D J L Mat

I Souchon

C Michon

S Le Feunteun
email: steven.le-feunteun@inra.fr

Gastro-intestinal in vitro digestions of protein emulsions monitored by pH-stat: influence of structural properties and interplay between proteolysis and lipolysis

Keywords: Food, Structure, Emulsion gel, Chemical-physics, Hydrolysis, Orlistat Liquid Continuous Phase, GCP: Gelled Continuous Phase, LFE: Liquid Fine Emulsion

This study describes an experimental design, based on pH-stat, to rapidly screen and assess food formulation effects on the degrees of hydrolysis (DH) of both proteins and lipids throughout in vitro gastro-intestinal digestions. This approach was used to quantitatively compare and hierarchize key structure parameters of protein emulsions. Six matrices (15 wt% whey proteins, 0 or 10 wt% oil), each differing by at least one structure characteristic, were studied. The physical state of the bulk and the oil droplet size were the major structural levers to modulate the hydrolysis of proteins (final DH between 51.7 and 58.3%) and lipids (final DH between 46.9 and 72.7%), with non-trivial interplays between proteolysis and lipolysis. Additionally, pH-stat measurements in presence of a pancreatic lipase inhibitor proved to be an efficient way to widen the scope of the proposed experimental approach to foods that are intrinsically made of both proteins and lipids.

Introduction

Food structure is characterized by different spatial scales, from nano to macroscopic levels.

Soluble molecules are typically in the nanometer scale and are generally embedded in structures organized at larger scales: biopolymer networks (with subunit-structure of about 10-100 nm), droplets (0.1-5 µm, typically), etc. To better understand how these structures break down during digestion and assess their impact on the release of nutrients, both in vivo and in vitro experiments can be undertaken. While in vivo studies are mandatory to unambiguously demonstrate that food structure can modulate the fate of nutrients in the host, in vitro experiments can be used to predict outcomes of in vivo digestions [START_REF] Bohn | Correlation between in vitro and in vivo data on food digestion. What can we predict with static in vitro digestion models?[END_REF] and more thoroughly investigate the underlying mechanisms.

When considering lipids, which are dispersed as oil droplets in most foods, both in vivo and in vitro studies have shown that smaller droplet size leads to faster lipolysis kinetics [START_REF] Armand | Digestion and absorption of 2 fat emulsions with different droplet sizes in the human digestive tract[END_REF][START_REF] Mcclements | Structured emulsion-based delivery systems: controlling the digestion and release of lipophilic food components[END_REF], since they develop a larger interfacial area for lipase adsorption. The type of emulsifiers at the interface is another key factor to consider as it may control not only the ability for lipases to access their substrate [START_REF] Mun | Influence of emulsifier type on in vitro digestibility of lipid droplets by pancreatic lipase[END_REF], but also the emulsion stability. In the changing conditions of the digestive tract, the composition of the interface may change and droplets interact with each other. This can lead to droplet flocculation and/or coalescence during both the gastric [START_REF] Golding | Impact of gastric structuring on the lipolysis of emulsified lipids[END_REF][START_REF] Sarkar | Behaviour of an oil-in-water emulsion stabilized by β-lactoglobulin in an in vitro gastric model[END_REF] and intestinal [START_REF] Giang | Dynamic modeling highlights the major impact of droplet coalescence on the in vitro digestion kinetics of a whey protein stabilized submicron emulsion[END_REF][START_REF] Giang | Dynamic modeling of in vitro lipid digestion: Individual fatty acid release and bioaccessibility kinetics[END_REF][START_REF] Li | Physicochemical behaviour of WPI-stabilized emulsions in in vitro gastric and intestinal conditions[END_REF][START_REF] Sarkar | Pancreatin-induced coalescence of oil-in-water emulsions in an in vitro duodenal model[END_REF] phases of digestion, and lower the rate of lipid absorption as estimated from the postprandial blood triglyceride concentrations [START_REF] Golding | Impact of gastric structuring on the lipolysis of emulsified lipids[END_REF][START_REF] Keogh | Slowly and rapidly digested fat emulsions are equally satiating but their triglycerides are differentially absorbed and metabolized in humans[END_REF].

The kinetics of protein digestion also depends on food structure. The macrostructure is of course a key parameter, with a slower rate of proteolysis generally observed for solid foods.

For instance, dairy proteins have been reported to be much more rapidly metabolized in mini-pigs when eaten in the liquid state compared to rennet gels of identical composition [START_REF] Barbé | The heat treatment and the gelation are strong determinants of the kinetics of milk proteins digestion and of the peripheral availability of amino acids[END_REF]. Nanostructure and microstructure effects are also important to consider. For example, when used as emulsifiers, dairy whey proteins show an enhanced susceptibility to hydrolysis by pepsin because of their modified conformation upon adsorption at the oil interface [START_REF] Macierzanka | Emulsification alters simulated gastrointestinal proteolysis of beta-casein and betalactoglobulin[END_REF]. Similarly, thermal denaturation and/or aggregation of β-lactoglobulin, the main protein of dairy whey, can lead to an improved hydrolysis by both pepsin [START_REF] Guo | Susceptibility of β-Lactoglobulin and sodium caseinate to proteolysis by pepsin and trypsin[END_REF][START_REF] Singh | Influence of heat and shear induced protein aggregation on the in vitro digestion rate of whey proteins[END_REF] and intestinal proteases [START_REF] Stănciuc | Denaturation impact in susceptibility of beta-lactoglobulin to enzymatic hydrolysis: a kinetic study[END_REF]. Differently structured whey protein gels can therefore lead to highly contrasting digestion profiles [START_REF] Macierzanka | The effect of gel structure on the kinetics of simulated gastrointestinal digestion of bovine β-lactoglobulin[END_REF].

