Caroline Dessal

Thomas Len

Franck Morfin

Jean-Luc Rousset

Mimoun Aouine

Pavel Afana- Siev

Laurent Piccolo

Keywords: Single-atom catalysis, Pt/-Al2O3, operando spectroscopies, XAS, DRIFTS O

Single-atom catalysts (SACs) are promising atom-efficient materials, with potentially superior performances with respect to their nanoparticulate counterparts. Owing to its practical importance and relative simplicity, CO oxidation on Pt/-Al2O3 is considered as an archetypal catalytic system. The efficiency of the corresponding SAC has recently been the subject of debate. In this work, in addition to systematic high-resolution scanning transmission electron microscopy, we have simultaneously monitored the Pt dispersion, oxidation state, and CO oxidation activity by operando fast X-ray absorption spectroscopy and diffuse reflectance infrared spectroscopy, both combined with mass spectrometry. It is shown that single Pt m+ atoms (m  2), resulting from the standard impregnation-calcination procedure of SAC preparation, are poorly active. However, they gradually but irreversibly convert into highly active ~1-nm-sized Pt + clusters ( < 2) throughout the heating/cooling reaction cycles, even under highly oxidizing conditions favorable to atomic dispersion. Increase in the Pt loading or the CO/O2 concentration ratio accelerates the clustering-reduction phenomena. This work not only evidences a gradual aggregation/activation process for an important catalytic system, but also highlights the power of operando spectroscopies to address stability issues in single-atom catalysis.

INTRODUCTION

In the quest for minimal consumption of precious metals, single-atom heterogeneous catalysis has recently attracted much attention because single-atom catalysts (SACs) expose all the virtually active metal atoms to the reactants. [START_REF] Qiao | Single-Atom Catalysis of CO Oxidation Using Pt1/FeOx[END_REF][START_REF] Li | Oxidation Catalyzed by Single Gold Atoms Supported on Aluminum Oxide Clusters[END_REF][START_REF] Wei | FeOx-Supported Platinum Single-Atom and Pseudo-Single-Atom Catalysts for Chemoselective Hydrogenation of Functionalized Nitroarenes[END_REF][START_REF] Yan | Single-Atom Pd1/Graphene Catalyst Achieved by Atomic Layer Deposition: Remarkable Performance in Selective Hydrogenation of 1,3-Butadiene[END_REF][START_REF] Jones | Thermally Stable Single-Atom Platinum-on-Ceria Catalysts via Atom Trapping[END_REF][START_REF] Caparrós | Remarkable Carbon Dioxide Hydrogenation to Ethanol on a Palladium/Iron Oxide Single-Atom Catalyst[END_REF][START_REF] Liu | Catalysis by Supported Single Metal Atoms[END_REF][START_REF] Wang | Heterogeneous Single-Atom Catalysis[END_REF][START_REF] Cui | Bridging Homogeneous and Heterogeneous Catalysis by Heterogeneous Single-Metal-Site Catalysts[END_REF][START_REF] Lang | Non Defect-Stabilized Thermally Stable Single-Atom Catalyst[END_REF] Moreover, with respect to supported nanoparticles, SACs can exhibit different performances in relation to their distinct electronic structure, onto which the support has a critical influence. [START_REF] Chen | Characterization, and Stability of Supported Single-Atom Catalysts[END_REF] These materials have shown promising performances in a number of reactions related to energy, environment and chemicals production. However, owing to the inherent tendency of single atoms to aggregate into larger entities such as nanoparticles, SAC stability may be the main issue in future developments. For example, several works have shown the agglomeration tendency of Pt atoms on various supports under reducing atmospheres. [START_REF] Bradley | Behavior of Pt Atoms on Oxide Supports During Reduction Treatments at Elevated Temperatures, Characterized by Aberration Corrected Stem Imaging[END_REF][START_REF] Sinkler | Aberration-Corrected Transmission Electron Microscopy and in Situ XAFS Structural Characterization of Pt/γ-Al2O3 Nanoparticles[END_REF][START_REF] Nagai | In Situ Redispersion of Platinum Autoexhaust Catalysts: An On-Line Approach to Increasing Catalyst Lifetimes?[END_REF][START_REF] Gänzler | Tuning the Structure of Platinum Particles on Ceria In Situ for Enhancing the Catalytic Performance of Exhaust Gas Catalysts[END_REF][START_REF] Duan | Stability Investigation of a High Number Density Pt1 /Fe2O3 Single-Atom Catalyst under Different Gas Environments by HAADF-STEM[END_REF][START_REF] Dessal | Atmosphere-Dependent Stability and Mobility of Catalytic Pt Single Atoms and Clusters on γ-Al2O3[END_REF] Nevertheless, whereas dynamical effects in catalysis have been investigated in detail for supported nanoparticles, [START_REF] Lira | Role and Evolution of Nanoparticle Structure and Chemical State during the Oxidation of NO over Size-and Shape-Controlled Pt/γ-Al2O3 Catalysts under Operando Conditions[END_REF][START_REF] Zhai | Fluxionality of Catalytic Clusters: When It Matters and How to Address It[END_REF] similar studies on SACs are still scarce. Noticeably, in many studies published so far, the metal dispersion (single atom or cluster/nanoparticle) during, or even after the reaction, is unknown, making the in situ/operando approaches [START_REF] Lira | Role and Evolution of Nanoparticle Structure and Chemical State during the Oxidation of NO over Size-and Shape-Controlled Pt/γ-Al2O3 Catalysts under Operando Conditions[END_REF][START_REF] Frenken | Operando Research in Heterogeneous Catalysis[END_REF][START_REF] Kaftan | Sensitivity of CO Oxidation toward Metal Oxidation State in Ceria-Supported Catalysts: An Operando DRIFTS-MS Study[END_REF][START_REF] Zlotea | Role of Hydrogen Absorption in Supported Pd Nanocatalysts during CO-PROX: Insights from Operando X-Ray Absorption Spectroscopy[END_REF][START_REF] Asakura | Dynamic Behavior of Rh Species in Rh/Al2O3 Model Catalyst during Three-Way Catalytic Reaction: An Operando X-Ray Absorption Spectroscopy Study[END_REF][START_REF] Zhang | Catalysis on Singly Dispersed Rh Atoms Anchored on an Inert Support[END_REF][START_REF] Liu | Evolution and Stabilization of Subnanometric Metal Species in Confined Space by in Situ TEM[END_REF] highly desirable. In this context, an atomic-scale understanding of SAC restructuring is expected to favor the design of stable SACs. [START_REF] Qin | Strategies for Stabilizing Atomically Dispersed Metal Catalysts[END_REF] A number of studies dedicated to Pt/Al2O3 SACs have been reported to date, with applications to CO oxidation, water-gas shift, and hydrocarbon processing. [START_REF] Kwak | Coordinatively Unsaturated Al 3+ Centers as Binding Sites for Active Catalyst Phases of Platinum on γ-Al2O3[END_REF][START_REF] Mei | Unique Role of Anchoring Penta-Coordinated Al 3+ Sites in the Sintering of γ-Al2O3-Supported Pt Catalysts[END_REF][START_REF] Zhai | Alkali-Stabilized Pt-OHx Species Catalyze Low-Temperature Water-Gas Shift Reactions[END_REF][START_REF] Moses-Debusk | CO Oxidation on Supported Single Pt Atoms: Experimental and Ab Initio Density Functional Studies of CO Interaction with Pt Atom on θ-Al2O3(010) Surface[END_REF][START_REF] Zhang | Thermally Stable Single Atom Pt/m-Al2O3 for Selective Hydrogenation and CO Oxidation[END_REF][START_REF] Cui | Synthesis of Single Atom Based Heterogeneous Platinum Catalysts: High Selectivity and Activity for Hydrosilylation Reactions[END_REF] CO oxidation on Pt/-Al2O3 can be considered as a reference model system due to its relative simplicity and practical relevance in e.g. air pollution control. [START_REF] Morfin | Synergy between Hydrogen and Ceria in Pt-Catalyzed CO Oxidation: An Investigation on Pt-CeO2 Catalysts Synthesized by Solution Combustion[END_REF] In a pioneer work on Pt1/-Al2O3, Narula and coworkers concluded from infrared/X-ray absorption spectroscopies and scanning transmission electron microscopy (STEM) that Pt single atoms are intrinsically active toward CO oxidation. [START_REF] Moses-Debusk | CO Oxidation on Supported Single Pt Atoms: Experimental and Ab Initio Density Functional Studies of CO Interaction with Pt Atom on θ-Al2O3(010) Surface[END_REF] A model based on density functional theory (DFT) suggested that Pt1 prefers to bond to O2 over CO, and that the reaction proceeds through a Pt carbonate. In contrast, Stair and coworkers concluded from infrared spectroscopy, temperature-programmed reaction and STEM that, unlike Pt nanoparticles, single Pt atoms supported on a variety of substrates including -Al2O3, are inactive for CO oxidation and water-gas shift reactions. [START_REF] Ding | Identification of Active Sites in CO Oxidation and Water-Gas Shift over Supported Pt Catalysts[END_REF] This was mostly ascribed to an excessively strong Pt1-CO interaction.

In a recent work, we have shown from environmental STEM, X-ray absorption spectroscopy and DFT investigations that single Pt atoms on alumina are stabilized in oxygen atmosphere through the formation of Pt-Oads-Alsupport bridges; in contrast, Pt atoms aggregate into mobile subnanometric clusters under hydrogen. [START_REF] Dessal | Atmosphere-Dependent Stability and Mobility of Catalytic Pt Single Atoms and Clusters on γ-Al2O3[END_REF] Here, we investigate the structural stability of Pt/-Al2O3 SACs throughout CO oxidation in correlation with their catalytic performances by using, in combination with STEM, state-of-the-art operando spectroscopies. We evidence that, even for relatively low Pt amounts and a priori favorable reaction conditions, single Pt atoms are prone to aggregate into clusters, which are much more active and then dominate the CO oxidation kinetics. This implies that, in metal/oxide SAC studies, even a low fraction of clusters or nanoparticles should not be neglected, as they may represent, depending on the considered reaction, the actual active species.

EXPERIMENTAL SECTION

Catalyst preparation

γ-Al2O3 powders (Sasol Puralox SCFa-140, >97.0 % Al2O3) were calcined in air at 600 °C for 3 h before use. For catalyst preparation by incipient wetness impregnation, 2.0 g of alumina was impregnated with 1.8 mL of an aqueous solution of Pt(NH3)4(NO3)2 (Alfa Aesar, >50 wt% Pt), dried in air at 60 °C for 5 h, and calcined in air (40 mL/min) at 300 °C for 2 h (2 °C/min ramp). The Pt loadings of the 0.3Pt and 1Pt samples were find equal to 0.31 wt% and 0.96 wt%, respectively, by inductively coupled plasma -optical emission spectroscopy (ICP-OES, Horiba Jobin Yvon).

Scanning transmission electron microscopy

The STEM-HAADF experiments were performed with a FEI Titan ETEM G2 electron microscope, equipped with a Cs image aberration corrector and operated at 300 keV. The extraction voltage, camera length, acceptance angles, STEM resolution and probe current were 4500 V, 245 mm, 29.2-146 °, 0.14 nm and <0.1 nA, respectively. For sample preparation, the powder was crushed and dispersed in ethanol through ultrasonication, dropped onto a holey carbon-coated 200 mesh Cu grid, and dried by a lamp. To avoid contamination during analysis and remove all residual carbon, the samples were Ar plasma-cleaned for 20 s (Plasma Prep 5, GaLa Instrumente). No filtering was applied on the STEM images.

