaterite.

Unlike to the other two polymers, PESA acts differently: no inhibition of crystal growth but strong inhibition of nucleation (see Fig. 8 ) is obtained. In addition, as for HA, XRD and SEM analyzes show that only calcite crystals are obtained. As pointed out by [START_REF] Pesonen | Density functional complexation study of metal ions with poly(carboxylic acid) ligands. Part 2. Poly(acrylic acidco-maleic acid), poly(methyl vinyl ether-co-maleic acid), and poly(epoxy succinic acid)[END_REF] , PESA is a very efficient chelator leading to a decrease of available Ca 2 + ions in solution and thus to a decrease of supersaturation. This could explain the inhibition of nucleation. Moreover, the simulation of [START_REF] Pesonen | Density functional complexation study of metal ions with poly(carboxylic acid) ligands. Part 2. Poly(acrylic acidco-maleic acid), poly(methyl vinyl ether-co-maleic acid), and poly(epoxy succinic acid)[END_REF] showed that the formed complexes are very stable.

However, this chelation effect does not explain the fact that when crystallization starts after the delay time, the consumption of reagent is of the same order of magnitude or higher than the one obtained without additives (higher growth rate). To explain this behavior, complementary free Ca 2 + titration experiments were performed, (see Fig. SM5 in Supplementary Material). These experiments were performed at 35 °C at constant polymer concentration (0.5 mgL -1 ) and different supersaturation ratio (from 7.6 to 12). The results show that whatever the supersaturation ratio, the free Ca 2 + concentration drops always to the same value around 8.310 -3 mol/L -1 and remains stable until the beginning of the crystallization. It is also important to notice that the crystallization delay time depends either on supersaturation either on polymer concentration. The corresponding ion activity product ( log = -7 . 42 . This result suggests that the addition of PESA induces the precipitation of an amorphous phase (for which we cannot state whether it is LIPS or solid) and this polymer stabilizes this metastable intermediate phase, precursor of calcite crystals. The formation of calcite is then either due to an abrupt transformation of ACC to calcite (secondary nucleation) either to deposition/aggregation of this amorphous ACC on the existing surface calcite seeds which explain the roughness of the observed crystals.

IAP = a C a 2+ a CO 2 - 3 = -7 .
The three additives tested in this study (HA, PASP and PESA) show three different action ways, that explain the synergetic effects found when two additives are mixed. For instance, mixing an inhibitor of crystal nucleation (PESA) and a crystal growth inhibitor (HA) allows to delay crystal nucleation (with the action of PESA), but also to decrease crystal growth rate, thanks to the adsorption of HA on the surface the newly formed crystals on the seeds. However, the synergetic effect of mixing these two additives is not as efficient as it can be expected. Indeed, even if the crystal growth rate (or the flow rate of reagent addition) is close to the one obtained with HA alone, the delay time observed in the case of the mixture (PESA + HA) is much lower than the one obtained with PESA alone. This could be due to molecular interaction or oligomerization between the two polyelectrolytes in solution that decreases the amount of free groups available for Ca 2 + complexation (smaller increase of pH at the beginning of the experiments compared to PESA alone). Surprisingly, the reverse is observed when PASP and PESA are mixed: even if the inhibition rate is almost the same (than in the previous case), the delay time of crystallization is at least twice the one obtained with PESA alone. By taking the assumption that the presence of PASP cancels the formation route of calcite (and thus promotes the formation of vaterite), then the ACC stabilized by the presence of PESA, cannot turn into calcite, which delays crystallization.

From a practical point of view, using a mixture of PESA and PASP provides a promising approach for scale inhibition. Under the experimental conditions applied in this study, maximum apparent delay time was nearly 19 hours, with inhibition rate close to 89% for a mixture of PESA at 1.5 mg •L -1 and PASP at 0.5 mg •L -1 . In comparison, mean residence time in industrial cooling circuits is around 24 hours; thus, a PESA-PASP mixture (both green additives) should significantly reduce scaling by acting on nucleation and crystal growth. In addition, the formation of aggregated vaterite particles with pumpkins shape make the deposit more porous and thus less adherent to the surface.

Conclusion

The scaling inhibition efficacy of three polymer additives, HA, PASP and PESA, was assessed under conditions representative of industrial cooling circuits using a constant composition method developed in our previous research. Technically, this approach allows to study, close to industrial conditions (water hardness, temperature, suspended matter…) the scaling behavior of real water. From a practical point of view, by understanding and measuring the influence of each additive alone on the crystallization of calcium carbonate, this approach has enabled to design efficient mixture of additives to avoid or delay scale formation. We have shown that by mixing two additives showing two different acting ways, it is possible either to combine their effects or to improve their inhibitory effect. For instance, nearly 19 hours, with inhibition rate close to 89% was found for a mixture of PESA and PASP even for a solution with relatively high supersaturation ratio and containing seeds of calcite. These findings open highly promising perspectives for preventive scaling treatment, improving the thermal efficacy of heat exchangers in industrial cooling circuits. -a series of 8 identical hermetically capped bottles with identical magnetic bars; -each bottle is filed with 200 mL solution at 7.6 supersaturation; -solution temperature is held at 35 °C; -in each bottle, a constant mass of calcite seed (20 mg) is introduced, for a concentration of 100 mg •L -1 ; -at t = 0, the test additive is added at concentrations from 0.1 to 1.2 mg •L -1 , a range set by the assay limit of quantification for residual additive at end of test; -after 6 days' contact, the solutions are filtered at 0.2 μm, collecting filtrate for assay of residual additive and of calcite seed for SEM characterization.