Few studies have also focused on food emulsions rich in proteins and on the interplay between lipolysis and proteolysis. Gelatin gels have been shown to slow down the rate of pancreatic lipolysis of embedded oil droplets because of a reduced lipase diffusion into the protein network [START_REF] Sarkar | Impact of protein gel porosity on the digestion of lipid emulsions[END_REF]. Others have investigated the influence of the structure of whey protein emulsion gels on lipid digestion, with interesting findings on the relations between their mechanical properties and their rate of disintegration upon simulated gastric contractions [START_REF] Guo | Modulating fat digestion through food structure design[END_REF]. Beyond confirming a slower lipolysis in presence of a surrounding protein network, it has been shown that the release of oil droplets from whey protein emulsion gels is delayed for hard gels compared to soft ones (Guo, Ye, Lad, Dalgleish, & Singh, 2014b), leading to a reduced rate of lipid hydrolysis (Guo, Bellissimo, & Rousseau, 2017;[START_REF] Guo | Impact of colloidal structure of gastric digesta on in-vitro intestinal digestion of whey protein emulsion gels[END_REF]. For emulsion gels of identical composition (10% whey proteins, 20% oil), the same team also observed that the size of the dispersed oil droplets can modify the structural and mechanical properties of the gels, and hence their digestion kinetics, with less coalescence and phase separation when embedded droplets were small (1 µm vs 6 and 12 µm) (Guo, Ye, Lad, Dalgleish, & Singh, 2014a). This literature on the digestion of emulsion gels remain quite recent and is still scarce, but nicely illustrates how protein digestion can govern the way lipids entrapped in a protein network are released. Such findings are interesting to gain a better view of the fate of complex foods in the gastro-intestinal tract. Nonetheless, most of these studies have been conduct from a lipid digestion perspective, with no concomitant measurements of the protein digestion extent to more thoroughly quantify the interplays between protein and lipid hydrolysis. Previous studies of our group have shown that pH-stat can be used to monitor the degrees of hydrolysis of proteins during the gastric phase of in vitro digestions [START_REF] Mat | Monitoring protein hydrolysis by pepsin using pH-stat: In vitro gastric digestions in static and dynamic pH conditions[END_REF], and of both proteins and lipids during the intestinal phase [START_REF] Mat | In vitro digestion of foods using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion kinetics of macronutrients, proteins and lipids[END_REF]. Building upon these previous developments, our present work intends to show how pH-stat can be used, as a rather simple and high throughput approach, to quantitatively assess food formulation effects on the gastro-intestinal in vitro digestion of both their protein and lipid contents.

More specifically, this study aimed at applying such approach to quantitatively compare the effects of three key structure parameters of protein emulsions: the state of proteins in the continuous phase (liquid and gelled state), the thickness and degree of denaturation of the protein layer at the oil/water interface, and the lipid droplet sizes (1 vs 20 µm). Additionally, because pH-stat is sensitive to both proteolysis and lipolysis in intestinal conditions, this method is generally considered inappropriate to the study of complex foods that are intrinsically made of both proteins and lipids. To overcome this issue, we also present a means, based on the use of a lipase inhibitor during pH-stat intestinal monitoring, that appears suitable to evaluate the contribution of each reaction with such complex foods.

Material and methods

Material

Whey protein isolate powder (Prolacta 95, 95 wt% of proteins on dry powder) was obtained from Lactalis, France. Rapeseed oil (Fleur de colza, Lesieur, France) was purchased at a local supermarket. Pepsin (P6887), pancreatin (P7545), pancreatic lipase (L3126) and bile extract (B8631), all of porcine origin, were obtained from Sigma-Aldrich, France, as well as the Orlistat lipase inhibitor (O4139). Enzyme activities and bile salts concentrations were determined according to the protocols described in [START_REF] Brodkorb | INFOGEST static in vitro simulation of gastrointestinal food digestion[END_REF][START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF]. Water was Milli-Q water and all other materials were of standard analytical grade.

Designed matrices

In total, 6 different matrices where designed for the purpose of this study: 4 protein emulsions and 2 protein-only matrices, the latter corresponding to the continuous phases of the emulsions. Their compositions are presented in Table 1, as well as their schematic structure and size characteristics.

A gelled coarse emulsion (GCE) was produced based on to the protocol previously described in [START_REF] Mat | In vitro digestion of foods using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion kinetics of macronutrients, proteins and lipids[END_REF]. In short, an emulsion was prepared with 0.3 wt% of whey proteins and 30 wt% of oil using a rotor-stator homogenizer (Polytron PT3100D, Kinematica AG, Switzerland) fitted with a PTDA32/2-B250 for 5 min at 10,000 rpm. It was then heated for 5 min at 70 °C. This preliminary step was performed to ensure a certain amount of denaturation of the adsorbed proteins, and to test if their partial cross-linking at the interface could influence lipolysis. The warm emulsion was mixed with a solution of 22.3 wt% of proteins prepared beforehand to achieve the final composition of 15 wt% of proteins and 10 wt% of oil. The preparation was then heated for 30 min at 80 °C in a water bath to perform gelation.

An equally-composed liquid fine emulsion (LFE) was prepared as described in [START_REF] Mat | In vitro digestion of foods using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion kinetics of macronutrients, proteins and lipids[END_REF]. In short, it consisted in emulsification by the same rotor-stator treatment, followed by sonication at 20 kHz and 130 W using a 13 mm probe (VCX 130, Sonic & Materials, UK) for 10 min (effective time, with on/off cycles of 10 s) in order to further reduce the sizes of the oil droplets. The mean temperature in the emulsion was maintained below 30 °C with an ice bath.

The corresponding lipid-free matrices consisted in a 15 wt% of whey protein solution for the liquid one (liquid continuous phase, LCP), while its gelled counterpart (gelled continuous phase, GCP) was obtained after a heat treatment (80 °C, 10 min) of the same solution.

The other two emulsions were prepared in similar ways as GCE with slight variations. On the one hand, a gelled coarse emulsion with a modified oil/water interface (GCEi) was made with omission of the first heat treatment (at 70 °C), meaning that whey proteins were not heat-denatured before the dispersion of the droplets in the protein solution. On the other hand, a gelled fine emulsion (GFE), in which the pre-emulsion was produced with a higher quantity of whey proteins (6.0 wt% instead of 0.3 wt%) and a prolonged sonication step of 15 min to produce oil droplets similar in size to those in LFE. These two additional emulsions were designed to change only one parameter at a time (interface structure and droplet size, respectively) when compared to GCE, and more properly assess the effect of the physical state of the continuous phase by comparing LFE with GFE.

Matrix characterization

The oil droplet size distributions in the emulsions were controlled with a laser light scattering particle size analyzer (Mastersizer 2000, Malvern, France), using refractive index of 1.47 and 1.33 for oil and water (dispersant), respectively. A value of 0.001 was set as the absorption of the emulsion. Droplet sizes ( The evolution of the rheological properties of the protein emulsions upon heating up to 80 °C was also measured in triplicate according to the protocol used in [START_REF] Mat | In vitro digestion of foods using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion kinetics of macronutrients, proteins and lipids[END_REF]. Final storage modulus values (G') are reported in Table 1. G' was similar for protein emulsions with large oil droplets (GCE and GCEi) and for their lipid-free counterpart (GCP), whereas it tended to be slightly higher for the protein emulsion containing small oil droplets (GFE)

because of the increased interactions between dispersed and continuous phases.

Gastro-intestinal in vitro digestions monitored by pH-stat

The digestion experiments were all performed in triplicate, and consisted in a 3-phase digestion based on the recommendations of the Infogest consortium [START_REF] Brodkorb | INFOGEST static in vitro simulation of gastrointestinal food digestion[END_REF][START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF], where details can be found on the composition of digestive fluids and enzyme activities. The only noticeable difference is that NaHCO3 salts were replaced by NaCl at the same molar ratio in all electrolyte solutions to avoid unwanted pH drift [START_REF] Mat | In vitro digestion of foods using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion kinetics of macronutrients, proteins and lipids[END_REF], meanwhile maintaining the same ionic strength. Gastro-intestinal digestions were carried out in a 200 mL jacketed beaker maintained at 37 °C by water circulation using a constant magnetic stirring (250 rpm). This set-up was mounted onto an automatic titration unit (Titroline7000, VWR, France).