Operando X-ray absorption spectroscopy

The local structure of Pt species was investigated by XAS in transmission mode on the ROCK beam line at the SOLEIL synchrotron. X-ray absorption spectra at the L3 edge of Pt were recorded. The powder samples were placed inside a pseudo plug-flow operando cell composed of a sample holder fitted into a heated block. [START_REF] Fontaine | X-Ray Absorption Spectroscopy and Heterogeneous Catalysis: Performances at the SOLEIL's SAMBA Beamline[END_REF] The cell was connected to a gas distribution system allowing for careful control of the gas flows at atmospheric pressure. In situ thermal treatments were applied from RT to 300 °C under air flow (10 5 Pa, 50 mL/min, 4 °C/min) or H2 flow (10 5 Pa, 30 mL/min, 5 °C/min). Under the reaction mixture at atmospheric pressure (total flow rate 50 mL/min), the sample was heated at 2 °C/min from RT to 300 °C, and cooled down at the same rate. The gas mixture consisted of CO:O2:He = 2:10:88% (COOX10 conditions) or CO:O2:He = 2:2:96% (COOX2 conditions). The reactor was flushed for 10 minutes under He between the oxidative and the reductive treatments. Data merging and preliminary analysis were carried out using ATHENA software. [START_REF] Ankudinov | Real-Space Multiple-Scattering Calculation and Interpretation of x-Ray-Absorption near-Edge Structure[END_REF] EXAFS fitting was performed using VIPER program. [START_REF] Klementev | Extraction of the Fine Structure from X-Ray Absorption Spectra[END_REF] The fit was performed alternatively in k and R spaces. When in the R space, it was done in the (Im + Module) mode, mathematically equivalent to the k-fit.

It should be noted that the error in Pt-Pt coordination number (CN) determination by EXAFS is high for these low-loaded and highly dispersed systems, because of the overlap between the Pt-Pt features and wiggles of the Pt-O FT signals. Thus, in the presence of several Pt-O bonds per Pt atom, determining the presence of less than one Pt neighbor is at the limit of the EXAFS technique. The second neighbor peak with a distance slightly above 3 Å is most probably due to the support Al atoms involved in the Pt-O-Al bonds. However, the high disorder of the Pt coordination environment makes it impossible to fit this contribution with an acceptable figure of merit.

Operando diffuse reflectance infrared Fourier transform spectroscopy

Operando infrared spectroscopy was performed using a Thermo Nicolet 6700 FTIR spectrophotometer equipped with a DRIFTS cell (Harrick HVC-DRP) and a high-sensitivity MCT detector. The cell, equipped with CaF2 windows, was connected to a gas handling system allowing in situ treatments with several gases at temperatures up to 500 °C. About 30 mg of the powder sample was placed in the cell sample holder. The sample was submitted to the following treatments or reactions at atmospheric pressure with a total flow rate of 25 mL/min: (i) 20% O2 in He, 2 °C/min, 350 °C, 2 h; (ii) 2% CO + 2 or 10% O2 in He, 2 cycles; (iii) H2, 2 °C/min, 350 °C, 2 h; (iv) identical to (ii); (v) identical to (i); (vi) identical to (ii). The spectra were recorded in the 1000-4000 cm -1 range at a resolution of 2 cm -1 by accumulating 64 scans. The Omnic software (Thermo) was used for initial data processing. Temperature cycles were composed of ramps (3 °C/min) and plateaus (20 min, for DRIFTS acquisition) every 50 °C. The reported temperature corresponds to that of the powder sample surface after calibration with a pyrometer. The gases exiting the DRIFTS cell were continuously monitored with a mass spectrometer (Aspec QMS from SRA Instruments). The experiments were also performed with bare -Al2O3 in order to subtract the signal of gaseous CO from the DRIFTS spectra.

Catalytic tests

The catalytic properties in CO oxidation were evaluated under atmospheric pressure in a continuous-flow fixedbed reactor which consisted of a cylindrical glass tube (10 mm inner diameter) equipped with a sintered glass filter supporting the catalyst powder. The same quantity of Pt (0.5 mg) was used in all tests. The reactor was placed in a ceramic furnace heated or cooled at 1 °C/min between 50 °C and 280 °C. The catalyst temperature was recorded through a thermocouple immersed in the catalytic bed. By using mass-flow controllers, the gases were mixed (CO:O2:He = 2:10:88% or 2:2:96%) and flowed through the reactor at a total rate of 50 Nml/min. The effluent gases were analyzed online using a Micro GC equipped with a TCD detector.

RESULTS AND DISCUSSION

Two Pt/-Al2O3 catalysts were investigated, with a Pt loading of 0.3 wt% (0.3Pt sample) or 1.0 wt% (1Pt sample). Fig. 1 and Figs. S1-S3 show STEM images of the samples. For the as-prepared (i.e., calcined) samples, while 0.3Pt contains exclusively single Pt atoms (Fig. 1a), a mixture of single atoms and small clusters is present on 1Pt (Figs. 1e and S2a), consistently with previous reports. [START_REF] Dessal | Atmosphere-Dependent Stability and Mobility of Catalytic Pt Single Atoms and Clusters on γ-Al2O3[END_REF][START_REF] Kwak | Coordinatively Unsaturated Al 3+ Centers as Binding Sites for Active Catalyst Phases of Platinum on γ-Al2O3[END_REF] In order to compare the performances of these catalysts with those of pre-reduced ones, the samples were submitted to a series of successive reactive treatments under flow conditions at atmospheric pressure: (i) calcination in air, to accurately reset the initial (oxidized) conditions; (ii) CO oxidation reaction; (iii) reduction in hydrogen; (iv) CO oxidation (identical to ii). All four treatments were performed from RT up to 300 °C, followed by cooling back to RT. For the reaction stages, two sets of conditions were used, with CO:O2 contents in helium of 2:10% (referred to as COOX10 conditions) or 2:2% (COOX2). Note that the COOX10 (more oxidizing) conditions were chosen to tentatively stabilize the SAC, given that it is stable under oxygen. [START_REF] Dessal | Atmosphere-Dependent Stability and Mobility of Catalytic Pt Single Atoms and Clusters on γ-Al2O3[END_REF] Similarly, pre-exposure to O2 was shown to limit the H2-induced sintering of Pt nanoparticles supported on alumina. [START_REF] Matos | In Situ Coarsening Study of Inverse Micelle-Prepared Pt Nanoparticles Supported on γ-Al2O3: Pretreatment and Environmental Effects[END_REF] This protocol was similarly applied in a conventional flow-fixed-bed catalytic reactor, in operando DRIFTS-MS experiments, and in synchrotron-based operando XAS-MS experiments. In conventional testing (Fig. Figure 2 displays the results of such an investigation for the 0.3Pt sample under oxygen-rich COOX10 reaction conditions. In addition to Fig. 2b, a conventional view of the Pt-carbonyl region of DRIFTS spectra is shown in Fig. S8 (left panels). The corresponding data for 1Pt COOX10 (Fig. S9) and 0.3Pt COOX2 (Fig. S10) are reported in Supporting Information, together with XANES and EXAFS references (Fig. S11), and additional XAS raw data and analyses (Figs. S12-S15, Tables S1-S3).

Pre-oxidized single Pt atoms

In the as-prepared (air-calcined) SAC, the EXAFS Fourier transform only shows a Pt-O contribution (r  2.0 Å, Fig. 2a) without any Pt-Pt one, confirming the absence of metallic Pt clusters, in accordance with previous works. [START_REF] Dessal | Atmosphere-Dependent Stability and Mobility of Catalytic Pt Single Atoms and Clusters on γ-Al2O3[END_REF][START_REF] Moses-Debusk | CO Oxidation on Supported Single Pt Atoms: Experimental and Ab Initio Density Functional Studies of CO Interaction with Pt Atom on θ-Al2O3(010) Surface[END_REF] Comparison of the XANES of the SAC with those of bulk Pt, bulk PtO2, and pure Pt(NH3)4(NO3)2 precursor references (Fig. S11a) suggests an oxidation degree of the single Pt atoms comprised between II (Pt precursor) and IV (PtO2).

During the first CO oxidation cycle (bottom of Fig. 2a), the main EXAFS contribution remains the Pt-O one. At first sight, the first CO oxidation run (heating) is performed on single Pt atoms, which show a moderate activity with a half-conversion temperature T1/2  230 °C in a conventional flow-fixed-bed reactor (Figs. 2c andS4a). Upon cooling, T1/2 decreases to ca. 200 °C. The resulting hysteresis is mainly explained in the literature by mass or heat diffusion effects, low-temperature poisoning of Pt by CO, and different oxidation states of Pt in light-off and extinction stages, which can be coupled to each other. [START_REF] Morfin | Synergy between Hydrogen and Ceria in Pt-Catalyzed CO Oxidation: An Investigation on Pt-CeO2 Catalysts Synthesized by Solution Combustion[END_REF][START_REF] Carlsson | A Transient in Situ FTIR and XANES Study of CO Oxidation over Pt/Al2O3 Catalysts[END_REF][START_REF] Casapu | Origin of the Normal and Inverse Hysteresis Behavior during CO Oxidation over Pt/Al2O3[END_REF] Looking closely at the EXAFS evolution, small Pt-Pt features are visible at 150-200 °C during heating. Then, the Pt-Pt feature becomes more clearly visible during cooling, in the 150 °C-RT temperature region. The presence of Pt-Pt features is correlated to minima in the XANES white line intensity (WLI), represented horizontally in Fig. 2a. The Pt-Pt feature appearance in EXAFS and WLI decrease in XANES indicate Pt clustering and reduction.

Another indication of Pt clustering comes from DRIFTS monitoring (Figs. 2b andS8). While the main peak at 2106-2100 cm -1 is ascribed to CO linearly adsorbed on single Pt m+ cations (m  2), the shoulder at 2090-2070 cm -1 , present at ca. 150 °C on heating and at 150-50 °C on cooling, can be ascribed to partially oxidized Pt clusters (Pt + ,  < 2). [START_REF] Ding | Identification of Active Sites in CO Oxidation and Water-Gas Shift over Supported Pt Catalysts[END_REF][START_REF] Carlsson | A Transient in Situ FTIR and XANES Study of CO Oxidation over Pt/Al2O3 Catalysts[END_REF] Unlike the main band, this shoulder shifts towards higher wavenumbers as temperature decreases, due to the increased dipole-dipole coupling between CO adsorbates. [START_REF] Primet | Electronic Transfer and Ligand Effects in the Infrared Spectra of Adsorbed Carbon Monoxide[END_REF] Furthermore, the disappearance of this contribution -and this contribution only-throughout the full CO conversion period suggests that the precursor of the active species is Pt + . This small fraction of Pt clusters would coexist with poorly active single Pt cations, the DRIFTS signature of which appears indeed essentially unaffected by the reaction. Indeed, no decrease of the Pt m+ -CO band intensity was observed at high CO conversion (Fig. 2b). However, a slight decrease in this single-atom contribution was observed at high CO conversion in the second CO oxidation cycle (Fig. S5), and more markedly under oxygen-poor COOX2 conditions (Fig. S7). these cases the Pt clusters are majority species, and the decreasing amounts of SAs could be expected. Nevertheless, unlike the Pt + -CO (clusters), the Pt m+ -CO species (SAs) never disappear completely, thus the depletions can be ascribed to desorption and not necessarily to reaction. Further, the ~2100 cm -1 band (SAs) actually increases on heating, and decreases back at the end of the cycle (Fig. 2b). Such an evolution indicates that single Pt atoms can hardly accommodate both CO and oxygen and that the adsorption competition is favorable to oxygen at low temperature and to CO at higher temperatures, at which the reaction proceeds on the clusters. This partly reconciles the results of Moses DeBusk et al. and Ding et al., who found that single Pt atoms prefer to accommodate oxygen or CO, respectively. [START_REF] Moses-Debusk | CO Oxidation on Supported Single Pt Atoms: Experimental and Ab Initio Density Functional Studies of CO Interaction with Pt Atom on θ-Al2O3(010) Surface[END_REF][START_REF] Ding | Identification of Active Sites in CO Oxidation and Water-Gas Shift over Supported Pt Catalysts[END_REF] Pt clustering appears even more marked during the second DRIFTS cycle (Fig. S5), and it correlates with a higher activity (lower T1/2, Fig. S4a). However, at the end of the CO oxidation cycles, a large proportion of single Pt atoms remains, consistently with STEM analyses (Fig. 1b).