Residual additive concentration (C e ) in the solution at 7.6 supersaturation at end of each test was assayed on UV-Visible spectrophotometry using 2 reagents: benzethonium chloride and trisodium citrate dihydrate. The assay principle is based on proportionality between turbidity and additive concentration. Turbidity is caused by the reaction between benzethonium chloride and the functional groups of the polymer additive. Trisodium citrate dihydrate is needed to complex the free calcium ions in the sample in order to reduce interference resulting from complexation by polymer additive molecules. All assays were performed on a doublebeam Shimadzu 1800 spectrophotometer with 10 cm quartz optical trajectory tanks, with working wavelength set at 306 nm. This tank length reduces the limit of quantification to 0.1 mg •L -1 , enabling assay of low polymer additive concentrations after the adsorption tests.

Fig. 1 .

 1 Fig. 1. Laboratory constant composition set-up for kinetic study of calcium carbonate precipitation under conditions representative of industrial cooling circuits.

Fig. 2 .

 2 Fig. 2. Typical curve of constant composition crystallization experiment. The step in the volume curve ( V) corresponds to the volume resolution of the burette, pH = 0.001 corresponds to the resolution of the pH probe. The time between two pH oscillations corresponds to the time resolution of the experiment. The typical blue curve consists of an initial plateau, characteristic of the delay time and of an increase of the volume added corresponding to crystal growth. In some cases, a change of the injected volume is observed and is characteristic of a change in the crystallization mechanism (growth, nucleation or different phase crystallization).

  found 45 ± 4 kJ •mol -1 and Rodriguez Blanco et al. (2011) found 66 ± 2 kJ •mol -1 . In the recent work of Cheap-Charpentier et al. (2018) the activation energy (22 kJ •mol -1 ) is related to a scaling process on an immobilized CaCO 3 layer.

Fig. 3 .

 3 Fig. 3. Temporal evolution of pH and added reagent volume during constant composition experiments performed at T = 35 °C and = 7.6 with (a) HA (b) PASP (c) PESA.

Fig. 4 .

 4 Fig. 4. SEM pictures of grown calcium carbonate seeds with additives: (A) HA; (B) PASP; (C) PESA, at 35 °C and 7.6 supersaturation ratio.

Fig. 5 .

 5 Fig. 5. Evolution of calcium carbonate growth inhibition rate according to temperature for HA, PASP and PESA additives: = 7.6, C additi v e = 0.5 mg •L -1 , calcite seeds C seeds = 100 mg •L -1 .

Fig. 7 .

 7 Fig. 7. HA and PASP concentration effects on crystal growth inhibition. = 7.6, at 35 °C and 100 mg •L -1 calcite seeds.

Fig. 6 .

 6 Fig. 6. Temporal evolution of pH and added reagent volume during constant composition experiments performed at T = 35 °C and = 7.6 with concentrations of PESA ranging from 0.1 to 1 mg •L -1 .

Fig. 8 .

 8 Fig. 8. Temporal evolution of pH and added reagent volume during constant composition experiments performed at T = 35 °C and = 7.6 with different mixtures of (a) PESA and PASP (b) PESA and HA.

Fig. 9 .

 9 Fig. 9. SEM images for mixtures of various PESA concentrations with PASP at 0.5 mg •L -1 .

  56 ) is closed to the solubility product of ACC phases (I and II) calculated by Gebauer et al.(2009) log k AC C I = -7 . 51 and log k AC C II

Fig. A1 .

 A1 Fig. A1. Adsorption test bench for polymer additive molecules in solution at constant supersaturation in presence of calcite seeds at constant concentration.

Table 1

 1 Characteristics of polymer additives used for calcium carbonate growth inhibition performance tests.

Table 2

 2 Operating conditions for constant composition precipitation of calcium carbonate experiments.

	Parameter		Value
	pH		8.5
	Ionic Strength (mol •L -1 )	4.24 × 10 -2
	Supersaturation	=

a C a 2+ •a CO 2 - 3 Ks a C a 2+ and a CO 2 - 3 activity coefficients 7.6 Supersaturation ratio is equivalent to a river-water quality with a mean hardness from 20 to 25 °f (200 to 250 mg CaCO 3

Table 3

 3 Experimental growth rates at constant supersaturation = 7.6 and constant calcite seeds 100 mg •L -1 .

	Temperature ( °C)	25 °C	35 °C	45 °C
	Growth rate (mol •m -2 •min -1 )	3.07 × 10 -5	6.07 × 10 -5	11.90 × 10 -5

Table 4

 4 Effect of type of polymer additive on calcium carbonate crystal growth inhibition rate and apparent delay time.

	Polymer additive	Apparent latency time (min)	R 92.1
	Polyaspartic (PASP)	13.3	7.45 × 10 -6	87.7
	Polyepoxysuccinic (PESA)	190.3	6.02 × 10 -5	1.0
	by a reaction HCO 3

a (mol •m -2 •min -1 ) Inhibition rate (%) Homopolymer of acrylic acid (HA) 5.7 4.79 × 10 -6 -⇔ CO 2gasseous + OH -(

Table 6

 6 Adsorption characteristics on adsorption bench test in a solution with 7.6 supersaturation at 35 °C in presence of calcite seeds at 100 mg •L -1 .

	Adsorption characteristics	HA	PASP
	Q

Table 7

 7 Effect of PESA concentration in a PESA + PASP mix on various performance indicators, at 7.6 supersaturation at 35° C in presence of calcite seeds at 10 0 mg• L -1 .

	PESA + PASP mix	Apparent delay time (min)	R a (mol •m -2 •min -1 )	Inhibition rate (%)
	PESA 0.3 + PASP 0.5	191.0	6.39 × 10 -6	89.5
	PESA 0.5 + PASP 0.5	665.6	7.98 × 10 -6	86.9
	PESA 1.0 + PASP 0.5	1072.3	9.05 × 10 -6	85.1
	PESA 1.5 + PASP 0.5	1128.6	6.92 × 10 -6	88.6

Table 8

 8 Effect of PESA concentration in a PESA + HA mix on various performance indicators, at 7.

Acknowledgments

The authors wish to thank Marie-Line de Solan-Bethmale from the Laboratoire de Génie Chimique (LGC) for the SEM analyses and Cédric Charvillat from the Centre Interuniversitaire de Recherche et d'Ingénierie des Matériaux (CIRIMAT) for the powder XRD measurements.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.watres.2020.116334 .