Oral phase: Solid matrices were demolded and grinded with a domestic kitchen food chopper (Braun Turbo, 600W, type 4191, Spain) for 3 s at maximum power to produce submillimeter particles. 7.5 g of the grinded matrix were then mixed with 7.5 mL of simulated salivary fluid in the jacketed beaker and let to reach temperature equilibrium. This oral phase was only carried out for electrolyte concentration considerations, with no added enzymes at this stage.

Gastric phase and pH-stat measurements:

The pH probe and the titration cone of the titration unit were put in place. 13.5 mL of gastric electrolytes were added and the pH was adjusted to 3.0 using HCl 1 N. Once pH and temperature equilibria were achieved, 1.5 mL of a pepsin solution (to reach 2,000 U/mL in the final mixture) were added and titration was immediately turned on in a pH-stat mode. It was programmed to maintain a constant pH value of 3.0 for 2 h using HCl 0.3 N as a titrant.

Intestinal phase and pH-stat measurements: At the end of the gastric phase, the beaker was reconnected to another titration unit (same reference). The volume of the chyme was completed with water up to 33 mL in order to always start the intestinal phase with the same volume, regardless of the volume of titrant added previously. 25.5 mL of intestinal electrolytes containing a pre-established amount of NaOH 1 N, to bring the pH to 7.0, were first added. 2.5 mL of bile solution (prepared beforehand by melting bile extract in a 55 °C water bath) were then added to the mix. Pre-established amounts of pancreatin and pancreatic lipase powders (to achieve a trypsin activity of 100 U/mL and a lipase activity of 2,000 U/mL in the final mixture), conserved at -20 °C in a tube, were rapidly rehydrated with an intestinal electrolyte solution containing a pre-established amount of NaOH 1 N to bring the pH of the solution to 7.0. The tube was left in the water bath to reach 37 °C, and after 5 min, the pH was checked, and adjusted to 7.0 if needed. 5 mL of the enzymes solution were then added into the jacketed beaker to complete the intestinal fluid (33 mL). The titration program was immediately turned on for 2 h in a pH-stat mode to maintain a pH value of 7.0 using NaOH 0.2 N as a titrant.

Three blank intestinal digestions, with no food, were also conducted as it appeared that the mixing of the intestinal solutions induced a small pH-stat signal, possibly induced by interactions between bile extract constituents and pancreatic enzymes. This contribution was subtracted from all the titration curves obtained during the intestinal digestion of the studied matrices.

Intestinal phase in presence of Orlistat: In order to test whether protein hydrolysis could be solely monitored during the intestinal digestion of GCE and LFE matrices (i.e. despite their high lipid content and the lipolytic activity of pancreatin), the action of a lipase inhibitor (tetrahydrolipstatin, branded as Orlistat) was tested during additional digestions experiments. For these experiments, the gastric phase was carried out as described above.

However, 660 µL of the intestinal electrolyte solution were substituted by 660 µL of an Orlistat solution (4 mg/mL in DMSO) in order to achieve a final concentration of 40 µg/mL.

Moreover, pancreatic lipase was not added in complement to pancreatin in order to limit the lipolytic activity of the intestinal secretions, while maintaining the same proteolytic activity.

Determination of the degree of hydrolysis

Gastric proteolysis: The degree of hydrolysis of proteins during the gastric phase ( _ )

was estimated according to the relation previously described in [START_REF] Mat | Monitoring protein hydrolysis by pepsin using pH-stat: In vitro gastric digestions in static and dynamic pH conditions[END_REF]:

_ = 100 × × × ℎ × 1 1 - (1)
where V is the volume of added HCl (mL), is the normality of the acid, is the mass of proteins (g), ℎ = 8.8 meqv/g is the total number of peptide bonds in whey proteins [START_REF] Spellman | Proteinase and exopeptidase hydrolysis of whey protein: Comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis[END_REF], and = 0.080 is the mean degree of dissociation of the peptide carboxylic groups at pH = 3.0 and 37 °C [START_REF] Mat | Monitoring protein hydrolysis by pepsin using pH-stat: In vitro gastric digestions in static and dynamic pH conditions[END_REF].

Intestinal proteolysis:

The degree of hydrolysis of proteins during the intestinal phase ( _ ) was estimated for the lipid-free matrices (LCP and GCP) and for LFE and GCE in presence of Orlistat, according to the following relation:

_ = 100 × × × ℎ × 1 ! " + _ (%&') (2)
where V is the volume of added NaOH (mL), _ (%&') is the degree of hydrolysis at the end of the gastric phase, and ! " is the mean degree of dissociation of the α-amino groups. The value of ! " was estimated to be 0.1118 and 0.1111 from the results obtained with LCP and GCP, respectively, using the same procedure as in [START_REF] Mat | In vitro digestion of foods using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion kinetics of macronutrients, proteins and lipids[END_REF]) that relies on independent determinations of the degree of hydrolysis of end samples, i.e. collected at the end of the experiments, using the OPA (ortho-phthalaldehyde) method. A value of ! " = 0.1114 was therefore used in Eq. ( 2).

Intestinal lipolysis:

The degree of hydrolysis of lipids during the intestinal phase ( ) ) was estimated according to the following relation:

) = 100 × * × × + ) ) × 2 × 1 (3)
where ΔV (mL) is the difference between the volumes of NaOH added for the studied emulsion and its lipid-free counterpart (LCP or GCP),

)
is the oil mass (g), + ) is the molar weight of the triglycerides in the oil (calculated as 930 g/mol), and is the mean degree of dissociation of the free fatty acids' carboxylic group. was estimated to be 0.77 according to complementary 12h long intestinal digestions, performed on reduced quantities (1.5 g) of a liquid emulsion made with 10 wt% of rapeseed oil and 0.1 wt% of whey proteins in order to reach 100% release of fatty acids (controlled by the appearance of a sustained plateau during pH-stat measurements).

Statistical analysis

One way ANOVA was used to compare the degree of hydrolysis between two matrices, or two categories of matrices (e.g. liquid vs gelled). The initial reaction rates and the final extent of hydrolysis were compared 3 min and 120 min after the start of the gastric and intestinal phases, respectively. Statistically significant effects were accepted at the 95% level. All statistical analyses were conducted using the statistics toolbox of Excel TM .

Results & Discussion

Effects of structural properties on gastric proteolysis

Fig. 1 shows the kinetics of protein hydrolysis by pepsin at pH = 3.0 for the 6 studied matrices measured by pH-stat during gastric digestion [START_REF] Mat | Monitoring protein hydrolysis by pepsin using pH-stat: In vitro gastric digestions in static and dynamic pH conditions[END_REF]. Results are spitted into Fig. 1A and 1B for legibility, and those obtained for the gelled coarse emulsion (GCE) are duplicated in both subfigures for comparison purposes. The degree of hydrolysis (DH) of proteins measured after 3 and 120 min of reaction are also reported in Table 2.