Remarkably, some authors have proposed, using DFT calculations, CO oxidation mechanisms on Pt/Al2O3(010) SACs, which involve either Pt(CO3) [START_REF] Moses-Debusk | CO Oxidation on Supported Single Pt Atoms: Experimental and Ab Initio Density Functional Studies of CO Interaction with Pt Atom on θ-Al2O3(010) Surface[END_REF] or Pt (O-O-C=O) [START_REF] Gao | CO Oxidation Mechanism on the γ-Al2O3 Supported Single Pt Atom: First Principle Study[END_REF] species. The former has also been considered by Newton et al. to interpret their transient experiments on commercial Pt/Al2O3 catalysts. [START_REF] Newton | Room-Temperature Carbon Monoxide Oxidation by Oxygen over Pt/Al2O3 Mediated by Reactive Platinum Carbonates[END_REF][START_REF] Newton | Kinetic Studies of the Pt Carbonate-Mediated, Room-Temperature Oxidation of Carbon Monoxide by Oxygen over Pt/Al2O3 Using Combined, Time-Resolved XAFS, DRIFTS, and Mass Spectrometry[END_REF] Our simultaneous monitoring of the catalyst state and light-off activity, based for the first time on operando experiments, shows that the involvement of Pt carbonates is not required (though not ruled out) to explain our data. In our conditions, the oxidation of CO to CO2 can proceed through the conventional Langmuir-Hinshelwood mechanism over the fraction of Pt clusters formed in situ within the initial SAC. The increase of this cluster fraction throughout CO oxidation cycles leads to an increase in the CO oxidation activity. Our results are consistent with those of Ding et al. [START_REF] Ding | Identification of Active Sites in CO Oxidation and Water-Gas Shift over Supported Pt Catalysts[END_REF] and of Lou and Liu, [START_REF] Lou | CO Oxidation on Metal Oxide Supported Single Pt Atoms: The Role of the Support[END_REF] who concluded on the inactivity or poor activity, respectively, of Pt single atoms supported on -Al2O3,. In connection to these findings, the superiority of subnanometric clusters overs single atoms was previously reported for CO oxidation over Ir/FeOx. [START_REF] Lin | More Active Ir Subnanometer Clusters than Single-Atoms for Catalytic Oxidation of CO at Low Temperature[END_REF]

Pre-reduced Pt clusters

The second part of the experiments consisted in a reducing treatment in hydrogen in order to obtain a majority of Pt clusters, [START_REF] Dessal | Atmosphere-Dependent Stability and Mobility of Catalytic Pt Single Atoms and Clusters on γ-Al2O3[END_REF] followed by an additional CO oxidation cycle (or two in the cases of DRIFTS and conventional tests). Fig. 2a shows the gradual disappearance of the Pt-O EXAFS feature during the hydrogen treatment, together with the decrease in the XANES WLI and the appearance of a small Pt-Pt contribution (r  2.6 Å). As previously reported, such a treatment of a Pt/-Al2O3 SAC leads to a dominant fraction of 0.9 nm-sized Pt clusters [START_REF] Sinkler | Aberration-Corrected Transmission Electron Microscopy and in Situ XAFS Structural Characterization of Pt/γ-Al2O3 Nanoparticles[END_REF][START_REF] Dessal | Atmosphere-Dependent Stability and Mobility of Catalytic Pt Single Atoms and Clusters on γ-Al2O3[END_REF] (see also Fig. S2c,d for 1Pt sample). The Pt-Pt CN was close to 6 (Table S1), in agreement with the calculations of Jentys for 13-atom spherical clusters. [START_REF] Jentys | Estimation of Mean Size and Shape of Small Metal Particles by EXAFS[END_REF] The Pt-Pt CN remains roughly constant under CO/O2 at low temperature, up to 150 °C, i.e. before the light-off. DRIFTS supports the quasi exclusive presence of reduced Pt clusters in these conditions, as attested by the 2070-2050 cm -1 feature corresponding to linear Pt 0 -CO adsorption, with clear coverage-dependent band shift. [START_REF] Alexeev | Effects of Reduction Temperature and Metal-Support Interactions on the Catalytic Activity of Pt/γ-Al2O3 and Pt/TiO2 for the Oxidation of CO in the Presence and Absence of H2[END_REF] A fraction of partially oxidized Pt also exists (shoulder at ca. 2080 cm -1 , better seen in Fig. S8), which is assigned to Pt + multimers and/or cluster periphery sites. The sudden disappearance of the 2070-2050 cm -1 DRIFTS feature upon light-off (Fig. 2b andS8) shows again that the Pt clusters -now majority-are the catalytically active species. This pre-reduced catalyst is much more active than the pre-calcined one, with a decrease of T1/2 by 52 °C (Figs. 2c andS4). The amplification of activity is ascribed to the increased fraction of Pt clusters. The level of the XANES WLI under reaction conditions (Fig. 2a) suggests that these active Pt clusters are oxidized. However, no Pt-Pt contribution is present in the EXAFS signal during the 100% CO conversion period, while the Pt-O component (r  2.0 Å) and the WLI are reinforced (Fig. 2a). This is ascribed to the oxidation of the Pt clusters into amorphous PtOx. Indeed, when 100% CO conversion is achieved, in the major part of the catalyst bed, the gaseous atmosphere contains only inert gas and O2, thus being highly oxidative. In order to confirm this hypothesis, as opposed to an oxidative redispersion of the Pt clusters into single Pt cations, a quenching experiment was carried out during conventional catalytic tests (on the 1Pt sample, which shows a similar behavior as 0.3Pt, see Fig. S9). At the end of the COOX10 heating ramp, after a plateau of 25 min at 280 °C, the reactor was quickly water-cooled to RT under air and the sample was analyzed by STEM. Fig. S2e,f clearly shows Pt clusters with an average size of 1.1 ± 0.3 nm, which supports the hypothesis that the Pt clusters are not redispersed but oxidized during the reaction at high temperature, i.e. at 100% CO conversion.

Upon return to low temperature under CO oxidation conditions, the reinforcement of the DRIFTS feature at ca. 2100 cm -1 (Figs. 2b and S8, top panels), which had appeared at 150 °C upon heating, is most likely due to CO adsorption on remaining single Pt m+ cations. Although present in much smaller fraction after reduction, these species behave similarly as before reduction towards CO adsorption. Indeed, as previously mentioned, an increase of the 2100-2110 cm -1 feature (single Pt cations after calcination) upon heating up to 300 °C and subsequent cooling down to 150 °C was observed (Figs. 2b and S8, bottom panels).

However, most of the signal (2080-2050 cm -1 ) is ascribable to Pt clusters. Unlike before the reduction treatment (cycles 1 and 2), after reduction two successive CO oxidation cycles produce similar DRIFTS signals (Fig. S5, cycles 3 and 4), consistently with the catalytic tests (Fig. S4). This indicates that the clustering process is complete. At the end of the whole process, Pt is thus mostly returned into the form of Pt 0 and, to lower extent, Pt + clusters (low XANES intensity and CN = 6, see Table S1). However, a significant proportion of single Pt atoms (Pt m+ ) remains, as seen from EXAFS and DRIFTS (Fig. 2a) and post-reaction STEM (Figs. 1c andS1d-f). In DRIFTS experiments, after the first two stages (post-calcination and post-reduction), the catalyst was further calcined in situ under air flow and two additional CO oxidation cycles (5 and 6, Fig. S5) were performed. The DRIFTS data overall appear very similar to those of the post-reduction stage. This implies that the clustering process, which begins after the initial calcination, and completes after reduction, is irreversible.

Effect of Pt loading and reaction conditions

When comparing the results for 0.3Pt COOX10 (Fig. 2) with those for 1Pt COOX10 (Fig. S9) and 0.3Pt COOX2 (Fig. S10), all three processes look qualitatively similar. However, DRIFTS evidences that the fraction of metallic Pt clusters in the first reaction stage is more important at higher Pt loading (1Pt COOX10), and even more under less oxidizing conditions (0.3Pt COOX2). Consistently with the above conclusion that Pt clusters are more active than single atoms, T1/2 is decreased by 24 °C at higher Pt loading under COOX10 conditions (Fig. S4). In contrast, after the reduction treatment leading to a majority of Pt clusters in all cases, the DRIFTS data are almost identical for 0.3Pt and 1Pt, and accordingly the COOX10 activities are now similar. The activity of 0.3Pt is lower under COOX2 conditions (T1/2 = 239 °C, vs. 226 °C for COOX10) because of the limited supply in O2 induced by strong CO adsorption at low temperature, i.e. negative reaction order in CO, similarly to what is known for larger Pt particles within the Langmuir-Hinshelwood mechanism. [START_REF] Engel | Elementary Steps in the Catalytic Oxidation of Carbon Monoxide on Platinum Metals[END_REF] In all the cases, as shown by Figs. S5-S7 (cycles 3-6 vs cycles 1-2), the adsorption of CO as measured by DRIFTS is much more prominent on prereduced catalysts (Pt clusters) than on pre-oxidized ones (single Pt atoms). Fig. 3 summarizes the results and allows a quantitative comparison of the evolutions of the white line intensities (a) and Pt-Pt coordination numbers (b) between the three cases. Both show large variations throughout the experiment. The WLI is maximum under highly oxidizing conditions, i.e. calcination treatment and high reaction temperature (high CO conversion). It decreases under less oxidizing conditions, i.e. reduction treatment and low reaction temperature. The WLI decrease corresponds to concomitant decrease of Pt-O CNs in EXAFS, from more than 5 to less than 1 (Fig. S12 and Tables S1-S3). As expected, the WLI and the Pt-O CN show very similar trends (Fig. S12). Overall, Fig. 3 evidences clear Pt reduction (a) and Pt clustering (b) tendencies. Switching from 0.3Pt-COOX10 conditions to 1Pt-COOX10 or 0.3Pt-COOX2 leads to a decrease in the WLI, i.e. both the increase in Pt loading and the decrease in oxygen concentration have Pt reduction effects. The Pt-Pt bonds corresponding to few-atom clusters are already detected in EXAFS from ~100 °C upon heating in 1Pt-COOX10 and 0.3Pt-COOX2 experiments, whereas for 0.3Pt-COOX10 the Pt clusters appear only below 200 °C on cooling. However, the COOX2 conditions induce a larger increase in CN in the first reaction cooling stage, with a maximum CN of 5 vs. 3-4 for the COOX10 conditions. Therefore, more reducing (COOX2) conditions lead to a larger number of Pt atoms in the clusters.

CONCLUSIONS

In this work, in addition to conventional catalytic testing and STEM, we have used operando XAS and DRIFTS spectroscopies to investigate the oxidation of CO over a 0.3 wt% Pt/γ-Al2O3 SAC prepared by incipient wetness impregnation-calcination. The SAC behavior has been compare d to its pre-reduced and 1 w t% counter parts under both standard (CO:O2 = 2:2 wt%) and highly oxidizing (CO:O2 = 2:10 wt%) conditions.

It is shown that the initially single Pt cations gradually aggregate into partially oxidized Pt clusters in the course of the reaction light-off/light-out cycles. The single atoms are strongly coordinated to oxygen at room temperature, and to CO at higher temperature. Such a strong adsorption prevents the SAs from catalyzing the reaction. As the clusters are more active than the single atoms, their formation during the reaction, and upon hydrogen treatment, leads to increases in the CO oxidation activity. As compared to the low-loaded SAC, the 1 wt% Pt catalyst shows a larger proportion of clusters, a lower average oxidation state, and a higher Pt-specific activity. Similarly, exposing the SAC to less oxidizing conditions deeply increases the cluster fraction and decreases the oxidation state.