Appendix: Adsorption bench test and experimental conditions

Three models are widely used for isotherms: Langmuir, Freundlich and Redlich-Peterson [START_REF] Liu | Adsorption isotherm, kinetic and mechanism studies of some substituted phenols on activated carbon fibers[END_REF]. For calcium carbonate crystal growth inhibition by polymer additives, the Langmuir model is the most widely used to describe additive molecule adsorption on growth site surfaces [START_REF] Dimova | Binding of polymers to calcite crystals in water: characterization by isothermal titration calorimetry[END_REF][START_REF] Dalas | The inhibition of calcium carbonate crystal growth by the cysteine-rich Mdm2 peptide[END_REF][START_REF] Zhang | Water adsorption on kaolinite and illite after polyamine adsorption[END_REF][START_REF] Lisitsin | Modeling the effect of anti-scalant on CaCO 3 precipitation in continuous flow[END_REF][START_REF] Klepetsanis | The inhibition of calcium carbonate formation by copolymers containing maleic acid[END_REF].

The model is based on 3 assumptions:

• The adsorbent exposes a limited number of adsorption sites: i.e., has a maximum adsorption capacity, Q m . • All adsorption sites are identical and receive only 1 molecule at a time. • At binding, the energy between site and adsorbed molecule is constant, unaffected by adsorption at neighboring sites.

Under controlled temperature, adsorption isotherms depend on the type of adsorbent (calcite seeds or suspended matter) and on the type of adsorbed molecules (polymer additive molecules). This dependence is represented by an equation relating quantity of adsorbed polymer (Q e ) to additive molecules equilibrium concentration [START_REF] Liu | Adsorption isotherm, kinetic and mechanism studies of some substituted phenols on activated carbon fibers[END_REF][START_REF] Blachier | Adsorption of polyamine on clay minerals[END_REF].

where Q e is the quantity of polymer adsorbed by the adsorbent (mol •g -1 ), V is the solution volume (L), C add and C e are respectively the initial and the residual post-equilibrium polymer concentration (mol •L -1 ), and m is the adsorbent (seeds) mass (g).

The Langmuir adsorption isotherm model is a representation of Q e = f(C e ) with the following non-linear expression:

where Q m is the maximum adsorption capacity (in mole polymer per g calcite seeds), and K a is the Langmuir adsorption affinity constant (L •mol -1 ). Constants Q m and K a can be determined experimentally by linearizing relation

To consolidate the adsorption characteristics obtained on the constant composition set-up, an adsorption test bench was implemented ( Fig. A1 ).

The test bench comprised:

-a thermostatic bath at 35 °C in which a magnetic stir table is immersed;


Noémie Le Tallec-Estève

Chloé Rousseau

Benoit Desrues

Olivier Loréal

Ronan Thibault
email: ronan.thibault@chu-rennes.fr

Prof Thibault Ronan

Transferrin saturation is independently associated with the severity of obstructive sleep apnea syndrome and hypoxia among obese subjects

Keywords: obesity, metabolic syndrome, iron, obesity surgery

Introduction and aims: Obstructive sleep apnea syndrome (OSAS) is a frequent complication of obesity. Intermittent chronic hypoxia which frequently results from OSAS could modulate the systemic control of iron metabolism and alter serum iron parameters, especially among obese patients. Aims: to evaluate whether serum parameters of iron bioavailability and storage (primary), as well as age, waist circumference, arterial hypertension and tobacco use (secondary) are associated with OSAS severity and/or hypoxia. Methods: design: a singlecenter retrospective study with prospective data collection; inclusion criteria: consecutive patients referred for initial assessment for obesity underwent nocturnal respiratory polygraphy and iron status serum assessment within a 3-month period. The adjusted analyzes were performed using ANOVA and reported as adjusted means and 95% confidence interval (95% CI). Results: 13 men and 56 women were included. OSAS prevalence : 72% (n=50). Ferritin (mean±SD, 260±276 vs. 111±89 μg/l, p=0.01) and transferrin saturation (31±10 vs. 24±9%, p=0.002) were significantly higher in case of moderate/severe OSAS than in absent/mild OSAS, independently from gender and tobacco use. Serum iron (19.4 μg/l [CI95%, vs. 16.2 μg/l ([14.1-18.2], p=0.056) and transferrin saturation (31.5 % [26.3-36.7]) vs. 25.3%

[21.6-29.1], p=0.043) were higher when time under oxygen saturation <90% was >15%. Age (mean±SD, 51±11 vs. 41±12 yr, p=0.001), waist circumference (136±18 vs. 123±12 cm, p=0.003), arterial hypertension (59% (n=13/22) vs. 23% (n=11/47), p=0.004) and tobacco use (64% (n= 14/22) vs. 32% (n= 15/47), p=0.01) were significantly greater in moderate/severe OSAS than in absent/mild OSAS. Conclusions. Transferrin saturation was associated with OSAS severity and time under hypoxia. This suggests a relationship between OSAS-induced hypoxia and iron metabolism among obese patients.