A high initial reaction rate followed by a progressive slowdown was observed for all foods.

Results also shows that the beginning of the reaction was slightly slower (P < 0.001 at t = 3 min, Table 2) for 4 gelled matrices (GCP, GCE, GCEi, GFE) than for the 2 liquid ones (LCP and LFE). This can be directly related to the physical state of the protein phase since the substrate is readily accessible to pepsin in solutions, whereas its accessibility is initially limited to the external surface of gel fragments for solid matrices. It also appears that the slowdown was more pronounced for liquid matrices than for the solid ones, leading to final DH values (Table 2) significantly higher (P < 10 -6 ) for solid matrices (5.5-6.0%) than for LCP and LFE (3.4-3.8%). This can be explained by an enhanced susceptibility of denatured proteins to peptic hydrolysis. Indeed, β-lactoglobulin, the major constituent of whey proteins, has been reported to be rather resistant to pepsin in its native form [START_REF] Astwood | Stability of food allergens to digestion in vitro[END_REF], and more sensitive to pepsinolysis after denaturation by heat treatments above 70 °C [START_REF] Reddy | Structural and conformational basis of the resistance of β-lactoglobulin to peptic and chymotryptic digestion[END_REF]. Overall, the degrees of hydrolysis we measured are in line with the values found in the literature for pepsin digestion of whey proteins: 1.7% after 4 h with native proteins [START_REF] Asselin | Effects of in vitro proteolysis on the allergenicity of major whey proteins[END_REF], between 3 to 10% after 2 h with heat-treated proteins [START_REF] Kim | Peptic and tryptic hydrolysis of native and heated whey protein to reduce its antigenicity[END_REF], or 7.9% after 3 h with whey protein gels [START_REF] Luo | Digestion of protein and protein gels in simulated gastric environment[END_REF].

Fig. 1A and 1B also show that the trends observed for protein emulsions closely followed those observed for their lipid-free counterparts (LCP and GCP), with undistinguishable kinetics and final degree of hydrolysis (P > 0.7) for the 4 solid matrices (Table 2). This suggests that, whatever their size or interface (Table 1 and Fig. 1B), embedded oil droplets had a negligible influence on the peptic digestion of our emulsion gels. The final DH was slightly (by 0.4% on average) but statistically (P = 0.045) higher for LCP than for LFE, however. This difference, which appeared in the early stages of the reaction (Fig. 1A), could reflect a decreased pepsin-substrate meeting probability in oil-droplet containing solutions, but additional data would be needed to confirm such an effect.

In summary, these results confirm the significant effect of whey protein denaturation on their hydrolysis by pepsin, and show a lesser influence of embedded oil droplets, whatever their size or interface, on our protein-rich emulsions (15 wt% proteins and 10 wt% oil).

Effects of structural properties on intestinal proteolysis as inferred from lipidfree matrices

The cumulative volume of NaOH recovered by pH-stat during the subsequent intestinal digestion are presented in Fig. 2A for all matrices. However, in the neutral conditions of intestinal digestion, both proteolysis and lipolysis reactions contribute to the pH-stat signal. This is the reason why the volume of added titrant was much larger for lipid-containing matrices.

To address the impact of the protein structure on the proteolysis kinetics, one should therefore focus on the comparison between LCP and GCP. In a similar but more substantial way than during the gastric phase (Fig. 1A), the reaction was initially faster (P = 10 -5 ) for LCP than GCP (Fig. 2A), illustrating again the effect of substrate accessibility. By the end of the experiments, a sustained plateau was clearly reached with both matrices, meaning that the reaction was complete in each case. The final volumes of added titrant were similar, though they tended (P = 0.08) to be higher for GCP (2.79 ± 0.05 mL) than for LCP (2.66 ± 0.03 mL).

The overall quantity of peptide bonds hydrolyzed during the intestinal phase were thus about the same for denatured and native whey proteins. This small difference, if any, is consistent with the previous report that heat denaturation of whey proteins has a much less pronounced effect on chymotrypsin action (major constituent of pancreatic proteases) than pepsin action [START_REF] Reddy | Structural and conformational basis of the resistance of β-lactoglobulin to peptic and chymotryptic digestion[END_REF]. It is noteworthy that we previously found a bigger difference in a previous work on comparable matrices [START_REF] Mat | In vitro digestion of foods using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion kinetics of macronutrients, proteins and lipids[END_REF]. We since figured out that protons entrapped in too large gel fragments (e.g. > 1 mm) during the gastric phase can slowly release during the intestinal phase, hence leading to an overtitration by pH-stat and an overestimation of the proteolysis extent. Special care was therefore taken in the present study to avoid such effects by grinding the solid matrices into submillimeter pieces (averaged minimal Feret diameters of 0.5 mm, data not shown) and wait enough time for pH adjustment at the transition between gastric and intestinal phases.

The evolution of the intestinal DH of proteins, which accounts for the hydrolysis achieved during the gastric phase (i.e. DH of 5.7% for GCP and of 3.8% for LCP at t = 0 min of the intestinal phase), are presented in Fig. 2B. The final DH obtained for LCP (51.7 ± 0.4%) was smaller (P = 0.024) than the one measured for GCP (57.4 ± 2.7%), hence confirming the higher overall susceptibility of gelled whey proteins (GCP) than native proteins (LCP) to digestive proteases. We may highlight in here that LCP and CGP matrices have been shown

to induce contrasted impacts on both the gastrointestinal physiology and the intestinal microbiota in rats, associated to a higher protein content reaching the caecum for LCP [START_REF] Beaumont | Structure of protein emulsion in food impacts intestinal microbiota, caecal luminal content composition and distal intestine characteristics in rats[END_REF][START_REF] Oberli | Lipo-protein emulsion structure in the diet affects protein digestion kinetics, intestinal mucosa parameters and microbiota composition[END_REF]. A higher resistance of this native protein solution to protease action therefore appears consistent with these in vivo findings.

From Fig. 2A, we may finally note that the titration curves for GCE (pre-heated initial emulsion) and GCEi (unheated initial emulsion) were indistinguishable, hence suggesting that the higher quantity of proteins and cross-links at the oil droplet interface of GCE was of negligible influence on the overall digestion kinetics. We may indeed expect both liquid matrices, on the one hand, and the 4 solid matrices, on the other hand, to behave similarly from a pancreatic proteolysis point of view, as demonstrated during the gastric phase. However, it is not possible to ensure this statement from the analyses of the results of Fig.