While demonstrating the clustering dynamics of Pt atoms from a systematic multi-technique investigation, this work enriches the debate on the CO oxidation activity of

Figure 1 .

 1 Figure 1. Representative STEM images of Pt/-Al2O3 samples throughout the catalysis experiments. (a-c) 0.3 wt% Pt/Al2O3 catalyst after calcination (a), subsequent CO oxidation cycle with CO:O2 = 2:10% (b), and after full experiment (c). Average size of the clusters (such as that pointed by the arrow) in (c): 1.0 ± 0.3 nm. (d) 0.3 wt% Pt/Al2O3 catalyst after full experiment with CO:O2 = 2:2%. Mostly Pt clusters are present (average size 1.9 ± 1.0 nm). (e-f) 1.0 wt% Pt/Al2O3 catalyst, as-prepared (e) and after full experiment with CO:O2 = 2:10% (f). Mostly single Pt atoms are present in (e) and mostly Pt clusters (average size 1.0 ± 0.2 nm) are present in (f). Some of the single atoms are circled in yellow.

Figure 2 .

 2 Figure 2. Monitoring of Pt species and their CO oxidation activity (0.3 wt% Pt/-Al2O3, CO:O2 = 2:10%). (a) EXAFS color map showing the evolution with time (from bottom to top) of the FT throughout the calcination/reaction/reduction/reaction procedure. The full color scale corresponds to 0-2.13 Å -4 in FT module. Two FT signals are shown in classical view. The radial distance is phase-corrected. The white disks represent the XANES white line intensity in the same experiment. The intensity values for Pt, Pt(NH3)4(NO3)2 and PtO2 references (Fig. S11a) are reported on the bottom axis. (b) DRIFTS color map showing the evolution of the C-O absorption band(s) in the Pt carbonyl wavenumber region during post-calcination (bottom panel) and post-reduction (top panel) reaction steps. The full color scales correspond to 0-0.10 (bottom) and 0-0.18 (top) in absorbance. Three spectra corresponding to the horizontal lines (at 150 °C during heating and 100 °C during cooling) on the DRIFTS maps are plotted in classical view. The 100% CO conversion region, as derived from mass spectrometry, is indicated. In (a) and (b), the Y-axes show temperatures in °C. (c) CO oxidation light off curves obtained in a conventional flow-fixed-bed reactor following a similar protocol as in operando experiments.

Figure 3 .

 3 Figure 3. Evolution of XANES white line intensities (a) and Pt-Pt coordination numbers derived from EXAFS analysis (b)during the calcination/reaction/reduction/reaction sequence on 0.3 wt% Pt/-Al2O3 (COOX10 and COOX2 conditions) and 1.0 wt% Pt/-Al2O3 (COOX10). The total duration of each experiment was ~20 h, except in COOX2 for which only the first half was carried out. The drawings represent the catalyst state. The ball color represents the oxidation state of Pt, going from highly cationic (light blue) to slightly cationic (medium blue) and reduced (dark blue). Orange arrows represent CO oxidation. The double black arrows at the bottom of the figure indicate the approximate full-CO-conversion temperature regions.

  Pt1/γ-Al2O3 SACs. These results are likely to be qualitatively applicable to other catalytic systems and demonstrate the value of operando investigations to assess the intrinsic activity-stability of SACs. Supporting Information. Additional STEM, DRIFTS, XAS, and CO oxidation data. This material is available free of charge via the Internet at http://pubs.acs.org. * E-mail: Laurent.Piccolo@ircelyon.univ-lyon1.frThe authors declare no competing financial interests. S. Belin (SOLEIL), C. Zlotea and A. Malouche (ICMPE) are acknowledged for support with XAS experiments at the ROCK beamline of SOLEIL synchrotron (proposal 20170160). L. Roiban (MATEIS) is acknowledged for help in TEM experiments. N. Cristin and P. Mascunan (IRCELYON) are acknowledged for ICP analyses. Agence Nationale de la Recherche (Ul-traCat project, ANR-17-CE06-0008), Région Auvergne-Rhône Alpes (CMIRA 2016 project) and LABEX iMUST ANR-10-LABX-0064/ ANR-11-IDEX-0007 are acknowledged for financial support. The XAS work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (ANR-10-EQPX45). CLYM is acknowledged for access to the FEI Titan microscope.

	(absorbance)	2.5	O 2	CO+O 2	H 2	CO+O 2	a
	XANES white line intensity	1.5 2.0	0.3Pt COOX10 1.0Pt COOX10 0.3Pt COOX2
		EXAFS Pt-Pt coordination number	2 4 6					b
	300	°C 0
	Temp.			100% CO conversion		100% CO conversion
		RT
					Time


Cinzia Signorini

Claudio De Felice

Thierry Durand

Jean-Marie Galano

Camille Oger

Silvia Leoncini

Lucia Ciccoli

Marisa Carone

Monica Ulivelli

Caterina Manna

Alessio Cortelazzo

Chung-Yung Lee

Joussef Hayek

Relevance of 4-F4t-neuroprostane and 10-F4t-neuroprostane to neurological diseases

Keywords:

Introduction

Isoprostanoids, the metabolites of non-enzymatic lipid peroxidation of polyunsaturated fatty acid (PUFA), are measured to assess in vivo oxidative stress status [START_REF] Leung | Current development in non-enzymatic lipid peroxidation products, isoprostanoids and isofuranoids, in novel biological samples[END_REF][START_REF] Milne | Roberts 2nd, The isoprostanes-25 years later[END_REF]. Among all the metabolites from PUFAs, 15-F 2t -isoprostane (15-F 2t -IsoP) derived from arachidonic acid (AA) has been largely studied in medical research [START_REF] De Las Heras-Gómez | Potential applications of lipid peroxidation products -F(4)-neuroprostanes, F(3)-neuroprostanes(n-6 DPA), F(2)-dihomo-isoprostanes and F(2)-isoprostanes -in the evaluation of the allograft function in renal transplantation[END_REF][START_REF] Zhang | Plasma 15-F2t-isoprostane in idiopathic pulmonary arterial hypertension[END_REF][START_REF] Cracowski | Postocclusive reactive hyperemia inversely correlates with urinary 15-F2t-isoprostane levels in systemic sclerosis[END_REF]. Also, 15-F 2t -IsoP (also referred to as 8-iso-PGF 2α ) has been identified to be the most abundant F 2 -IsoP isomer in the free radical-induced AA oxidation [START_REF] Waugh | Identification and relative quantitation of F2-isoprostane regioisomers formed in vivo in the rat[END_REF] and elevated levels are closely related for example, to inflammation and cardiovascular diseases. As a result, 15-F 2t -IsoP is quantified by scientists to unveil pathological mechanisms associated to oxidative stress and recently, the measurement of 15-F 2t -IsoP was classified in a meta-analysis for its role in several specific diseases associated to oxidative damage [START_REF] Van 't Erve | Classifying oxidative stress by F(2)-isoprostane levels across human diseases: a meta-analysis[END_REF].

Despite this, 15-F 2t -IsoP appears to be a non-specific metabolite to be determined in diseases related to neuronal damage [START_REF] Galano | Isoprostanes, neuroprostanes and phytoprostanes: an overview of 25years of research in chemistry and biology[END_REF]. One reason is the difference in PUFA content where docosahexaenoic acid (DHA) is more abundant compared to AA in the brain, in particular the gray matter. Non-enzymatic DHA oxidation generates 128 isomers from 8 regioisomer series (4, 7, 10, 11, 13, 14, 17 or 20) of which 4-series or F 4 -neuroprostanes (F 4 -NeuroPs) [START_REF] Galano | Isoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF][START_REF] Roberts 2nd | The biochemistry of the isoprostane, neuroprostane, and isofuran Pathways of lipid peroxidation[END_REF][START_REF] Galano | Non-enzymatic cyclic oxygenated metabolites of adrenic, docosahexaenoic, eicosapentaenoic and α-linolenic acids; bioactivities and potential use as biomarkers[END_REF] is the most abundant. Moreover, because of the extra methylene bonds in the structure, DHA is more prone to oxidation than AA. F 4 -NeuroPs are very promising isoprostanoids and might have paramount applications in medical field, taking into account the abundance of DHA in the central nervous system [START_REF] Greco | Isoprostanes as biomarkers and mediators of oxidative injury in infant and adult central nervous system diseases[END_REF][START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF][START_REF] Signorini | MECP2 duplication syndrome: evidence of enhanced oxidative stress. A comparison with rett syndrome[END_REF]. In this regard, it was shown that NeuroPs and not IsoPs are the critical metabolites for neuronal damage. Among all the F 4 -NeuroP molecules, only a handful of the isomers are characterized in neuro-pathological conditions and so far, 4-F 4t -NeuroPs and 10-F 4t NeuroPs are considered to be the most represented [START_REF] Galano | Isoprostanes, neuroprostanes and phytoprostanes: an overview of 25years of research in chemistry and biology[END_REF][START_REF] García-Flores | Snapshot situation of oxidative degradation of the nervous system, kidney, and adrenal glands biomarkersneuroprostane and dihomo-isoprostanes-urinary biomarkers from infancy to elderly adults[END_REF][START_REF] Yen | Levels of F2-isoprostanes, F4-neuroprostanes, and total nitrate/nitrite in plasma and cerebrospinal fluid of patients with traumatic brain injury[END_REF][START_REF] Gladine | Lipid profiling following intake of the omega 3 fatty acid DHA identifies the peroxidized metabolites F4-neuroprostanes as the best predictors of atherosclerosis prevention[END_REF][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][START_REF] García-Flores | Assessment of oxidative stress biomarkers -neuroprostanes and dihomo-isoprostanes -in the urine of elite triathletes after two weeks of moderate-altitude training[END_REF][START_REF] Medina | Antiepileptic drugs affect lipid oxidative markers-neuroprostanes and dihomo-isoprostanes-in patients with epilepsy[END_REF][START_REF] Garcia-Flores | Lipidomic approach in young adult triathletes: effect of supplementation with a polyphenols-rich juice on F4neuroprostanes and F2-dihomo-isoprostane markers[END_REF][START_REF] De Las Heras-Gomez | Potential applications of lipid peroxidation products-F4-neuroprostanes, F3-neuroprostanesn-6 DPA and F2-dihomo-isoprostanes and F2-isoprostanes-in the evaluation of the allograft function in renal transplantation[END_REF][START_REF] García-Flores | Snapshot situation of degradation of the nervous system biomarkers -neuroprostanes and dihomo-isoprostanes-from infancy to elderly[END_REF][START_REF] Cháfer-Pericás | Development of a reliable analytical method to determine lipid peroxidation biomarkers in newborn plasma samples[END_REF][START_REF] Kuligowski | Urinary lipid peroxidation byproducts: are they relevant for predicting neonatal morbidity in preterm infants?[END_REF].