INTRODUCTION

Obstructive sleep apnea syndrome (OSAS) is defined by recurrent episodes of apnea or hypopnea during sleep that result from complete or partial upper airway collapse. It leads to chronic intermittent hypoxia [START_REF] Jordan | Adult obstructive sleep apnoea[END_REF]. OSAS affects about 4% of the general population [START_REF] Punjabi | The epidemiology of adult obstructive sleep apnea[END_REF], but its prevalence can reach 30 to 50% among obese patients [START_REF] Tufik | Obstructive sleep apnea syndrome in the Sao Paulo Epidemiologic Sleep Study[END_REF]. Identifying and treating OSAS among obese patients is essential, because OSAS facilitates the onset of metabolic and cardiovascular diseases [START_REF] Mesarwi | Metabolic dysfunction in obstructive sleep apnea: A critical examination of underlying mechanisms[END_REF], and favors weight gain and aggravation of obesity [START_REF] Pillar | Abdominal fat and sleep apnea: the chicken or the egg?[END_REF][START_REF] Quan | Is there a bidirectional relationship between obesity and sleep-disordered breathing?[END_REF][START_REF] Ong | The reciprocal interaction between obesity and obstructive sleep apnoea[END_REF].

Iron metabolism is finely tuned. Serum iron is mainly provided by macrophages during the erythrophagocytic process that enables iron recycling and by enterocytes in the course of digestive absorption of iron from nutrients. Serum concentration of iron and transferrin iron saturation levels are indicators of the iron that is bioavailable for cells. Hepcidin controls iron homeostasis [START_REF] Ganz | Hepcidin and iron regulation, 10 years later[END_REF][START_REF] Brissot | Haemochromatosis[END_REF]. Hepcidin is a peptide mainly synthetized by hepatocytes and secreted into the plasma. It modulates the expression of ferroportin, the only known iron exporter from cells towards plasma. An abnormal decrease in hepcidin levels in plasma increases the serum iron concentration and transferrin saturation, exposing the patient to iron overload, which, in turn, increases serum levels of ferritin, the protein involved in the storage of excess iron [START_REF] Brissot | Haemochromatosis[END_REF][START_REF] Pietrangelo | Hemochromatosis: an endocrine liver disease[END_REF]. An abnormal increase in hepcidin levels decreases transferrin saturation and exposes the subject to iron deficiency [START_REF] Ganz | Hepcidin and iron regulation, 10 years later[END_REF]. Hepcidin expression is increased by iron storage and inflammation [START_REF] Ganz | Hepcidin and iron regulation, 10 years later[END_REF][START_REF] Brissot | Haemochromatosis[END_REF]. Conversely, hepcidin expression is decreased by anemia and hypoxia, through mechanisms that could involve erythropoietin, hypoxia inducible factor [START_REF] Peyssonnaux | Regulation of iron homeostasis by the hypoxia-inducible transcription factors (HIFs)[END_REF][START_REF] Piperno | Modulation of hepcidin production during hypoxia-induced erythropoiesis in humans in vivo: data from the HIGHCARE project[END_REF] and erythroferrone [START_REF] Kautz | Identification of erythroferrone as an erythroid regulator of iron metabolism[END_REF] which is synthetized by erythroblasts during erythropoiesis [START_REF] Pinto | Erythropoietin mediates hepcidin expression in hepatocytes through EPOR signaling and regulation of C/EBPalpha[END_REF]. In case of hypoxia, the down-regulation of hepcidin expression becomes a priority, irrespective of body iron levels [START_REF] Tandara | Iron metabolism: current facts and future directions[END_REF]; this leads to iron mobilization and an increase in transferrin saturation, in order to provide adequate amounts of iron for the erythropoiesis process, which is activated to compensate for the hypoxia.

A c c e p t e d m a n u s c r i p t

OSAS has been reported to be associated with polycythemia [START_REF] Solmaz | Is obstructive sleep apnoea syndrome really one of the causes of secondary polycythaemia?[END_REF] and increased hematocrit levels [START_REF] Choi | Does obstructive sleep apnea increase hematocrit?[END_REF]. In addition, erythropoietin plasma concentrations are higher among patients with OSAS [START_REF] Zhang | Erythropoietin levels in patients with sleep apnea: a meta-analysis[END_REF]. Therefore, we hypothesize that among obese patients with OSAS, an increase in iron transferrin saturation could be a marker for the severity of OSAS and hypoxia. The aims of our study were to evaluate whether serum parameters of iron bioavailability and storage (primary), and age, waist circumference, arterial hypertension and tobacco use (secondary) are associated with OSAS severity and/or hypoxia.

PATIENTS AND METHODS

Study design and population

This was a single-center retrospective study based on prospective data collection. The 69 patients were consecutively recruited from June 1 st , 2016 to April 23, 2017

Obesity-related diseases and therapy

Patients were considered to have type 2 diabetes in the following cases: fasting blood glucose ≥7.0 mmol/l, prior history of diabetes, oral antidiabetic drug use and/or insulin therapy.

Fasting hyperglycemia was defined as fasting blood glucose ≥5.6 mmol/l. Arterial hypertension was defined as systolic (SBP) or diastolic (DBP) blood pressures ≥140 or ≥90 mmHg, respectively, or if patients were under antihypertensive therapy. Metabolic syndrome was defined according to the International Diabetes Foundation 2005 criteria (4). History of tobacco use, respiratory diseases (chronic obstructive pulmonary disease, asthma), or chronic inflammatory diseases (systemic, rheumatic or gastrointestinal) were reported. Drugs that can interfere with iron metabolism, such as proton pump inhibitors (PPIs), nonsteroidal antiinflammatory drugs (NSAIDs), aspirin, or that affect sleep, such as psychotropes (antidepressants, benzodiazepines, hypnotics), were identified.