2A, nor to investigate the possible effects of oil droplets on the intestinal protein hydrolysis kinetics, because of the contribution of lipid hydrolysis.

pH-stat monitoring of intestinal digestion in the presence of a lipase inhibitor

Most foods are intrinsically made of both proteins and lipids, with no simple way of manufacturing a lipid-free equivalent, as performed in this study, to evaluate the contributions of proteolysis and lipolysis to the pH-stat signal in intestinal conditions. To overcome this limitation, we investigated another experimental strategy that relies on the use of a lipase inhibitor, tetrahydrolipstatin, branded as Orlistat. This molecule has been studied both in vitro and in vivo and proved very efficient in inhibiting the human pancreatic lipase [START_REF] Carrière | Inhibition of gastrointestinal lipolysis by Orlistat during digestion of test meals in healthy volunteers[END_REF]Tiss, Lengsfeld, Carrière, & Verger, 2009;Wilcox, Brownlee, Richardson, Dettmar, & Pearson, 2014). It prevents the hydrolysis of triglycerides by covalently reacting with the active site of the lipase. According to the specificity of its mechanism of action, Orlistat is also assumed to solely inhibit lipases and leave protease action unhindered.

Fig. 3A and 3B present the titration kinetics obtained when Orlistat was added into the reaction mixture during intestinal digestions of LFE and GCE, respectively. Results were very close to the ones obtained with the lipid-free matrices, LCP and GCP, hence confirming that Orlistat is a very good inhibitor of intestinal lipolysis, with no, or very little, effects on proteases. This strategy therefore constitutes a very interesting alternative, which should be suitable to all types of food, to unveil the contributions of intestinal proteolysis and lipolysis when using pH-stat. However, the lipid hydrolysis reaction cannot be perfectly prevented, as evidenced by the slow but progressive increase towards the end of the experiments.

Nonetheless, present results clearly show that this approach enables to reach the same general conclusions as those obtained from the lipid-free matrices strategy. The present approach can moreover provide a means to estimate the effects of lipids on the hydrolysis of proteins, as illustrated by the slower initial rate of proteolysis for GCE than for GCP (P < 0.001 at t = 3 min) during the first hour (Fig. 3B).

Effects of structural properties on intestinal lipolysis

The lipolysis contribution to the intestinal titration was determined by subtracting the volumes of titrant added for the lipid-free matrices. The resulting curves were then converted into DH values, as presented in Fig. 4 and Table 2.

The intestinal titration curves were the same for GCE and GCEi (Fig. 2A), leading to similar final DH (46.9 ± 8.8% and 48.1 ± 2.2%, respectively). Alongside to our conclusion on protein hydrolysis, the higher quantity proteins and of cross-links at the oil droplet interface of GCE also appeared to be of negligible influence on the lipolysis kinetics. This finding is also in excellent agreement with the previously reported limited effect of interfacial β-lactoglobulin cross-linking on the in vitro intestinal digestion of liquid emulsions [START_REF] Sandra | Effect of interfacial protein cross-linking on the in vitro digestibility of emulsified corn oil by pancreatic lipase[END_REF].

Compared to GCE and GCEi, both LFE and GFE showed marked enhanced lipolysis profiles (Fig. 4, P < 0.001 at 120 min). The mean diameter of oil droplets in GFE and LFE was almost 10 times smaller than in GCE and GCEi (Table 1), hence demonstrating the major influence of the interfacial surface on the lipid hydrolysis rate even when oil droplets are embedded within a protein gel. The most rapid initial reaction rate was obtained with LFE (P < 0.05 from 3 to 9 min) most certainly because of the higher accessibility of oil droplets in this liquid matrix when compared with GFE. However, the subsequent slowdown of lipid hydrolysis was less pronounced for GFE than for LFE, hence leading to statistically comparable final DH (P = 0.19). This suggests that the physical state of the continuous phase also has an influence on lipid hydrolysis. The entrapment of the oil droplets in the protein gel may help stabilizing them throughout the intestinal reactions, whereas droplets in LFE can more easily enter into contact, coalesce and cream, thereby almost disappearing from a reaction point of view [START_REF] Giang | Dynamic modeling highlights the major impact of droplet coalescence on the in vitro digestion kinetics of a whey protein stabilized submicron emulsion[END_REF]. This was visually witnessed as no oil layer was formed at all with GFE, which contrast with what was observed with LFE, GCE, and SECc. The solid fine emulsion thus appeared to enable an initially more gradual, but overall comparable, release of lipolysis products than its liquid counterpart. Such finding might be of interest to better understand nutrient interactions within the gut, or in the formulation of lipid-based delivery systems.

Conclusions

In this study an integrated method of in vitro static digestion, respecting the Infogest recommendations, was presented. It relies on the use of the pH-stat titration during both the gastric and intestinal phases of digestion. The method proved efficient at determining the kinetics of gastric proteolysis, intestinal lipolysis and intestinal proteolysis on both liquid and solid matrices, which may be considered as good models of complex foods rich in both proteins and lipids. The physical state of the proteins (particularly in the bulk) and the size of the oil droplet were identified as major structural parameters to modulate protein and lipid hydrolysis. Overall, pH-stat proved to be a simple, rapid, and very efficient method to quantitatively monitor gastro-intestinal proteolysis and lipolysis of food products. The use of Orlistat as an efficient inhibitor of lipolysis makes it possible to study the proteolysis of food matrices in which lipids are naturally present.

  Table 1) are given as volume-weighted mean diameters, calculated using d , = ∑n i d /∑n i d . Similar d , values were obtained for LFE

	and GFE: 1.22 ± 0.06 µm and 1.37 ± 0.01 µm, respectively; and for GCEi and GCE: 19.13 ±
	0.03 and 18.83 ± 0.64 µm, respectively.

Acknowledgements

This work received support from the French National Research Agency under the "Investissements d'Avenir" program (reference No. ANR-11-IDEX-0003-02). Tiss, A., Lengsfeld, H., Carrière, F., & Verger, R. (2009). Inhibition of human pancreatic lipase by tetrahydrolipstatin: further kinetic studies showing its reversibility. Journal of Molecular Catalysis B: Enzymatic, 58, 41-47. https://doi.org/10.1016/j.molcatb.2008.11.003 Wilcox, M. D., Brownlee, I. A., Richardson, J. C., Dettmar, P. W., & Pearson, J. P. (2014). The modulation of pancreatic lipase activity by alginates. Food Chemistry, 146, 479-484. https://doi.org/10.1016/j.foodchem.2013.09.075


Julie Gennesseaux
email: jgennesseaux@chu-reims.fr

Guillaume Giordano Orsini
email: orsini@chu-reims.fr

Sophie Lefour
email: slefour@chu-reims.fr

Serge Bakchine
email: sbakchine@chu-reims.fr

Quentin Marion
email: qmarion@chu-reims.fr

Coralie Barbe
email: cbarbe@chu-reims.fr

Stéphane Gennai
email: sgennai@chu-reims.fr

Early management of transient ischemic attack in emergency departments in France

Keywords: transient ischemic attack, emergency department, organization, primary care

Introduction

Emergency departments play a key role in the diagnosis and treatment of transient ischemic attacks, but limited data are available about the early management of such patients in emergency wards.

