Vladimir Sladkov

Mingjian He

Pawel Jewula

Marie-José Penouilh

Stéphane Brandès

Christine Stern

Jean-Claude Chambron

Michel Meyer

A solution-and gas-phase study of uranyl hydroxamato complexes

Keywords: Benzohydroxamic acid, cyclic hydroxamic acid, U=O bond activation, speciation, capillary electrophoresis

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Introduction

Uranium mines have been exploited in France from 1945-2001 [START_REF]The extraction of uranium in France: Data and key figures[END_REF]. During this period, 5.2  10 7 tons of ore have been extracted together with 2  10 8 tons of barren rocks to recover 8  10 4 tons of uranium by lixiviation, either in situ (static lixiviation), or in factories (dynamic lixiviation). Barren rocks and residues from static lixiviation have been used for conditioning and warranting the safety of old mining sites. These materials are considered long-lived, weakly or very weakly radioactive (10 3 -10 5 Bq kg -1 ). Before pitchblende (UO2) deposits were discovered in the Limousin area, the first mines exploited contained ores of the autunite family (M2(UO2)2(PO4)2, M = Ca, Cu, etc.) in which uranium is incorporated in the form of watersoluble uranyl (UO2 2+ ) salts. Therefore, understanding the physico-chemical interactions of uranyl with the minerals and biological materials contained in the soils is an important issue [START_REF] Crançon | [END_REF][3][4][5]. Among the latter, siderophores occupy a prominent position [6][7][8][9][10][11]. These natural chelators are low molecular weight compounds that are produced and excreted by microorganisms in order to capture Fe 3+ from insoluble iron oxo-hydroxides present in the pedological layer [START_REF] Crumbliss | Coordination Chemistry of Siderophores: Thermodynamics and Kinetics of Iron Chelation and Release[END_REF][START_REF] Stintzi | Siderophore Chemistry[END_REF][START_REF] Dertz | Biochemical and Physical Properties of Siderophores[END_REF]. Fe 3+ being hexacoordinate, most siderophores incorporate three bidentate chelating units, which are often derived from hydroxamic acids. The latter coordinate metal cations in the deprotonated, hydroxamate form, as oxygen-based (O,O) -anionic chelates. The simple hydroxamic acids NMAH and PIPOH (Fig. 1) have been used as pertinent models of the linear and cyclic hydroxamic acid subunits incorporated into siderophores [START_REF] Brandès | [END_REF][16][17]. In addition to these low molecular weight compounds, more complex aquatic humic substances, which derive from the natural transformation of lignin, have been recognized as responsible for the bioavailability of Fe 3+ in seawater [18,19]. It was proposed that, as siderophores, humic substances could complex Fe 3+ thanks to hydroxamate residues [20]. As a matter of fact, simple benzohydroxamic acids were recently used as model compounds for humic substances, able to supply iron to algae [21].

The hypothesis that hydroxamic acid-containing natural compounds could be involved in the complexation of uranyl relies on the similarity of the electronic properties of Fe 3+ and UO2 2+ , which are both Lewis acidic cations [22]. In order to get further insight into this important issue, we compare in this paper the binding properties towards uranyl of a primary hydroxamic acid, namely benzohydroxamic acid (BHAH) taken as a model of aquatic humic substances, with those of N-methylacetohydroxamic acid (NMAH) [START_REF] Brandès | [END_REF]22] and 1-hydroxypiperidine-2-one (PIPOH) [16,17] (Fig. 1), both in the gas phase as well as in aqueous solution. These two 3 secondary hydroxamic acids of linear (NAMH) and cyclic (PIPOH) structures can be considered as models for the binding groups incorporated in many siderophores, such as those of the desferrioxamine and exochelin groups, respectively. Fig. 1 Molecular formulae (cis isomer) and acronym of monohydroxamic acids discussed herein.

Experimental

The detailed description of the synthesis and characterization of the isolated Vibrational spectroscopy (MIR and Raman) provides a convenient mean to probe the chemical environment of the uranyl cation in its equatorial plane [START_REF] Nakamoto | Infrared and Raman Spectra of Inorganic and Coordination Compounds[END_REF][START_REF] Brandès | [END_REF]17]. Indeed, both the symmetric (s) and antisymmetric (as) U=O stretching modes, respectively Raman and IR active, are significantly red-shifted upon chelation with hydroxamate ligands with respect to crystalline [UO2(NO3)2(H2O)2]4H2O (s = 869 cm -1 and as = 941 cm -1 ). Both complexes give rise to strong IR absorptions at 906 cm -1 , while the Raman shift occurs at 830 cm -1 for [UO2(BHA)2(H2O)]0.85H2O and 835 cm -1 in the case of [UO2(PIPO)2(H2O)], as typically observed for dihydroxamato uranyl complexes [START_REF] Brandès | [END_REF][START_REF] Chakraborty | [END_REF][25][26]. A broad feature around 3200 cm -1 also attests to the presence of water.