Anthropometric data

Weight, height, body mass index, and waist and hip circumferences were measured in all subjects the day of the nocturnal respiratory polygraphy. After one night fasting, body composition was measured at 8 am using bioimpedance analysis (Bodystat 1500, Bodystat ltd, Isle of Man, UK). Patients were advised to restrict themselves to routine physical activity the day before. After skin swabbing with 70% ethanol, four adhesive electrodes (3M Red Dot T, 3M Health Care, Borken, Germany) were placed on the dorsal side of the left hand, left wrist, left foot, and left ankle while the patient was supine with palms facing inwards. An electrical current of 0.8 mAmp was produced at 50 KHz for five seconds by the bioimpedance analyser.

The following data was collected: fat mass and fat-free mass (% of weight and kg), and total body water (%).

Study endpoints

The primary endpoint was the severity of OSAS, as defined by the AHI severity categories described above in the 'Nocturnal respiratory polygraphy' section. The secondary endpoints were the percentage of time under hypoxia, i.e. oxygen saturation <90%, and the lowest oxygen saturation value, serum iron parameters, anthropometric data, body composition, obesity-related diseases, age, tobacco use, and the metabolic syndrome parameters (waist circumference, arterial pressure, plasma glucose, triglycerides, and HDL-cholesterol). The numbers of patients in each category are shown in Tables 2,3 & 4.

Statistical analysis

Quantitative variables are described as numbers (percentage) and means ± standard deviation (SD). The groups were compared using Student's test (2 groups) or ANOVA tests (>2 groups) when the distribution was normal, or using Mann-Whitney or Wilcoxon (two groups) nonparametric tests otherwise. For qualitative variables, the numbers and percentage are presented for each category. The groups were compared using χ2 parametric or Fisher nonparametric tests (theoretical number <5). The adjusted analyses were performed using ANOVA and presented as adjusted means and 95% confidence interval (95% CI). The significance threshold was 5% for all analyses. The analyses were carried out on SAS software, version 9.4.

A c c e p t e d m a n u s c r i p t

RESULTS

Patients' enrollment and characteristics

The study flow chart is shown in Figure 1. Among the 69 patients included, 13 were men (19%) and 56 were women (81%). The patient characteristics are summarized in Table 1.

Among the eight patients with type 2 diabetes, five were treated with metformin, five with glucagon-like peptide (GLP)-1 analog, and two with insulin. Their mean glycated hemoglobin was 6.97% (range 6.30-8.40).

OSAS prevalence and severity

The overall prevalence of OSAS was 72% (n=50/69), including 40% (n=28) with mild OSAS, 13% (n=9) with moderate OSAS, and 19% (n=13) with severe OSAS (Table 1). The mean oxygen saturation was 91.6±3.0%. Twenty-seven percent (n=19) of the patients had minimum oxygen saturation ≤75%, and 32% (n=22) had oxygen saturation below 90% for more than 15% of the recording time (Table 1).

Relationships between iron parameters, OSAS severity, and hypoxia

Bio-clinical data according to the severity of OSAS is presented in Table 2. Serum iron concentration, transferrin saturation, and ferritin were significantly higher in the moderate/severe OSAS group than in the absent/mild OSAS group. After excluding the patients on PPIs, NSAIDs, aspirin, or psychotropes (n=27), the differences regarding serum iron parameters remained statistically significant (data not shown). Plasma CRP and hemoglobin were not statistically different. Transferrin saturation was associated with both the severity of OSAS (evaluated according to AHI) (Table 3) and with time with oxygen saturation <90% ( 3). Iron and CRP concentrations were higher when time under oxygen saturation <90% was >15% (Table 4). Transaminases, prothrombin time, transferrin and albumin levels were not statistically related to OSAS severity nor to time in hypoxia (Tables 3 and4).

Other variables associated with OSAS and its severity

As expected, patients with moderate/severe OSAS had more frequent and more marked hypoxia and more time with oxygen saturation <90% (Table 2). Patients with moderate/severe OSAS were significantly older, had larger waist circumference, more frequently presented arterial hypertension, and had higher fasting blood glucose and triglyceride levels. They were also more often smokers than patients with absent or mild OSAS. The group with moderate/severe OSAS was more likely to be on anti-hypertensive drugs than the absent/mild OSAS group (11 (50%) vs. 10 (21%); p=0.016). There was no difference in the use of anti-diabetic treatment, NSAIDs, PPIs, psychotropes, statins or fibrates between the absent/mild OSAS and moderate/severe OSAS groups.

DISCUSSION

In a group of obese patients eligible for obesity surgery, the severity of OSAS, defined by AHI and the time in hypoxiai.e. peripheral oxygen saturation <90% -were both independently associated with an increase in transferrin iron saturation, regardless of gender, tobacco use, biological inflammation, metabolic syndrome parameters, or drug therapy.

Serum iron levels were also related to time in hypoxia, whereas the serum transferrin concentration was not, suggesting that the increase in serum transferrin saturation is mostly related to an increase in serum iron.

A c c e p t e d m a n u s c r i p t

The relationship between iron metabolism, metabolic syndrome, obesity and insulin resistance is well known. Whether iron metabolism disturbances participate in metabolic syndrome or are one of its consequence remains debated [START_REF] Britton | Iron and non-alcoholic fatty liver disease[END_REF]. Our study suggests that hypoxia secondary to OSAS could have an important role in the relationship between iron metabolism, and metabolic syndrome/overweight/obesity. Among patients with OSAS, obesity and/or insulin resistance, sub-clinical inflammation could lead to a decrease in both serum iron and transferrin saturation levels through IL6 and an increase in serum hepcidin [START_REF] Kanbay | A new prognostic marker for obstructive sleep apnea: hepcidin[END_REF], the key regulator of iron metabolism. This hypothesis is encouraged by the finding of increased iron storage, plasma ferritin and liver iron content together with increased hepcidin levels in hepatosiderosis patients [START_REF] Ruivard | Iron absorption in dysmetabolic iron overload syndrome is decreased and correlates with increased plasma hepcidin[END_REF]. Among severely obese patients, a lower transferrin saturation level is associated with an increase in hepcidin expression in adipose tissue macrophages [START_REF] Bekri | Increased adipose tissue expression of hepcidin in severe obesity is independent from diabetes and NASH[END_REF].