Therefore, we aimed to evaluate emergency physicians' management of transient ischemic attack and analyze variations factors.

Methods

A multicenter survey among emergency physicians of the Grand Est region network (Est-RESCUE) was conducted from January 28 th to March 28 th , 2019. Medical and administrative data were collected by the same network and the national directory of medical resources.

Results

Among 542 emergency physicians recipients, 78 answered (14%) and 71 were finally included, practicing in 25 public hospitals homogeneously distributed across the territory, including 3 university hospitals. A cerebral magnetic resonance imaging was obtained for 75-100% of patients by 4.3% of responders, 36.4% of which were performed within more than 24 hours. A cardiac monitoring was prescribed in 75-100% of cases by 32.4% of responders. A neurologic consultation was routinely requested by 84.6% of responders practicing in a university hospital and 36,8% of responders practicing in a community hospital (p=0.02). Patients were hospitalized in a neurovascular unit in 75-100% of cases by 17.4% of responders, which happened more likely in university hospitals (p<0.001).

Conclusion

Transient ischemic attack suffers from management disparities across territories, due to limited access to technical facilities and neurologic consultations. Therefore, international recommendations are too often not followed. Implementation of territorial neurovascular tracks may help to standardize the management of these patients.

INTRODUCTION

Transient ischemic attack (TIA) is one of the most important factor of acute ischemic stroke (AIS) [START_REF] Amarenco | org Investigators. One-year risck of stroke after transient ischemic attack or minor stroke[END_REF].

Emergency departments (ED) play a major role in its early management [START_REF] Oostema | Emergency department management of transient ischemic attack: a survey of emergency physicians[END_REF].

In 2009, the American Heart Association and the American Stroke Association (AHA/ASA) agreed on a new definition of TIA, defined by the absence of a cerebral tissue lesion, and no more by the time from the onset [START_REF] Easton | Definition and evaluation of transient ischemic attack: a scientific statement for healthcare professionals from the American Heart Association/American Stroke Association Stroke Council; Council on Cardiovascular Surgery and Anesthesia; Council on Cardiovascular Radiology and Intervention; Council on Cardiovascular Nursing; and the Interdisciplinary Council on Peripheral Vascular Disease. The American Academy of Neurology affirms the value of this statement as an educational tool for neurologists[END_REF]. This definition implicated a 24/7 access to magnetic resonance imaging (MRI), not provided in several hospitals. Consequently, some experts suggested to revise the previous definition of TIA, reducing the time from the onset to one hour, and considering that symptoms lasting more than 24 hours were related to an AIS, leaving the unanswered question of TIA lasting from 1 to 24 hours [START_REF] Mohr | History of transient ischemic attack definition[END_REF]. Therefore, TIA diagnosis remains difficult, certitudes depending on anamnesis, neurologic semiology, and MRI findings.

Moreover, to find an etiology and to instore adequate treatments, guidelines suggest performing a carotid imaging and searching for an atrial fibrillation (AF) within the first 24 hours [START_REF] Lo | Clinical policy: critical issues in the evaluation of adult patients with suspected transient ischemic attack in the emergency department[END_REF], to limit further AIS. When TIA is diagnosed, antiplatelet agents are strongly recommended, as they reduce the risk of disabling and of fatal AIS by 80% [START_REF] Rothwell | Effects of aspirin on risk and severity of early recurrent stroke after transient ischaemic attack and ischaemic stroke: timecourse analysis of randomised trials[END_REF]. Aspirin stands as the first choice, but some recent clinical trials outlined the interest of its association with Clopidogrel to prevent AIS at 3 months [START_REF] Wang | Clopidogrel with aspirin in acute minor stroke or transient ischemic attack[END_REF]. In all cases, Aspirin must be administrated within 24 hours following the diagnosis of TIA. However, its ideal posology, has not been determined yet.

Emergency physicians (EP) are the cornerstone of the early management of TIA. Their implication must stick to the guidelines, while facing structural limitations such as the availability of specific imaging, neurovascular and cardiologic consultations. To help EP urged to select patients to hospitalize while discharging the others, decision tools have been created to identify high risk patients for further AIS, but their poor reliability to identify low risk patients, failed to achieve this goal [START_REF] Lo | Clinical policy: critical issues in the evaluation of adult patients with suspected transient ischemic attack in the emergency department[END_REF].

The best place to perform all necessary medical tests to patients presenting a TIA has not been established yet. However, TIA clinics demonstrated their ability to reduce further AIS [START_REF] Lavallée | TIA clinic: a major advance in management of transient ischemic attacks[END_REF], but they are exceptions in France, TIA remaining managed in public hospitals. Also, the major issue seems more likely to organize a fast checkup rather than the place where it occurs [START_REF] Chang | Can I send this patient with stroke home? Strategies managing transient ischemic attack and minor stroke in the emergency department[END_REF].

To identify strengths and weaknesses in the early management of TIA in the Grand Est region in France, we created a survey on behalf of the Est-RESCUE network, made up of EP practicing in the same region.

The first objective was to evaluate medical purposes regarding the early management of TIA.

Secondary objectives were to compare medical practices depending on physicians' experience, hospital type (university or peripheral) and specificities (number of TIA consultations, imaging availability, existence of neurovascular and cardiologic consultations).

METHODS

A descriptive observational multicenter study was conducted, based on a survey created with help from neurologists, public health and EP implicated in the early management of TIA, and the regional neurovascular physician referee.

All EP registered in the Est-RESCUE network and working in a public or private ED admitting adults, were recipients of the survey. Data about health institutions of the Grand Est region were extracted from the Est-RESCUE network and the national directory of medical resources. EP working only in prehospital settings were excluded.

The survey was sent by E-mail on January 28 th , 2019. It included 27 items form with questions about the medical demography, health institution, ED where physicians practiced, available and employed diagnosis tools, the possibility of a neurovascular and a cardiologic consultation, treatments started in ED, patient orientation (hospitalization ward or discharge), referring to EP habits. EP satisfaction regarding the management of their TIA patients, was collected using a scale from 0 (completely unsatisfied) to 10 (completely satisfied). An electronic reminder was sent every two weeks until the end of the data collection in March 28 th , 2019.

Statistical analysis was realized with SAS software® version 9.4 (SAS Institute Inc., Cary, NC, USA). Data were described by mean and standard deviation for quantitative variables, and by number and percentage for qualitative variables. Associations between parameters were studied by univariate analysis using Wilcoxon tests, Kruskal Wallis tests, Chi2 or Fisher exact tests, as appropriate. A p value < 0.05 was considered statistically significant.

RESULTS

Among 542 EP recipients, 78 (14%) answered the survey. Seven were excluded due to exclusive prehospital practice. Finally, 71 EP were included. They worked among 25 of the 64 health institutions of the Grand Est region offering an ED admitting adults.