Previously, our group showed altered levels of plasma F 4 -NeuroPs in Rett syndrome (RTT) [START_REF] Signorini | F 4 -neuroprostanes mediate neurological severity in Rett syndrome[END_REF], and Down syndrome (DS) patients [START_REF] Manna | Increased non-protein bound iron in Down syndrome: contribution to lipid peroxidation and cognitive decline[END_REF], as well as in RTT experimental mice models [START_REF] De Felice | Oxidative brain damage in Mecp2-mutant murine models of Rett syndrome[END_REF] and in a rodent model of neonatal hypoxic-ischemic encephalopathy [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]. Moreover, in RTT and DS diseases, elevated levels of F 4 -NeuroP have been shown to be intimately related to neurological severity [START_REF] Signorini | F 4 -neuroprostanes mediate neurological severity in Rett syndrome[END_REF] and cognitive performance [START_REF] Manna | Increased non-protein bound iron in Down syndrome: contribution to lipid peroxidation and cognitive decline[END_REF], respectively. Other studies showed that elevated plasma F 4 -NeuroPs are in smokers [START_REF] Seet | Biomarkers of oxidative damage in cigarette smokers: which biomarkers might reflect acute versus chronic oxidative stress? Free Radic[END_REF], type 2 diabetes [START_REF] Seet | Oral zinc supplementation does not improve oxidative stress or vascular function in patients with type 2 diabetes with normal zinc levels[END_REF], ischemic-stroke [323], tick-borne encephalitis [START_REF] Łuczaj | Tick-borne encephalitis-lipid peroxidation and its consequences[END_REF] and neuroborreliosis [START_REF] Moniuszko-Malinowska | Lipid peroxidation in the pathogenesis of neuroborreliosis[END_REF] but its chemical formation, the preferential formation of the type of F 4 -NeuroP isomers and the relevance to the type of diseases are not well characterized.

In this study, we aimed i) to optimize mass spectrometric analysis for 4(RS)-4-F 4t -NeuroP (4-F 4t -NeuroP) and 10(RS)-10-F 4t -NeuroP (10-F 4t -NeuroP) that were chemically synthesized as standards and in oxidized DHA liposome, and ii) to investigate the relevance of 4-F 4t -NeuroP and 10-F 4t -NeuroP in different types of neurological diseases, i.e. multiple sclerosis (MS), Down syndrome (DS), autism spectrum disorders (ASD), and Rett syndrome (RTT), where oxidative injury and fatty acid oxidation take part in the pathogenesis [START_REF] Manna | Increased non-protein bound iron in Down syndrome: contribution to lipid peroxidation and cognitive decline[END_REF][START_REF] Ljubisavljevic | The validation study of neurofilament heavy chain and 8-hydroxy-2'-deoxyguanosine as plasma biomarkers of clinical/paraclinical activity in first and relapsing-remitting demyelination acute attacks[END_REF][START_REF] Bartova | Changes in levels of oxidative stress markers and some neuronal enzyme activities in cerebrospinal fluid of multiple sclerosis patients[END_REF][START_REF] Kemp | Oxidative injury in multiple sclerosis cerebellar grey matter[END_REF][START_REF] Kałużna-Czaplińska | Chromatographic and mass spectrometric techniques in studies on oxidative stress in autism[END_REF][START_REF] Wong | Prenatal exposure to common environmental factors affects brain lipids and increases risk of developing autism spectrum disorders[END_REF][START_REF] Ciccoli | Red blood cells in Rett syndrome: oxidative stress, morphological changes and altered membrane organization[END_REF][START_REF] Rossignol | Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism[END_REF][START_REF] Valenti | Mitochondrial dysfunction as a central actor in intellectual disability-related diseases: an overview of Down syndrome, autism, Fragile X and Rett syndrome[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][START_REF] De Felice | The role of oxidative stress in Rett syndrome: an overview[END_REF][START_REF] Shichiri | The role of lipid peroxidation in neurological disorders[END_REF][START_REF] Pagano | Oxidative stress and mitochondrial dysfunction in Down syndrome[END_REF][START_REF] Deon | Oxidative stress in patients with X-linked adrenoleukodystrophy[END_REF][START_REF] Berger | Pathophysiology of X-linked adrenoleukodystrophy[END_REF].

Materials and methods

Subjects

A total of 122 subjects were enrolled in this study. Of them, 16 patients (46.6 ± 12.1 years old, male to female ratio 7:9) were relapsing-remitting MS [START_REF] Miller | Multiple Sclerosis[END_REF], 20 were RTT (10.3 ± 10.3 years old, all female) with proven MECP2 mutations and typical clinical presentation [START_REF] Neul | Rett syndrome: revised diagnostic criteria and nomenclature[END_REF] (Child Neuropsychiatry Unit, University Hospital, Siena Italy), 9 were autistic patients (13.5 ± 4.6 years old, male to female ratio 7:2) diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition [START_REF]Diagnostic and Statistical Manual of Mental Disorders[END_REF], and 16 were DS subjects (24.9 ± 4.7 years old, male to female ratio 9:7) who had cariotipically confirmed trisomy 21. In addition, a total of 61 of age-and gender-matched healthy control subjects were recruited. Written consent form was obtained by all the subjects enrolled, or by the patient guardian. This study was approved by the institutional review boards and was carried out in accordance to the rules expressed in the Declaration of Helsinki Ethical Principles for Medical Research involving Human Subjects (Brazil, 2013).

In addition, depending on the disease, different types of clinical tests were conducted on the patients, clinical severity was assessed by the Rett Clinical Severity Score (RCSS) [START_REF] Neul | Specific mutations in methyl-CpG-binding protein 2 confer different severity in Rett syndrome[END_REF] (disability range: 7-30) and Childhood Autism Rating Scale (CARS) [START_REF] Schopler | Toward objective classification of childhood autism: childhood autism rating scale (CARS)[END_REF] for ASD (disability range: 36-48). Expanded Disease Status Scale Score (EDSS) [START_REF] Kurtzke | Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)[END_REF] for MS (disability range: 0.0-4.0), and Raven's Colored Progressive Matrices (CPM) [START_REF] Raven | Coloured Progressive Matrices[END_REF] as measure of cognitive impairment for DS (disability range: 3-22) were also used.

Sample preparation

Platelet poor plasma samples were obtained by centrifugation (2400×g for 15 min at 4 °C) of blood aliquots collected in heparinized tubes. As an antioxidant, butylated hydroxytoluene (BHT) (90 μM prepared in absolute ethanol) was added to each plasma samples, mixed and stored at -70 °C for "free" NeuroPs (i.e. not esterified to phospholipids) determination.

To identify the existence of the NeuroPs isomers in a diseased model, brain tissues of RTT mice (Mecp2 stop/y model) and its wildtype were extracted [START_REF] De Felice | Oxidative brain damage in Mecp2-mutant murine models of Rett syndrome[END_REF].

All mice (n = 8; age 12-17 weeks) were obtained from Dr. J. Guy, Wellcome Centre for Cell Biology, University of Edinburgh, United Kingdom. The aggregate score of phenotypic severity of the mice was performed on weekly basis for symptoms arising from Mecp2 deficiency. [START_REF] Guy | Reversal of neurological defects in a mouse model of Rett syndrome[END_REF] After transcardial perfusion with saline, the mice were sacrificed and the brain tissues were removed and bisected on the sagittal plane. The brain hemispheres were immediately frozen in dry ice and stored at-80 °C until assay. In circulation, NeuroPs are found as "free" and "esterified" form but in tissues most of the NeuroPs are esterified to phospholipids. Therefore, the samples required a hydrolysis process. The tissue was homogenized (10% w/v) in phosphate-buffered saline, pH 7.4 with BHT. To an aliquot (1 ml) of the brain homogenate, aqueous KOH (1 mM, 500 μl) was added. After incubation at 45 °C for 45 min, the pH was adjusted to 3 by adding HCl (1 mM, 500 μl). Each sample was spiked with the internal standard, tetradeuterated prostaglandin F 2α (PGF 2α -d 4 , 500 pg in 50 μl ethanol) and then ethyl acetate (10 ml) was added to extract the lipid portion by vortex-mixing and centrifugation at 1000 × g for 5 min at room temperature.

In vitro oxidation of DHA

Following a model of free radical-induced oxidation process reported [START_REF] Nourooz-Zadeh | F4-isoprostanes: a novel class of prostanoids formed during peroxidation of docosahexaenoic acid (DHA)[END_REF], DHA was oxidized by 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH). DHA (20 mg) was vortexed with 20 ml of phosphate-buffered saline (10 mM potassium phosphate, 10 mM sodium chloride, pH 7.4, PBS). The dispersion was then ultrasonicated for 3 min and incubated in presence of 10 mM AAPH for 24 h at 37 °C. [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]. The synthesis of the two series of 4-and 10-F 4t -NeuroPs [START_REF] Oger | Stereocontrolled access to isoprostanes via a bicyclo[3.3.0]octene framework[END_REF][START_REF] Oger | The handy use of Brown's catalyst for a skipped diyne deuteration: application to the synthesis of a d4-labeled-F4t-neuroprostane[END_REF][START_REF] Guy | Oxygenated metabolites of n-3 polyunsaturated fatty acid as potential oxidative stress biomarkers: total synthesis of 8-F3t-IsoP, 10-F4t-NeuroP, and [D4]-10-F4t-NeuroP[END_REF] is summarized in Scheme 1. Starting from the commercially available 1,3cyclooctadiene 1, the two key bicyclic intermediates 2 and 3 were obtained in 10 and 5 steps respectively giving 8.8% (for intermediate 2) and 18% (for intermediate 3) yields. The introduction of α and ω chains was performed by using regioselective protections/deprotections, oxidations, Wittig elongation and cross metathesis coupling reactions as the main steps. The final step is the saponification of the methyl esters in the presence of LiOH to obtain free acids. The 4(RS)-4-F 4t -NeuroP was obtained starting from intermediate 3 that followed 16 steps of the main synthesis giving 9% yield while 10(S)-10-F 4t -NeuroP and its 10epimer were obtained in 13 steps from intermediate 2 giving 6.7% yield.

Extraction of F 4 -NeuroPs

F 4t -NeuroPs were extracted from the plasma using the following procedures. The internal standard (PGF 2α -d 4 , 500 pg contained in 50 μl ethanol) was added to each plasma sample (1 ml) before extraction process. Thereafter, a volume of 2 ml acidified water (pH 3) were added to each sample and then applied on a C 18 solid phase extraction (SPE) cartridge followed by a NH 2 SPE cartridge for a series of clean-up procedures using known methods [START_REF] Guy | Reversal of neurological defects in a mouse model of Rett syndrome[END_REF][START_REF] Guy | Oxygenated metabolites of n-3 polyunsaturated fatty acid as potential oxidative stress biomarkers: total synthesis of 8-F3t-IsoP, 10-F4t-NeuroP, and [D4]-10-F4t-NeuroP[END_REF]. Briefly, the C 18 cartridge (500 mg Sorbent per Cartridge, 55-105 µm Particle Size, 6cc, Waters, USA) was preconditioned with methanol (5 ml) and water (5 ml), and sequentially washed after loading the sample with 10 ml water (pH 3), and 10 ml water: acetonitrile (85:15, v/v). Hexane: ethyl acetate: propan-2-ol (30:65:5 v/v/v, 5 ml) mix was used for the final eluate. Afterwards, the eluate was transferred to an NH 2 cartridge (500 mg Sorbent per Cartridge, 55-105 µm Particle Size, 6cc, Waters, USA), which was preconditioned with hexane (5 ml). After loading the eluate, it was sequentially washed with 10 ml of hexane: ethyl acetate (30:70, v/v), 10 ml acetonitrile: water (9:1, v/v) and 10 ml acetonitrile. The final elution was a mix of ethyl acetate: methanol: acetic acid (10:85:5, v/v/v, 5 ml). The collected eluate was first evaporated under nitrogen at 40 °C prior derivatization process.

The same procedures were used for the extraction of the oxidized DHA liposome. As for the brain tissue samples, the lipid portion obtained by extraction with ethyl acetate (as reported in Sample preparation paragraph) was directly transferred to an NH 2 cartridge and the subsequent procedures were the same as plasma samples.