Our data show that both serum iron and transferrin saturation levels are increased alongside a slight increase in CRP in moderate/severe OSAS, confirming an inflammatory context for these patients. It is also possible that hypoxia could have counteracted the expected decrease in transferrin saturation related to inflammation and subsequent increase in hepcidin level. Indeed, hypoxia is a well-known repressor of hepatic hepcidin expression [START_REF] Renassia | New insights into the links between hypoxia and iron homeostasis[END_REF]. Hypoxia could reduce the transcription of the Hepcidin AntiMicrobial Peptide (HAMP) gene that encodes for hepcidin by inducing Hypoxia inducible factor 2 (HIF-2) [START_REF] Rankin | Hypoxia-inducible factor-2 (HIF-2) regulates hepatic erythropoietin in vivo[END_REF][START_REF] Mastrogiannaki | Hepatic hypoxia-inducible factor-2 down-regulates hepcidin expression in mice through an erythropoietin-mediated increase in erythropoiesis[END_REF].

Unfortunately, because of the retrospective study design, hepcidin serum levels were not measured. However, our study results could point to a hypothetical role of hepcidin in the way in which hypoxia and inflammation act on iron metabolism among obese patients with OSAS.

A decrease in hepcidin plasma levels favored by hypoxia could in turn favor the increase in transferrin saturation. The level of hepcidin expression is controlled by numerous factors, especially at transcriptional level [START_REF] Roth | Regulators of hepcidin expression[END_REF]. Physiopathological events occurring during OSAS can Our data shows that among patients with OSAS registering more than 15% of the time with oxygen saturation under 90%, there is a significant increase in transferrin saturation, alongside an increase in CRP. This finding supports the hypothesis that the degree of hypoxemia could counteract the effect of inflammation, favoring iron release from cells.

Hypoxia could blunt the stimulating effect of inflammation on hepcidin expression (Figure 2). Abakay et al reported a decrease in serum iron and an increase in hepcidin level among OSAS patients compared to non-OSAS patients [START_REF] Abakay | Relationship between hepcidin levels and periodic limb movement disorder in patients with obstructive sleep apnea syndrome[END_REF]. They also showed that AHI was negatively correlated with serum iron levels and positively with hepcidin levels. Among patients with the highest hepcidin levels, two clusters in the data distribution were observed. However, the authors did not analyze the relationship between hypoxia and iron parameters/hepcidin. Among Chinese patients with and without OSAS, Liu et al reported a slight increase in hepcidin serum levels among patients with severe OSAS [START_REF] Liu | Association of serum hepcidin levels with the presence and severity of obstructive sleep apnea syndrome[END_REF]. Unfortunately Liu et al did not assess serum iron metabolism parameters or inflammatory status, so that the relationships between inflammatory parameters and AHI cannot be studied, nor can the impact of time in hypoxia and maximum hypoxemia.

Our hypothesis that hypoxia could explain the increase in transferrin saturation in OSAS requires confirmation. The precedence of hypoxia over other regulators of iron metabolism has been previously reported among patients with anemia secondary to chronic diseases [START_REF] Nemeth | Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein[END_REF] and among beta-thalassemic patients [START_REF] Gardenghi | Ineffective erythropoiesis in beta-thalassemia is characterized by increased iron absorption mediated by down-regulation of hepcidin and up-regulation of ferroportin[END_REF][START_REF] Camaschella | Iron metabolism and iron disorders revisited in the hepcidin era[END_REF]. In these extreme conditions, hypoxia takes precedence over the effect of active inflammation or the increase in iron storage, resulting in promotion rather than inhibition of iron release from macrophages and enterocytes and and Divalent Metal Transporter 1 (DMT1) genes encoding respectively for a reductase and a transporter of iron involved in non-heminic iron uptake from the digestive lumen [START_REF] Peyssonnaux | Regulation of iron homeostasis by the hypoxia-inducible transcription factors (HIFs)[END_REF][START_REF] Gruber | Acute postnatal ablation of Hif-2alpha results in anemia[END_REF]. The transcriptional expression of the SLC40A1 gene encoding for the ferroportin protein, the iron exporter from enterocytes toward plasma, is also increased by HIF-2.

Except for γGT, the markers of liver diseases did not differ between absent/mild and moderate/severe OSAS groups. Thus, cytolysis or hepatic insufficiency are not likely to be involved in the increases in serum iron and transferrin saturation.

After adjustment on potential confounding factors, we found no difference in serum ferritin concentrations according to the severity of OSAS or hypoxia. This is consistent with previous studies [START_REF] Thorarinsdottir | Serum ferritin and obstructive sleep apnea-epidemiological study[END_REF]. Despite the statistically significant relationship between transferrin saturation level and severity of OSAS, serum transferrin saturation remained within the normal values. However, as found in metabolic syndrome patients [START_REF] Lan | Redox active plasma iron in C282Y/C282Y hemochromatosis[END_REF], we cannot rule out that, among patients with severe OSAS, the increase in transferrin saturation could favor the appearance in plasma of small amounts of non-transferrin-bound iron, an abnormal molecular form of iron rapidly taken up by the liver, pancreas and heart, which could participate in the cell damage related to oxidative stress (40). During OSAS it has been reported that its severity, defined by AHI, was not associated with systemic oxidative stress levels, but the minimum oxygenation desaturation level was associated with a decrease in antioxidant potential (41). This suggests an involvement of anti-oxidant defenses in condition of hypoxia during OSAS.