Socio-professional characteristics of responders

Participants were male (n=39, 54.9%), of mean age 38.9 ± 8.8 y/o, attending physicians (59.2%), and head of ED (18.3%). Mean clinical practice was 10.4 ± 9.0 years. They either worked in both prehospital and hospital settings (n=60, 84.5%) or in hospital settings only (n=11, 18.3%).

Hospital characteristics

The 25 hospitals were homogeneously distributed in the Grand Est region: 7 in Champagne-Ardenne, practiced. Mean number of beds was 620 ± 367. Mean monthly TIA ED admissions in 2017 was 10.7 ± 8.2, with a 67.1% hospitalization rate.

All responders' hospitals had a short stay unit (< 24 hours), among which 20 (80%) permitted a cardiac monitoring. Nineteen hospitals (76%) had a cardiology ward, 12 (48%) a neurology ward and 9 (36%) a neurovascular unit. All responders' hospitals had access to a head computed tomography (CT) 24/7, 4 (16%) had no MRI and 8 (32%) were not able to perform a complete cardiovascular workup including a transthoracic sonography (TTS) and a duplex ultrasound of cervical vessels (DUS).

Professional practice for patients presenting a transient ischemic attack, patient emergency exit terms, and physician satisfaction regarding their management

Paraclinical exams

Fifty-seven (80.3%) responders obtained head CT for 75-100% of patients (Figure 1). CT angiography (CTA) was much less obtained, and 3/70 (4.3%) responders obtained MRI for 75-100% of patients.

Reported delay of MRI and CT access was respectively > 24 hours for 16/44 responders (36.4%) and < 3 hours for 40/68 responders (58.8%) (Figure 2).

An electrocardiogram (ECG) was performed by 70 (98.6%) responders within 3 hours. A cardiac monitoring was prescribed in 75-100% of cases by 32.4% of responders. Also, 52 (75.4%) and 45 (64.3%) responders declared that 0-25% of patients had a TTS and a Doppler ultrasound (DUS) performed in ED, respectively. When applicable, the delay to obtain a TTS or DUS was > 24 hours for 15/35 (42.9%) and 18/40 (45.0%) responders, respectively.

Specialist advice

A neurologic advice was systematically obtained by 32 (45.7%) responders, 13 (18.6%) declared never obtaining it. It was given in < 3 hours for 44/56 (78.6%) responders. Cardiologic advice was never obtained for 36/69 (52.2%) responders.

Medication

An antiplatelet agent was prescribed in 68/70 (97.1%) cases in the absence of AF or current anticoagulant treatment. Among these 68 responders, aspirin was systematically used. In AF condition, 58/70 (82.7%) responders prescribed an anticoagulant treatment. Heparin was mostly used (47/54, 87.0%), more frequently than new oral anticoagulants (24/53, 45.3%) and antivitamin K (5/48, 10.4%).

Responders prescribed an antihypertensive agent and statins from ED in 19/70 (27.1%) and 2/70 (2.9%) cases, respectively.

Use of a predictive score of recurrence

Decisions were supported by ABCD 2 score calculation for 25/70 (35.7%) responders. No other decision tool was used for 69/70 (98.6%) responders.

Post-emergency department evolution of patients

Hospitalization was requested in 75-100% of cases by 31/70 (44.3%) responders. Patients were hospitalized in 75-100% of cases in a neurovascular unit for 12/62 (19.4%) responders, general neurology ward for 4/55 (7.3%) responders, cardiology for 6/53 (11.3%) responders, other medical ward for 7/57 (12.3%) responders or a short stay unit for 8/67 (11.9%) responders.

Physician satisfaction

EP satisfaction regarding the management of their TIA patients was 5.1 ± 2.5.

Comparison of professional practice

According to physician' seniority

Analyzing the answers according to physician' seniority (≥ 2 years versus < 2 years) did not reveal any difference in terms of cerebral imaging and cardiovascular tests prescriptions (TTS and DUS), or treatment (Table 1). More experienced physicians (≥ 2 years) tended to request more frequently a systematic neurologic advice than less experienced one's (28/55, 50.9% vs 4/15, 26.7%; p=0.09).

Responders prescribing a cardiac monitoring in 75-100% of cases tended to be more frequently experienced physicians (21/55, 38.2% versus 2/16, 12.5%; p=0.05). Responders declaring that 25-100% of patients were hospitalized in neurovascular unit were more frequently less experienced physicians (13/14, 92.9% vs 28/48, 58.6%; p=0.02). Satisfaction was not associated with physician's experience (5.1 ± 2.3 for experienced physicians versus 5.1 ± 2.0; p=0.97).

According to the type of hospital (university versus community hospital)

CT were more often realized in < 3 hours in university hospitals (36/54, 66.7 versus 4/14, 28.6; p=0.01) (Table 2). Responders prescribing a cardiac monitoring in 75-100% of cases were more frequent in community hospitals (0/14, 0% versus 23/57, 40.4%; p=0.003). Neurologic advice was more often systematically requested in university hospitals (11/13, 84.6% versus 21/57, 36.8%; p=0.002).

Responders declaring a hospitalization in a neurovascular unit in 25-100% of cases were more frequent in university hospitals (13/13, 100% versus 28/49, 57.1%; p=0.003). Satisfaction tended to be higher for EP practicing in university hospitals (6.1 ± 2.0 in university hospitals versus 4.9 ± 2.2 in community hospitals, p=0.08).

According to hospital specificity

Responders prescribing a CTA in 75-100% of cases worked in ED admitting less TIA (4.0 ± 1.4 versus 11.2 ± 8.3; p=0.01). Responders prescribing a cardiac monitoring in 75-100% of cases worked in ED admitting less TIA (7.3 ± 6.4 versus 12.3 ± 8.6; p=0.0007). The mean number of TIA admitted in ED did not impact CT, MRI, DUS or TTS ED rates (p=0.28, p=0.30, p=0.20 and p=0.41, respectively) (Table 3).

The timeframe to obtain a CT or CTA was longer in hospitals admitting more TIA (p=0.001 and p=0.002, respectively). A systematic neurologic advice was more frequent in hospitals including a neurovascular unit (24/38, 63.2% versus 8/32, 25.0%; p<0.001) or a neurology ward (24/41, 58.5% versus 8/29, 27.6%; p<0.001).

DISCUSSION

Implementation of guidelines for TIA admitted in ED is still difficult. However, TIA remains a diagnosis and therapeutic emergency, with high risk of recurrence, especially within the first 48 hours. AHA/ASA guidelines insist on MRI and etiologic screening as fast as possible for every patient. Yet, we showed that only 4.3% of EP had access to MRI for 75-100% of TIA patients, compared to 80.3% for CT, and when MRI is realized, it is often with a delay over 24 hours. Nonetheless, to date, no study specified the optimal timeframe to realize cerebral imaging.