Derivatization of F 4 -NeuroPs

In the derivatization process, the carboxylic group of the F 4 -NeuroPs or PGF 2α -d 4 was converted into pentafluorobenzyl ester while the hydroxyl group was converted to trimethylsilyl ethers [START_REF] Signorini | Ion trap tandem mass spectrometric determination of F2-isoprostanes[END_REF]. To achieve this, the following procedures were made where 40 μl of pentafluorobenzyl bromide (10% in acetonitrile) and 20 μl of diisopropylethylamine (10% in acetonitrile) were added to each dried extract. It was incubated for 45 min at 40 °C and subsequently, the solvent was evaporated under a stream of nitrogen. Then, 50 μl of N,O-bis (trimethylsilyl)trifluoroacetamide with 5 μl of diisopropylethylamine (10% in acetonitrile) were added and incubated at 45 °C for 1 h. The samples were dried and re-suspended in 50 μl of undecane containing bis(trimethylsilyl)trifluoroacetamide (10%) for gas chromatography/ negative-ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) analysis. The same procedure was carried out for the derivatization of chemically synthesized 4-F 4t -NeuroP and 10-F 4t -NeuroP, the oxidized DHA liposome, and plasma or brain tissue samples.

Gas chromatography-mass spectrometry determination of F 4 -NeuroPs

The derivatized F 4 -NeuroPs and PGF 2α -d 4 were determined using GC/NICI-MS/MS with ion trap. The prepared samples (2 μl) were injected into the gas chromatograph (Trace GC and PolarisQ, Thermo/ Finnigan, USA) set at splitless mode (2 min). The injector temperature was 250 °C and the oven temperature was held at 175 °C for 3 min and then increased to 270 °C (30 °C/min). The transfer line was heated to 280 °C and the ion source temperature was maintained at 200 °C. Helium was used as the carrier gas, (1 ml/min) and the chromatography was performed using a SPB 1701 GC capillary column (Supelco, 30 m × 0.25 mm i.d., 0.25 µm film thickness). The reagent gas for the chemical ionization was methane set to 2.0 ml/min flow rate. The precursor ion isolation width was 1.5 Da, the multiplier was set to 1500 V, and the automatic gain control was performed. The collision energy was 1.3 eV and the scan event time was 0.87 s with maximum ion as 25 ms, and the number of microscans per ion was 9.

The mass ions determined were the product ions at m/z 323 and m/z 303 derived from the [M-181] -precursor ions of NeuroPs (m/z 593) and PGF 2α -d 4 (m/z 573) respectively, that corresponds to the loss of CH 2 C 6 F 5 from the derivatization process. Quantification of the NeuroPs were determined by relating the analyte/internal standard peak area ratio (F 4t -NeuroP/PGF 2α -d 4 ) to the calibration curve constructed.

Calibration curve, accuracy and precision of the method

The calibration curves were constructed by measuring seven concentration points (in triplicates) ranging from 1 to 500 pg/ml of the synthesized 4-F 4t -NeuroP and 10-F 4t -NeuroP with 500 pg PGF 2α -d 4 as the internal standard. Following the procedure described above, all samples were purified by SPE, derivatized, and analyzed by GC/NICI-MS/MS. The coefficients of correlation were above 0.99. Based on the standard deviation of the area ratio (F 4t -NeuroP/PGF 2α -d 4 ) for the lowest concentration point and the curve slope, the limits of detection (LOD) and quantification (LOQ) were 0.36-1.21 and 0.33 -1.08 pg/ ml, for 4-F 4t -NeuroP and 10-F 4t -NeuroP, respectively. The intra-assay and inter-assay coefficients of variability, as well as the accuracy of the method, were determined by analyzing three samples containing 500 pg/ml of synthesized 10-F 4t -NeuroP and 500 pg/ml of PGF 2α -d 4 .

Data analysis

Differences between groups were evaluated by either one-way analysis of variance (ANOVA), Levene's test for equality of error variances, and Scheffé test for all pairwise comparisons. The association between variables were tested using the Spearman rank correlation at 95% confidence intervals (95% C.I.). The efficiency of 4-F 4t -NeuroP and 10-F 4t -NeuroP in discriminating ASD, DS, MS or RTT from either healthy control subjects, or in discriminating each neurological disease from each other, was evaluated using receiver operating characteristic (ROC) curve analysis. A two-tailed P < 0.05 was considered to indicate statistical significance. The MedCalc ver. 12.0 statistical software package (MedCalc. Software, Mariakerke, Belgium) was used for data analysis.

Results

Method optimization of F 4t -NeuroPs

In this study, the pentafluorobenzyl bromide-esters and trimethylsilyl-ether derivatives of the chemically synthesized extremely good detection level by the method set up. However, the S/N ratio of the oxidized DHA ion chromatogram was lower [START_REF] De Las Heras-Gomez | Potential applications of lipid peroxidation products-F4-neuroprostanes, F3-neuroprostanesn-6 DPA and F2-dihomo-isoprostanes and F2-isoprostanes-in the evaluation of the allograft function in renal transplantation[END_REF] as it contains a mixture of non-purified isomers. The ions produced through the loss of trimethylsilyl hydroxide (HOTMS) were predominant in the MS/MS spectrum of F 4 -NeuroPs. In particular, the ions at m/z 503, 413 and 323 were attributed by the elimination of one, two, or three trimethylsilyl hydroxide (HOTMS) groups, respectively, from [M-181] - ions and an example of 10-F 4t -NeuroP is depicted in Fig. 2. As the most abundant product ion was m/z 323, a narrower scan range was set at m/ z 322.5-323.5 for F 4 -NeuroPs and m/z 302.5-303.5 for PGF 2α -d 4 which was adequate to ensure high selectivity and sensitivity of the MS/MS method. The precision of the applied method was determined by the measurement of intra-assay coefficient of variability from three replicated measurements of 10-F 4t -NeuroP in healthy control plasma and was found to be 4.80 ± 0.35%. The inter-assay coefficient of variability was measured by comparing three samples containing specific amounts of chemically synthesized 10-F 4t -NeuroP and was found to be 5.51 ± 0.36%. The quantification accuracy that is defined as closeness of the measured value to the true quantity value was 96.89 ± 2.02% for 10-F 4t -NeuroP. The reference values for F 4 -NeuroP levels in plasma samples ranged from 0 to 0.9 pg/ml (n = 61, healthy control subjects) and in the investigated neurological diseases, the plasma values ranged from 1.1 to 29.8 pg/ml (n = 61).

Accuracy and precision of F 4t -NeuroPs measurement in plasma

Levels in healthy controls and neurological disease patients

As compared to the corresponding healthy controls, plasma 10-F 4t -NeuroP levels were significantly higher in the patients. On the other hand, plasma 4-F 4t -NeuroP levels were found to be significantly higher only in MS and RTT patients (Fig. 4, panels A and B). Furthermore, the significant positive correlation (r = 0.676; 95% C.I: 0.566-0.762; n =122; P < 0.0001) between the plasma levels of 10-F 4t -NeuroP and 4-F 4t -NeuroP demonstrate that the two isomers share the same pathway of DHA oxidation.

Even though 4-F 4t -NeuroP and 10-F 4t -NeuroP were detectable in all the plasma samples, 10-F 4t -NeuroP level was more abundant in the ASD, DS, and RTT patients compared to the control group. Particularly, 10-F 4t -NeuroP levels were greater compared to 4-F 4t -NeuroP levels in all ASD and RTT patients, and in 13 out of 16 DS patients. However, in MS patients, 12 out of 16 plasma samples showed a major amount of 4-F 4t -NeuroP. Moreover, 4-F 4t -NeuroP and 10-F 4t -NeuroP formation was shown to be influenced by MS disease progression. Specifically, 10-F 4t -NeuroP levels were positively correlated with the onset age of the disease (r = 0.533; 95% C.I.: 0.0509 to -0.814; n =16; P = 0.034), while the ratio of 10-F 4t -NeuroP to 4-F 4t -NeuroP was negatively related to the disease duration (r = -0.517; 95% C.I.: -0.806 to -0.0286; n =16; P = 0.04).

Receiver operating characteristic (ROC) curves indicate that the ratio of F 4t -NeuroP isomer, i.e. 10-F 4t -NeuroP/4-F 4t -NeuroP amounts, and the amount of individual 10-F 4t -NeuroP and 4-F 4t -NeuroP could be the discriminant between the brain disease group (ASD, DS, MS and RTT) from the healthy control subjects (Tables 1 and2). Moreover, 10-F 4t -NeuroP/4-F 4t -NeuroP ratio, as well as 10-F 4t -NeuroP and 4-F 4t -NeuroP amounts were discriminants for different clinical conditions. Nevertheless, the comparisons between ASD and DS were not significant. As compared to DS, 10-F 4t -NeuroP/4-F 4t -NeuroP ratio and 4-F 4t -NeuroP amounts in RTT were found to be discriminants, but not for 10-F 4t -NeuroP levels (Table 3).

Significant positive relationships were observed between relative disease clinical severity and 4-F 4t -NeuroP or 10-F 4t -NeuroP levels (r = 0.469; 95% C.I.: 0.312-0.601; n =114; P < 0.0001; r = 0.757; 95% C.I.: 0.666-0.826; n =114; P < 0.0001, respectively). Furthermore, the relative clinical severity was significantly correlated with 10-F 4t -NeuroP/4-F 4t -NeuroP ratio (r = 0.820; 95% C.I.: 0.726-0.883; n =72; P < 0.0001) and 4-F 4t -NeuroP/10-F 4t -NeuroP ratio (r = -0.328; 95% C.I. -0.513 to -0.114; n = 78; P = 0.0033).

Levels in RTT mice brain tissue

The amount of 4-F 4t -NeuroP and 10-F 4t -NeuroP in brain tissue of RTT mice was significantly higher than the wild-type. Unlike plasma levels in RTT patients, the amount of 4-F 4t -NeuroP and 10-F 4t -NeuroP were similar in the brain tissues of RTT mice. Moreover, in our observation, 4-F 4t -NeuroP and 10-F 4t -NeuroP levels were significantly correlated to the phenotypic severity of the RTT mice (Table 4).

Discussion

In this study, it was apparent that NeuroPs, specifically 4-F 4t -NeuroP and 10-F 4t -NeuroP i) were biologically synthesized in vivo in different human neurological conditions, ii) were related to clinical severity, iii) were useful to identify shared pathogenetic pathways in distinct brain diseases, and iv) their relative plasma abundance appeared to be a distinctive feature for different neurological conditions.

Insofar, 15-F 2t -IsoP derived from AA, is the most studied in medical research related to oxidative stress and disease progression. This is probably due to the availability of the standard and well characterized protocols using mass spectrometry. However, many of the findings were not relevant to neurological diseases as described in recent reports [START_REF] Galano | Isoprostanes, neuroprostanes and phytoprostanes: an overview of 25years of research in chemistry and biology[END_REF][START_REF] Van 't Erve | Classifying oxidative stress by F(2)-isoprostane levels across human diseases: a meta-analysis[END_REF] Therefore, investigations on F 4 -NeuroPs derived from DHA were worthy to be further explored, especially when taking into account their potential relevance in neurological disorders. As compared to F 2 -IsoPs, F 4 -NeuroPs could be related to the intrinsic mechanisms of brain diseases associated to more specific pathway [START_REF] Signorini | F 4 -neuroprostanes mediate neurological severity in Rett syndrome[END_REF] and it is well established that different oxidative stress biomarkers do not necessarily measure identical aspects of the disease progression [START_REF] Seet | Biomarkers of oxidative damage in cigarette smokers: which biomarkers might reflect acute versus chronic oxidative stress? Free Radic[END_REF].