Our study has several limitations. The most important is that, due to the retrospective design, we did not measure serum levels of hepcidin, the key regulator of iron metabolism. Measuring hepcidin among our obese patients with OSAS could have helped us to understand the A c c e p t e d m a n u s c r i p t

  in the Nutrition Unit of the University Hospital of Rennes (CHU Rennes), Rennes, France. The study included all patients eligible for obesity surgery according to the French recommendations, i.e. body mass index (BMI) ≥40 or BMI ≥35 kg/m² with obesity complications (cardiovascular diseases including arterial hypertension, OSAS, type 2 diabetes, disabling osteoarticular disease, nonalcoholic steatohepatitis ). They underwent a systematic nocturnal respiratory polygraphy to screen for OSAS as part of the initial assessment. The exclusion criteria were: previous diagnosis of OSAS, time between the nocturnal respiratory polygraphy and the plasma iron status assessment >3 months, history of obesity surgery (except gastric banding), ongoing iron supplementation, diseases known to be associated with iron metabolism dysregulation (genetic hemochromatosis, active chronic inflammatory disease), and pregnancy. Since this retrospective study was based on routine practice, informed consent was not required by French law.

  Nocturnal respiratory polygraphy was performed in all patients using the CID-Lx polygraph (CIDELEC, Angers, France). All graphs obtained during sleeping time were analyzed by a senior pneumologist specialized in sleep disorders (BD). Apnea was defined by a complete cessation of nasal-oral airflow for at least 10 seconds. The apnea-hypopnea index (AHI) was defined as the number of apneas or hypopneas per hour of sleep. OSAS was defined as AHI ≥5/hour. The severity of OSAS was defined as mild if 5≤AHI≤15, moderate if 15<AHI≤30, or severe if AHI>30. Hypoxia was defined as the percentage of time with arterial oxygen saturation <90% at 15% of sleeping time or more. The lowest oxygen saturation value was recorded.Iron serum parameters and blood analysesMeasurements of serum iron parameters were part of the systematic fasting blood analyses; the measurement methods are shown in brackets: blood cell count (impedance flow cytometry), prothrombin time (chronometry), glucose (hexokinase colorimetric assay), glycated hemoglobin (in mmol/mol hemoglobin according to the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), and in % according to the National Glycohemoglobin Standardisation Program), albumin (bromocresol green colorimetric assay), transthyretin (immunoturbidimetry), C-reactive protein (CRP) (latex enhanced immunoturbidimetry), alanine aminotransferase (ALT), aspartate aminotransferase (ALT), γ-glutamyl-transpeptidase (γGT) (all three by spectrophotometry according to the recommendations of the IFCC), triglycerides, total and HDL-cholesterol (colorimetry), and LDL-cholesterol (calculated from the Friedewald formula). Plasma iron parameters were assessed on heparinized plasma. Serum iron concentrations were determined by the colorimetric method (ferrozine assay), transferrin concentration, by

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  p t e d m a n u s c r i p t immunoturbidimetry, and ferritin by electrochemiluminescence (Cobas 8000 Roche). The transferrin saturation coefficient (TSC), and total transferrin binding capacity (TIBC) were calculated. The biological analyses were all performed in the Department of Biochemistry at CHU Rennes.

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  p t e d m a n u s c r i p t independently associated with the severity of OSAS (Table

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  p t e d m a n u s c r i p t generate at least two antagonist signals that could modulate hepcidin transcription: inflammation and hypoxia. Inflammation increases hepcidin expression through the IL6/STAT3 signaling pathway, thus limiting iron release from macrophages and enterocytes and reducing transferrin saturation (26-29).

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  p t e d m a n u s c r i p t increase in transferrin saturation. In addition, hypoxia could modulate the expression of iron metabolism genes, thus contributing to an increase in transferrin saturation. In enterocytes, HIF-2 can increase the transcriptional expressions of the Duodenal Cytochrome b (DCYTb)

  Ropert M, Le Lan C, Loréal O. Non-transferrin bound iron: a key role in iron overload and iron toxicity. Biochim Biophys Acta 2012;1820(3):403-10.41.Simiakakis M1, Kapsimalis F, Chaligiannis E, Loukides S, Sitaras N, Alchanatis M.Lack of effect of sleep apnea on oxidative stress in obstructive sleep apnea syndrome (OSAS) patients. PLoS One 2012;7(6):e39172.

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Figure 1 -

 1 Figure 1 -Study flow chart.

Figure 2 -

 2 Figure 2 -Diagram representing the potential mechanisms that could modulate systemic iron metabolism and parameters among obese patients with severe OSAS.

Figure 1 Assessment

 1 Figure 1

  Figure 2

Table 4 )

 4 , independently from gender, tobacco use, age, arterial hypertension, waist circumference, blood glucose and triglycerides. γGT levels were

Table 1 -Clinical and demographic characteristics of obese patients with obstructive sleep apnea syndrome (n=69).

 1

	Variables
	Women / Men -n (%)	56 (81) / 13 (19)
	Age (yr)	44.3 ± 12.6
	Gastric banding history -n (%)	4 (6)
	Tobacco use -n (%)	29 (42)
	Smoking cessation (N= 28)* -n (%)	13 (46)
	OSAS -n (%)	50 (72)
	Mild	28 (40)
	Moderate	9 (13)
	Severe	13 (19)
	Minimum oxygen saturation ≤ 75%	19 (27)
	Time with oxygen saturation < 90% >15% 22 (32)
	Anthropometric data
	Body mass index	42.8 ± 6.3
	Body composition
	Fat-free mass (%)	52.6 ± 6.5
	Fat mass (%)	47.4 ± 6.5
	Total body water (%)	39.5 ± 4.7
	Fat mass index (kg/m²)	20.4 ± 5.3
	Waist circumference (cm)	126.8 ± 15.2
	Waist/Hip ratio	1.0 ± 0.1
	Comorbidities -n (%)
	Type 2 diabetes	8 (12)
	Arterial hypertension	24 (35)

Table 2 -Univariate analysis of factors associated with obstructive sleep apnea syndrome and its severity (n=69).