Less than a quarter of responders had a cardiovascular workup in ED for their patients, and a large majority did not prescribe a cardiac monitoring. However, guidelines recommend washing out a carotid stenosis in the best timeframe possible (< 24 hours), because it is responsible for 15% of TIA and AIS and predicts recurrence [START_REF] Lo | Clinical policy: critical issues in the evaluation of adult patients with suspected transient ischemic attack in the emergency department[END_REF]. Thus, having a DUS for TIA patients is essential in ED. Without a complete cardiovascular workup (ECG, TTS, DUS), ECG is often the only exam completed in ED, allowing a cardio-embolic risk evaluation, but it usually does not make possible the diagnosis of paroxystic AF.

The question of a systematic cardiac monitoring remains. About therapeutics, we showed that 97.1% of EP prescribed an antiplatelet agent in ED (in the absence of AF or current anticoagulant treatment), preferring Aspirin to Clopidogrel, and both antihypertensive treatment and statins were poorly prescribed. This may be linked to the absence of blood pressure monitoring and exploration of lipid abnormalities in ED, and to guidelines recommending not to treat without a complete previous etiologic workup. Yet, in myocardial infarction, statins promote remodeling of damage tissue and their prescription are consensual. In AIS, a recent meta-analysis showed that an early treatment by statins is associated with lower final infarct volume [START_REF] Malhotra | Association of statin pretreatment with collateral circulation and final infarct volume in acute ischemic stroke patients: A meta-analysis[END_REF]. However, to date, there is no consensus on this topic.

A neurologic advice was systematically asked by less than 50% of responders, although a management in a neurovascular track is recommended. The absence of neurologists in the hospital where the ED stood was the main limit for such request.

The decision of hospitalization did not depend on decision tools such as ABCD 2 score, hardly used by responders anyway. Indeed, recent studies questioned its validity for high risk patients and suggested other scores such as ABCD 3 or ABCD 3 -I, which consider a potential carotid stenosis, a possible recurrence within seven days following the initial episode, and MRI findings [START_REF] Kiyohara | ABCD 3 and ABCD 3 -I scores are superior to ABCD 2 score in the prediction of short-and long-term risks of stroke after transient ischemic attack[END_REF]. Prevalence of AIS after TIA remains a major issue, predictive tools failing to improve the decision-making process in ED.

On top of that, we showed that the hospitalization rate in neurovascular units was low, even if in hospitals managing a high mean number of TIA, but this rate increases.

The principal limit of our study was inherent in its design (survey), with a 14% participation rate.

Nevertheless, this rate is most likely the same than other surveys [START_REF] Oostema | Emergency department management of transient ischemic attack: a survey of emergency physicians[END_REF], and collected data allow us to show a first original panorama of TIA management at the scale of a large French territory. Also, only EP working in public hospitals answered, preventing us to obtain data from private hospitals.

In the end, TIA management in French public hospitals is not optimal, with several discrepancies between hospitals. MRI, recommended in the first 24 hours, is poorly achieved compared to CT, and in delays often too long. The urgent cardiovascular workup is also little achieved, despite guidelines recommending searching for carotid stenosis and AF, to limit further thrombo-embolic risks. In addition, the absence of a systematic expert opinion often leaves the EP alone initiating a therapy and deciding the hospitalization, rarely in a neurovascular unit, due to the lack of such units in hospitals and the small amount of available beds.

Recently implanted in France, TIA clinics appear as way forward. They proved their efficiency to prevent the risk of recurrence after a first episode of TIA [START_REF] Lavallée | TIA clinic: a major advance in management of transient ischemic attacks[END_REF]. They allow a fast and complete neurologic and cardiologic workup in less than 48 hours (including cardiac monitoring) and provide therapeutic education. Besides this option, the creation of protocols adapted to territories between neurologists, cardiologists, radiologists and EP may help standardizing the management of TIA, combining medical will and available resources.

Figure 1 :Figure 2 :

 12 Figure 1: Number of emergency physicians according to the proportion of patients having imaging for transient ischemic attack CT: computed tomography

Figure 1 :Figure 2 :

 12 Figure 1: Number of emergency physicians according to the proportion of patients having imaging for transient ischemic attack

Table 1 :

 1 Comparison of professional practices according to emergency physicians' seniority

	Variables 1	Seniority ≥ 2 years	Seniority < 2 years	p
		(N=55)	(N=16)
	CT			0.55
	0-25%	3 (5.5)	1 (6.3)
	25-50%	2 (3.6)	2 (12.5)
	50-75%	5 (9.1)	1 (6.3)
	75-100%	45 (81.8)	12 (75.0)
	MRI			0.53
	0-25%	44 (81.5)	12 (75.0)
	25-50%	4 (7.4)	3 (18.8)
	50-75%	3 (5.6)	1 (6.3)
	75-100%	3 (5.6)	0 (0.0)
	TTS			0.62
	0-25%	38 (71.7)	14 (87.5)
	25-50%	4 (7.6)	1 (6.3)
	50-75%	5 (9.4)	1 (6.3)
	75-100%	6 (11.3)	0 (0.0)
	DUS			0.40
	0-25%	32 (59.3)	13 (81.3)
	25-50%	5 (9.3)	0 (0.0)
	50-75%	7 (13.0)	2 (12.5)
	75-100%	10 (18.5)	1 (6.3)
	Cardiac monitoring			0.16

Table 2 :

 2 Comparison of professional practices according to the type of hospital

	Variables 1	University hospital Community hospital	p
		(N=14)	(N=57)
	CT			0.93
	0-25%	1 (7.1)	3 (5.3)
	25-50%	0 (0.0)	4 (7.0)
	50-75%	1 (7.1)	5 (8.8)
	75-100%	12 (85.8)	45 (78.9)
	Time to obtain a CT			0.03
	< 3h	4 (28.6)	36 (66.7)
	3h-6h	8 (57.1)	14 (25.9)
	6h-24h	2 (14.3)	4 (7.4)
	MRI			0.72
	0-25%	12 (85.8)	44 (78.6)
	25-50%	1 (7.1)	6 (10.7)
	50-75%	0 (0.0)	4 (7.1)
	75-100%	1 (7.1)	2 (3.6)
	Time to obtain a MRI			0.11
	< 3h	1 (16.7)	12 (31.6)
	3h-6h	0 (0.0)	9 (23.7)
	6h-24h	0 (0.0)	6 (15.8)
	> 24h	5 (83.3)	11 (28.9)
	TTS			0.93
	0-25%	12 (85.8)	40 (72.7)

Table 3 :

 3 Influence of the number of patients monthly admitted in emergency departments for a transient ischemic attack on the timing of realization of cerebral imaging

		Mean	SD	p
	CT
	< 3h	7.8	± 5.7	0.005
	> 3h	15.2	± 9.6
	CTA
	< 3h	7.5	± 6.0	0.004
	> 3h	13.9	± 9.3
	MRI
	< 3h	8.2	± 5.9	0.057
	> 3h	12.0	± 8.2
	Legend
	CT: computed tomography
	CTA: computed tomography angiography
	MRI: magnetic resonance imaging
	SD: standard deviation