The interest in the study of F 4 -NeuroP was first initiated from the fact that they are generated by free radical/ROS oxidation of DHA, which is highly concentrated in the neuronal membrane, that allows neurons to work at their best by avoiding afflictions of the nervous system [START_REF] Dyall | Long-chain omega-3 fatty acids and the brain: a review of the independent and shared effects of EPA, DPA and DHA, Front[END_REF][START_REF] Mcnamara | Deciphering the role of docosahexaenoic acid in brain maturation and pathology with magnetic resonance imaging[END_REF][START_REF] Bradbury | Docosahexaenoic acid (DHA): an ancient nutrient for the modern human brain[END_REF][START_REF] Bazan | Endogenous signaling by omega-3 docosahexaenoic acid-derived mediators sustains homeostatic synaptic and circuitry integrity[END_REF]. The rate of oxidation is greater than AA due to extra methylene double bonds in the structure. Nevertheless, F 4 -NeuroPs have been quite recently indicated as a predictive variable of atherosclerosis in a mouse model, and displayed protective mechanism in cardiac ischemia-reperfusion injury [START_REF] Gladine | Lipid profiling following intake of the omega 3 fatty acid DHA identifies the peroxidized metabolites F4-neuroprostanes as the best predictors of atherosclerosis prevention[END_REF][START_REF] Roy | Non-enzymatic oxidized metabolite of DHA, 4(RS)-4-F (4t)-neuroprostane protects the heart against reperfusion injury, Free Radic[END_REF]. The selective role for the F 4 -NeuroPs in the brain tissue oxidative status was also evidenced by the major increase of F 4 -NeuroP levels, rather than F 2 -IsoPs, in brain injury [START_REF] Montine | Neuronal oxidative damage from activated innate immunity is EP2 receptor-dependent[END_REF][START_REF] Milne | Elevated oxidation of docosahexaenoic acid, 22:6 (n-3), in brain regions of rats undergoing ethanol withdrawal[END_REF]. Despite this, the number of scientific papers for F 4 -NeuroPs is relatively limited [START_REF] Galano | Isoprostanes, neuroprostanes and phytoprostanes: an overview of 25years of research in chemistry and biology[END_REF], and perhaps due to the lack understanding and international consensus in identifying the characteristic isomer among all the metabolites originating from DHA oxidation. Additionally, no commercial standards are available and no unanimous consensus has been reached for methodological protocol of F 4 -NeuroP analysis.

F 4 -NeuroPs are considered to be specific and accurate biomarkers when measurements are conducted in either brain tissue or cerebrospinal fluid [START_REF] Luczaj | Tick-borne encephalitis -lipid peroxidation and its consequences[END_REF]. For this reason, there is a limitation to further understand the NeuroP relevance in human neuro-pathology, and

Table 1

Discriminating power for 10-F 4t -NeuroP/4-F 4t -NeuroP ratio, and 10-F 4t -NeuroP or 4-F 4t -NeuroP content: summery of receiver operating characteristic (ROC) curve analysis.

Variable

Cut The neurological disease group (i.e., all MS, ASD, RTT and DS patients) (n = 61) was compared to the age-and gender-matched healthy control population (n=61). Plasma F 4t -NeuroPs were measured as "free" forms. +P.V.: positive predictive value; -P.V.: negative predictive value. subsequently, investigations could only reside to the availability of biological samples and in appropriate animal models. Our previous experiments on several RTT murine models corroborate the in vivo plasma F 4 -NeuroP results [START_REF] De Felice | Oxidative brain damage in Mecp2-mutant murine models of Rett syndrome[END_REF] and furthermore, in this report, we identified 4-F 4t -NeuroP and 10-F 4t -NeuroP in brain tissue whose levels were related to the phenotypic severity in RTT mice. Despite 4-F 4t -NeuroP and 10-F 4t -NeuroP brain levels in our observation was limited to one of the four pathological conditions examined in the patients, the data strongly emphasized one of the major key points which is the validity of plasma F 4 -NeuroPs levels as a mirror for the ongoing DHA oxidation process in the brain/nervous system, and its close relationship with symptoms of neurological abnormalities.

Our group has repeatedly explored the relevance of plasma levels of NeuroPs in RTT [START_REF] Signorini | F 4 -neuroprostanes mediate neurological severity in Rett syndrome[END_REF] and other authors have confirmed the importance of monitoring the plasma levels of NeuroPs in Parkinson's disease [START_REF] Seet | Oxidative damage in Parkinson disease: measurement using accurate biomarkers[END_REF] smokers [START_REF] Seet | Biomarkers of oxidative damage in cigarette smokers: which biomarkers might reflect acute versus chronic oxidative stress? Free Radic[END_REF], type diabetes [START_REF] Seet | Oral zinc supplementation does not improve oxidative stress or vascular function in patients with type 2 diabetes with normal zinc levels[END_REF], ischemic-stroke [START_REF] Seet | Oxidative damage in ischemic stroke revealed using multiple biomarkers[END_REF], tick-borne encephalitis [START_REF] Łuczaj | Tick-borne encephalitis-lipid peroxidation and its consequences[END_REF] and neuroborreliosis [START_REF] Moniuszko-Malinowska | Lipid peroxidation in the pathogenesis of neuroborreliosis[END_REF]. Also, NeuroPs have been determined in newborn serum plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry [START_REF] Cháfer-Pericás | Ultra high performance liquid chromatography coupled to tandem mass spectrometry determination of lipid peroxidation biomarkers in newborn serum samples[END_REF], thus reinforcing the relevance of systemic blood products as appropriate samples for the NeuroPs detection. However, the transport of F 4 -NeuroP levels in circulation to the brain, i.e. the ratio of plasma F 4 -NeuroPs to brain F 4 -NeuroPs, is still not known.

Although DHA is abundant in the brain, particularly in the gray matter, [START_REF] Glomset | Role of docosahexaenoic acid in neuronal plasma membranes[END_REF], the ability for the cerebral tissue to synthesize DHA from its omega-3 PUFA precursors are limited. Instead, most of the brain DHA is from the plasma transported by fatty acid-binding proteins and the crossing of blood-brain barrier [START_REF] Rapoport | Brain metabolism of nutritionally essential polyunsaturated fatty acids depends on both the diet and the liver, Prostaglandin Leukotrienes Essent[END_REF][START_REF] Innis | Dietary (n-3) fatty acids and brain development[END_REF][START_REF] Pan | Fatty acid-binding protein 5 at the blood-brain barrier regulates endogenous brain docosahexaenoic acid levels and cognitive function[END_REF], and it is expected that the oxidized DHA like NeuroPs will be transported in similar manner. In MS, the involvement of oxidative stress has been reported to affect both central and peripheral components [START_REF] Ohl | Oxidative stress in multiple sclerosis: central and peripheral mode of action[END_REF], where the relationship between oxidative DNA damage (8-hydroxy-2'-deoxyguanosine values) and index of blood-brain barrier permeability, clinical severity of disease, and demyelinated brain lesion volume were identified [START_REF] Ljubisavljevic | The validation study of neurofilament heavy chain and 8-hydroxy-2'-deoxyguanosine as plasma biomarkers of clinical/paraclinical activity in first and relapsing-remitting demyelination acute attacks[END_REF][START_REF] Fischer | NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury[END_REF]. Moreover, urinary 15-F 2t -IsoP levels were found to be elevated [START_REF] Guan | Patients with multiple sclerosis show increased oxidative stress markers and somatic telomere length shortening[END_REF], and the antioxidant capacity to be reduced [START_REF] Karlik | Markers of oxidative stress in plasma and saliva in patients with multiple sclerosis[END_REF].

Although, the participation of lipid peroxidation in MS cerebrospinal fluid is debatable [START_REF] Bartova | Changes in levels of oxidative stress markers and some neuronal enzyme activities in cerebrospinal fluid of multiple sclerosis patients[END_REF], evidences for oxidative injury, including fatty acid peroxidation, were reported in the cerebellar gray matter [START_REF] Kemp | Oxidative injury in multiple sclerosis cerebellar grey matter[END_REF]. Thus, elevated F 4t -NeuroP plasma levels could denote a gray matter damage in the MS pathogenesis in the early phase of the disease that could become more prominent at severe stage [START_REF] Wylezinska | Thalamic neurodegeneration in relapsing-remitting multiple sclerosis[END_REF][START_REF] Haider | Multiple sclerosis deep grey matter: the relation between demyelination, neurodegeneration, inflammation and iron[END_REF].

Correlations between the F 4 -NeuroP isomers and the relative severity scales were observed in this study. The data suggest that the relative abundance of the different F 4 -NeuroP isomers along the cascade formation is influenced by the pathological conditions and/or contribute to disease severity given that oxidative stress impairment is a well-known fact involved in the investigated neurological diseases [START_REF] Manna | Increased non-protein bound iron in Down syndrome: contribution to lipid peroxidation and cognitive decline[END_REF][START_REF] De Felice | The role of oxidative stress in Rett syndrome: an overview[END_REF][START_REF] Haider | Oxidative damage in multiple sclerosis lesions[END_REF]. These data are strongly against the hypothesis in the formation of F 4 -NeuroPs in vivo, which is random whenever DHA oxidation takes place. The major abundance of 4-F 4t -NeuroP in MS samples is in line with other previous observations; compared to the other series of the F 4 -NeuroP isomers, 4-and 20-series are different and could generate in greater amounts because the precursor cannot further cyclize in the chemical pathway [START_REF] Yin | Regiochemistry of neuroprostanes generated from the peroxidation of docosahexaenoic acid in vitro and in vivo[END_REF]. This topic is of paramount relevance to the prospect of F 4 -NeuroP quantification in DHA oxidant assessment in human clinical pathological conditions. Although our study did not investigate all the potential F 4 -NeuroP isomers, which could be derived from DHA oxidation, 10-F 4t -NeuroP and 4-F 4t -NeuroP synthesis appears to take place in the investigated neurological diseases, and is associated to the relative clinical severity, and able to discriminate pathological condition, suggesting their involvement in disease mechanisms. This view is supported by the receiver operating curve, which is widely considered a test of accuracy for discriminating between different population. Hence, it appears that most of the patients' groups can be distinguished with confidence on the basis of the 10-F 4t -NeuroP/4-F 4t -NeuroP ratio, as well as by the absolute concentration of the individual isomer. Further, the biological relevance for the investigated F 4 -NeuroP isomers is reinforced by their ability to discriminate the neurological disease. Intriguingly, ASD and DS (16-19% or higher) are easily distinguishable from the clinical phenotype, where a consistent proportion of the children with DS also shows ASD [START_REF] Warner | Autism characteristics and behavioural disturbances in ~500 children with Down's syndrome in England and Wales[END_REF]. Likewise, in RTT autistic features are transient and lasting weeks to several months [START_REF] Hagberg | A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: rett's syndrome: report of 35 cases[END_REF][START_REF] Percy | Rett syndrome: exploring the autism link[END_REF]. There is no doubt that the examined neurological conditions (i.e., RTT, ASD, MS, and DS) show a wide diversity in their neuropathological features. If we wished to identify a shared denominator among them, likely the loss of acquired neurological functions (i.e. "neuroregression") would be the communal event occurring in three of the considered conditions (RTT, ASD, MS) and in about a fifth of the DS patients with autistic traits.

Our data strongly suggest that in vivo DHA oxidation follows preferential chemical rearrangements according to different neurological diseases, and that 10-F 4t -NeuroP and 4-F 4t -NeuroP are able to identify pathogenetic pathways in distinct brain diseases, thus representing potentially new approach in biological disease biomarkers.

2. 4 . 4 (

 44 RS)-F 4t -NeuroP, and 10(R)-10-F 4t -NeuroP and 10(S)-10-F 4t -NeuroP synthesis

Scheme 1 .