 2 ALAT, alanine amino transferase; ASAT, aspartate amino transferase; COPD, chronic obstructive pulmonary disease; CRP, C-reactive protein; γGT, gamma-glutamyltranspeptidase.

	Variables	Absent/mild OSAS Moderate/severe P
		N=47	N=22
	Women / Men -n (%) Age (yr) -mean±SD	41 (87) / 6 (13) 41.0 ± 12.0	15 (68) / 7 (32) 51.5 ± 11.1	0.096 0.001
	Tobacco use -n (%)	15 (32)	14 (64)	0.013
	Smoking cessation (N= 28)-n(%) (%)	4 (27)	9 (69)	0.024
	Nocturnal polygraphy -mean±SD Minimum oxygen saturation (%) % time oxygen saturation <90% Oxygen saturation decrease (%) Oxygen saturation <90% / hour (n) Anthropometric data -mean±SD Body mass index (kg/m²) Waist circumference (cm) Total body water (%) Fat free mass (kg) Fat free mass (%) Fat mass (kg) Fat mass (%) Fat mass index (kg/m²)	83.5 ± 9.1 11.6 ± 25.6 4.1 ± 1.1 9.5 ± 5.5 41.6 ± 4.6 122.6 ± 11.6 39.0 ± 3.6 59.3 ± 10.3 52.8 ± 5.4 54.0 ± 9.9 47.2 ± 5.4 19.6 ± 3.6 a n u s c r i p t 71.0 ± 10.5 <0.0001 38.3 ± 30.4 <0.0001 7.9 ± 2.7 <0.0001 51.2 ± 27.3 <0.0001 45.6 ± 8.3 0.044 135.6 ± 18.3 0.003 40.5 ± 6.3 0.297 65.7 ± 13.0 0.031 52.1 ± 8.6 0.749 62.1 ± 20.6 0.089 47.9 ± 8.6 0.642 22.3 ± 7.5 0.335
	Comorbidities -n (%) Type 2 diabetes Arterial hypertension	4 (8.5) 11 (23) m	4 (18) 13 (59)	0.255 0.004
	Metabolic syndrome Asthma COPD Blood parameters -mean±SD Iron (µmol/l) Transferrin (g/l) Transferrine saturation (%) Ferritin (µg/l) Hemoglobin (g/dl) Hematocrit (%) CRP (mg/l) ALAT (IU/l) ASAT (IU/l) γGT (IU/l) Prothrombin time (%) A c c e p t e d 28 (60) 8 (17) 1 (2) 15.2 ± 4.8 2.6 ± 0.4 24.2 ± 8.8 111.5 ± 88.9 13.4 ± 1.2 40.6 ± 3.2 8.3 ± 5.6 28.8 ± 14.1 24.5 ± 8.6 31.6 ± 14.1 99.4 ± 12.8	16 (73) 5 (23) 1 (4.5) 18.7 ± 5.6 2.4 ± 0.4 31.5 ± 9.6 260.5 ± 276.3 13.5 ± 1.0 40.8 ± 3.0 9.5 ± 7.5 38.9 ± 20.5 28.1 ± 11.8 74.8 ± 101.7 98.8 ± 11.7	0.289 0.742 0.539 0.021 0.211 0.002 0.011 0.593 0.787 0.728 0.029 0.233 0.030 0.342
	Albumin (g/l)	41.9 ± 2.7	42.5 ± 3.3	0.412
	Glucose (mmol/l)	5.5 ± 1.0	5.9 ± 1.0	0.016
	Triglycerides (mmol/l)	1.3 ± 0.5	1.7 ± 0.8	0.007
	Total cholesterol (mmol/l)	4.8 ± 0.8	5.3 ± 1.5	0.127
	HDL cholesterol (mmol/l)	1.2 ± 0.3	1.1 ± 0.3	0.430
	LDL cholesterol (mmol/l)	3.0 ± 0.7	3.2 ± 1.4	0.501

Table 3 -Serum iron parameters independently associated with the severity of OSAS (n=69).

 3

	Variables	Absent OSAS (n=19)	Mild (n=28)	Moderate (n=9)	Severe (n=13)	P
						value
	Transferrin
	saturation (%)

Acknowledgements

We thank Dr Martine Ropert, Department of Biochemistry, CHU Rennes, Rennes, France, for all the biochemical analyses, including iron status. A c c e p t e d m a n u s c r i p t

mechanisms of the changes observed in iron parameters in relation to hypoxia and inflammation. A prospective study is clearly needed. Also because of the retrospective design, we did not assess antioxidant potential or non-transferrin-bound iron. Our hypothesis that hypoxemia could explain the increase in transferrin saturation in OSAS needs to be confirmed. Finally, we were not able to assess whether excessive alcohol intake could have played a role in γGT increase, as well as in the increase in serum ferritin [START_REF] Harrison-Findik | Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression[END_REF]. Indeed excessive alcohol intake is known to be associated with the severity of OSAS.

In conclusion, our data shows that transferrin iron saturation is independently associated with OSAS severity and the time in hypoxia. More studies are needed to evaluate the mechanisms involved in transferrin iron saturation increase among obese patients with OSAS, and their role in the natural course of OSAS. In view of our findings, transferrin iron saturation in plasma merits exploration as a longitudinal marker of the severity of OSAS or hypoxia among obese patients.

Statement of authorship

All authors have made substantial contributions: NLE, OL and RT conceived, and designed the study, analysed, and interpreted the data, and drafted the article; NLE, RT, and BD collected the data; CR analysed the data; all authors have approved the final version.

Conflict of interest statement

The authors have no conflict of interest related to this article to declare.