 1 Scheme 1. Chemical strategy for 4-F 4t -NeuroP and 10-F 4t -NeuroP.

  F 4 -NeuroPs were characterized using the GC/NICI-MS/MS by obtaining the mass [M-181] -(for the precursor ion) of F 4 -NeuroP molecules (m/z 593) and internal standard PGF 2α -d 4 (m/z 573). The detection of the product ions was carried out by microscans in the m/z range 322.5-323.5 (F 4 -NeuroP isomers) and 302.5-303.5 (PGF 2α -d 4 ). The retention time for the peaks produced were 19.54-19.75 min for 4-F 4t -NeuroP, and 21.27 and 21.44 min for the epimers of the 10-F 4t -NeuroP. Afterwards, the chromatographic spectrum of the oxidized DHA liposome was scanned for 4-F 4t -NeuroP and 10-F 4t -NeuroP and the peaks produced matched with the peaks of the chemically synthesized F 4t -NeuroPs (Fig. 1, panels B to E). The exact match between the chromatographic spectrum of the oxidized DHA for both 4-F 4t -NeuroP and 10-F 4t -NeuroP were further confirmed by the retention time of the internal standard, PGF 2α -d 4 , (Fig. 1, panel A). The relatively high signal-to-noise (S/N) ratio (103) of the 10-F 4t -NeuroP ion chromatogram (Fig. 1, panel D), indicate an

Fig. 3

 3 Fig. 3 displays a typical chromatogram of a plasma sample analysis where the peaks shown are related to 4-F 4t -NeuroP (19.59 min), and 10-F 4t -NeuroP (21.26 and 21.44 min) epimers with S/N ratio approximately 15 compared to the chromatographic peaks of chemically synthesized F 4t -NeuroP with higher S/N (approximately 100) (Fig. 1 panels C to E).The precision of the applied method was determined by the measurement of intra-assay coefficient of variability from three replicated measurements of 10-F 4t -NeuroP in healthy control plasma and was found to be 4.80 ± 0.35%. The inter-assay coefficient of variability was measured by comparing three samples containing specific amounts of chemically synthesized 10-F 4t -NeuroP and was found to be 5.51 ± 0.36%. The quantification accuracy that is defined as closeness of the measured value to the true quantity value was 96.89 ± 2.02% for 10-F 4t -NeuroP. The reference values for F 4 -NeuroP levels in plasma samples ranged from 0 to 0.9 pg/ml (n = 61, healthy control subjects) and in the investigated neurological diseases, the plasma values ranged from 1.1 to 29.8 pg/ml (n = 61).

Fig. 1 .

 1 Fig. 1. Chromatogram obtained using GC/NICI-MS/MS conditions by selecting the species [M-181] -as precursor ion. (A) internal standard PGF 2α -d 4 (303 m/z, precursor ion m/z 573); (B) in vitro oxidized DHA liposome (323 m/z, precursor ion m/z 593); (C) 4(RS)-4-F 4t -NeuroP (323 m/z, precursor ion m/z 593); (D) and (E); 10(S)-F 4t -NeuroP and 10(R)-F 4t -NeuroP, respectively; (323 m/z, precursor ion m/z 593). Retention times are indicated. The collision energy was 1.3 eV.

Fig. 2 .

 2 Fig. 2. MS/MS mass spectrum showing all the product ions of the derivatized 10-F 4t -NeuroP. As precursor ion, the [M-PFB] -ion (m/z 593) was selected. The m/z range was 300.0-520.0. The collision energy was 1.3 eV. Ions at m/z 503, 413 and 323 are attributed by the elimination of one, two, or three trimethylsilyl hydroxide, respectively. As the most abundant product ion is at m/z 323, a narrow range scan over m/z 322.5-323.5 was sufficient to ensure high selectivity and sensitivity of the MS/MS method.

Fig. 3 .

 3 Fig. 3. Chromatogram of GC/NICI-MS/MS for F 4t -NeuroPs obtained from plasma of a typical MS patient.

Fig. 4 .

 4 Fig. 4. 4-F 4t -NeuroP and 10-F 4t -NeuroP plasma levels in neurological disease patients and healthy controls. MS: multiple sclerosis; DS: Down syndrome; ASD: autism spectrum disorders; RTT: Rett syndrome; ASD-Ctrl, DS-Ctrl, MS-Ctrl, and RTT-Ctrl: age-and gender-matched control population of the respective disease. Panels A and A': *statistically different from ASD-Ctrl, DS-Ctrl, MS-Ctrl, RTT,-Ctrl ASD, DS and RTT; **statistically different from ASD-Ctrl, DS-Ctrl, MS-Ctrl, RTT-Ctrl, ASD, DS and MS. Panels B and B': * Statistically different from ASD-Ctrl, DS-Ctrl, MS-Ctrl, RTT-Ctrl, ASD, and DS; ** Statistically different from ASD-Ctrl, DS-Ctrl, MS-Ctrl, RTT-Ctrl; § Statistically different from ASD-Ctrl, DS-Ctrl, MS-Ctrl, RTT-Ctrl, and MS. Scheffé test for all pairwise comparisons: P < 0.05; Levene's test for equality of error variances: P < 0.001. One Way ANOVA test Significance level:P < 0.001.

Table 2

 2 Discriminating power for 10-F 4t -NeuroP/4-F 4t -NeuroP ratio, and 10-F 4t -NeuroP or 4-F 4t -NeuroP content for each neurological disease group -summary of receiver operating characteristic (ROC) curve analysis.Each neurological disease group was compared to the corresponding age-and gender-matched healthy control population. Plasma F 4t -NeuroPs were measured as "free" forms. N. S.: not significant, referred to the area under the ROC curve; +P.V.: positive predictive value; -P.V.: negative predictive value.

	Comparisons	Variable	Cut-off value	Sensitivity	Specificity	+ P.V.	-P.V.	P value
				(%)	(%)	(%)	(%)
	ASD vs Corresponding Healthy Control Group	10-F 4t -NeuroP/4-F 4t -NeuroP	> 1	100	100	100	100	< 0.0001
		10-F 4t -NeuroP	> 2.1 (pg/ml)	100	100	100	100	< 0.0001
		4-F 4t -NeuroP	> 0.5 (pg/ml)	44.44	91.67	66.7	81.5	N.S.
	DS vs Corresponding Healthy Control Group	10-F 4t -NeuroP/4-F 4t -NeuroP	> 1	100	100	100	100	< 0.0001
		10-F 4t -NeuroP	> 2.1 (pg/ml)	87.50	100	100	92,3	< 0.0001
		4-F 4t -NeuroP	> 0.9 (pg/ml)	50	100	81.8	75.9	0.0440
	MS vs Corresponding Healthy Control Group	10-F 4t -NeuroP/4-F 4t -NeuroP	> 0.4	100	60	80	100	0.0065
		10-F 4t -NeuroP	> 2.1 (pg/ml)	100	100	100	100	< 0.0001
		4-F 4t -NeuroP	> 0.9 (pg/ml)	100	100	100	100	< 0.0001
	RTT vs Corresponding Healthy Control Group	10-F 4t -NeuroP/4-F 4t -NeuroP	> 1	90	100	100	83.3	< 0.0001
		10-F 4t -NeuroP	> 2.1 (pg/ml)	100	100	100	100	< 0.0001
		4-F 4t -NeuroP	> 0.9 (pg/ml)	100	100	100	100	< 0.0001

Table 3

 3 Receiver operating characteristic (ROC) curve analysis for 10-F 4t -NeuroP/4-F 4t -NeuroP ratio, or 10-F 4t -NeuroP and 4-F 4t -NeuroP content.

	Pairwise Comparisons	Variable	Cut-off value	Sensitivity	Specificity	+ P.V.	-P.V.	P value
				(%)	(%)	(%)	(%)
	ASD vs MS	10-F 4t -NeuroP/4-F 4t -NeuroP	> 1.19	100	100	100	100	< 0.0001
	ASD vs MS	10-F 4t -NeuroP	≤ 11.9 (pg/ml)	100	75	69.2	100	< 0.0001
	ASD vs MS	4-F 4t -NeuroP	≤ 2 (pg/ml)	100	100	100	100	< 0.0001
	ASD vs RTT	10-F 4t -NeuroP/4-F 4t -NeuroP	> 1.57	100	100	100	100	< 0.0001
	ASD vs RTT	10-F 4t -NeuroP	≤ 9.5 (pg/ml)	77.78	66.67	50	87.5	0.05
	ASD vs RTT	4-F 4t -NeuroP	≤ 2 (pg/ml)	100	100	100	100	< 0.0001
	ASD vs DS	10-F 4t -NeuroP/4-F 4t -NeuroP	> 4.2	75	88.89	50	72.7	N.S.
	ASD vs DS	10-F 4t -NeuroP	> 5.8 (pg/ml)	88.89	37.5	44.4	85.7	N.S
	ASD vs DS	4-F 4t -NeuroP	≤ 2 (pg/ml)	100	31.25	45	100	N.S
	MS vs RTT	10-F 4t -NeuroP/4-F 4t -NeuroP	≤ 1.19	100	90	88.9	100	< 0.0001
	MS vs RTT	10-F 4t -NeuroP	> 12.8 (pg/ml)	75	71.43	66.7	78.9	0.0033
	MS vs RTT	4-F 4t -NeuroP	> 16.7 (pg/ml)	75	100	100	84	< 0.0001
	MS vs DS	10-F 4t -NeuroP/4-F 4t -NeuroP	≤ 1.19	100	100	100	100	< 0.0001
	MS vs DS	10-F 4t -NeuroP	> 9.9 (pg/ml)	93.75	75	78.9	92.3	< 0.0001
	MS vs DS	4-F 4t -NeuroP	> 4.9 (pg/ml)	100	100	100	100	< 0.0001
	RTT vs DS	10-F 4t -NeuroP/4-F 4t -NeuroP	≤ 1.57	100	88.89	95.2	100	< 0.0001
	RTT vs DS	10-F 4t -NeuroP	> 7.1 (pg/ml)	80.95	56.25	70.8	69.2	N.S.
	RTT vs DS	4-F 4t -NeuroP	> 4.9 (pg/ml)	80.95	100	100	80	< 0.0001
	Plasma F

4t -NeuroPs were measured as "free" forms. N. S.: not significant, referred to the area under the ROC curve; +P.V.: positive predictive value; -P.V.: negative predictive value.

Table 4 4

 4 -F 4t -NeuroP and 10-F 4t -NeuroP brain levels in symptomatic Mecp2 stop/y mice and their correlation to phenotype severity.

	Brain F 4t -	Mouse group	Median 95% C.I. for	P value
	NeuroP isomer		(ng/g)	median value	(two-
					tailed)
	4-F 4t -NeuroP	Mecp2 stop/y	7.85	4.85 to 13.4	0.02
		(n = 4)
		wt (n = 4)	1.4	0.5 to 2.0
	10-F 4t -NeuroP	Mecp2 stop/y	10.45	9.05 to 17.75	0.02
		(n= 4)
		wt (n= 4)	1.9	0.7 to 3.05
		Correlation with	95% C.I. for rho	P value
		phenotype severity b			(two-
					tailed)
	4-F 4t -NeuroP	0.944 a	0.72 to 0.99	0.0004
	10-F 4t -NeuroP	0.888 a	0.49 to 0.98	0.0032
	Brain F

4t -NeuroPs were measured as "free" plus "esterified" forms. a Spearman's rho correlations coefficient. C.I.: confidence interval; wt: wild type.

b

The phenotype severity was expressed as an aggregate score (Section 2).

Acknowledgments

In memory of Professor Mario Comporti, international pioneer in the exploration of oxidative stress in disease.

We thank all the subjects and their families for enrolling in the study.

The authors would like to thank the "Associazione Sindrome di Down, onlus" (Campania, Italy), and the Siena autism family association "Associazione Autismo Siena-Piccolo Principe" (Siena, Italy).

Our sincere acknowledgments to Dr. J. Guy, Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK for kindly providing Mecp2 stop/y mice brains (results included in Table 4) and reading the manuscript.

We thank the Administrative Direction of the Azienda Ospedaliera Senese for prior purchasing of the gas spectrometry instrumentation.

We also thank professional singer Matteo Setti for his artistic contributions to the exploration of the link between artistic stimuli and vital signs in Rett girls and his many and continued charity concert activities specifically dedicated to Rett syndrome.

Funding

The present work was funded by the Tuscan Region (Bando Salute 2009; "Antioxidants-omega-3 polyunsaturated Fatty Acids, lipoic acid-supplementation in Rett syndrome: A novel approach to therapy," RT no. 142).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.