Results and discussion

Synthesis and characterization of the bischelated uranyl complexes

Diffuse reflectance spectra of genuine powdered samples show in the UV range a broad feature corresponding likely to ligand-centered   * transitions at 252 nm in the case of [UO2(PIPO)2(H2O)] or at 274 nm in the case of [UO2(BHA)2(H2O)]0.85H2O. In the visible range, two broad and unstructured bands can be assigned to LMCT transitions from the filled  to the empty uranium-centered 5f orbitals [26]. Microcrystalline [UO2(PIPO)2(H2O)] gives rise to two well-defined maxima at 386 and 501 nm, while both bands of [UO2(BHA)2(H2O)]0.85H2O overlap to such an extent that only shoulders are visible around 350 and 470 nm. These characteristics are close to those reported for the red-colored {[UO2(FHA)2]}n coordination polymer formed with formohydroxamate (max = 370 (sh) and 475 nm) [26]. Noteworthy, both LMCT band maxima of [UO2(PIPO)2(H2O)] undergo hypsochromic shifts upon dissolution of the complex in methanol (max = 369 and 474 nm), whereas slight bathochromic shifts are observed for a methanolic solution of [UO2(BHA)2(H2O)]0.85H2O (max  360 and  460 nm). It can therefore be concluded that some structural rearrangements and/or partial dissociation of the complexes occur in solution.

In the crystal state, we have recently shown that [UO2(PIPO)2(H2O)] molecules assemble into a head-to-head chain oriented along the x direction through hydrogen bonds involving the protons of the bound water molecule and both hydroxamic ON atoms from the adjacent motif [17]. Disruption of these H-bonds by methanol might likely affect the coordination sphere around UO2 2+ and thus the electronic properties. Although no X-ray quality crystals of [UO2(BHA)2(H2O)] could be grown, it can be reasonably assumed that the isolated material has also a polymeric structure, like other uranyl complexes incorporating the primary formo- [26],

aceto- [27], or salicylhydroxamate chelates [28]. This assumption is further supported by the very low solubility in water and methanol. If so, slight modifications of the uranyl chromophore can be expected upon solubilizing the solid material, which should be reflected by shifts of the charge-transfer bands.

Gas phase properties of the bischelated uranyl complexes

Only sparingly soluble in pure methanol, the neutral [UO2(BHA)2(H2O)]0.85H2O complex readily dissolved upon addition of 15% v/v of DMSO. Thus, a 0.5 mM methanolic solution containing 0.2 mM of DMSO was analyzed by (+)-HR-ESI-MS (Fig. S3). Simulation of the isotopic patterns allowed us to assign without ambiguity all major manifolds appearing in the Accordingly, the lost fragment has a mass of 18.016 a.m.u., suggesting the release of a molecule of water albeit the parent cation is not hydrated. Hence, the abstracted oxygen atom found in the eliminated water molecule can originate either from one hydroxamic ligand or from the UO2 2+ cation upon U=O bond cleavage. In order to check the latter possibility and to discard the former, we prepared and analyzed likewise the 18 O-labelled complex [U 18 O2(PIPO)2(H2O)] obtained by reacting U 18 O2 2+ with PIPOH (Fig. S6). As expected, the signal assigned to [U 18 O2(PIPO)(PIPOH)2] + was shifted by 4 mass units with respect to the unlabeled complex (m/z = 618.227), while CID unambiguously revealed the elimination of a fragment of 20.014 a.m.u. corresponding to H2 18 O. This observation clearly ascertains that one "yl" oxygen atom from the UO2 2+ core was abstracted, suggesting that one U=O bond is broken during the collision process. This experiment definitively rules out the second conceivable waterelimination mechanism, whereby the oxygen atom would have originated from one coordinated ligand upon N-O or C-O bond cleavage.

Thus far, CID induced U=O bond activation has only rarely been reported in the literature [29][30][31][32], although Terencio et al. described very recently a similar situation for the related [UO2(NMA)(NMAH)2] + gas-phase complex [22]. Probed by infrared multiphoton dissociation spectroscopy (IRMPD) and DFT calculations, the most stable and thus plausible structure of that cation revealed the presence of only four oxygen atoms in the equatorial plane of the bound uranium center, two of them being provided by a bischelated NMA -ligand, while the two remaining ones are provided by carbonyl groups of the monodentate NMAH moieties. One N-OH function was found interacting with one "yl" oxygen atom and the second hydroxyl group was also hydrogen bonded to the former N-OH oxygen atom. Evidence for U=O bond activation was clearly provided by the disappearance of the characteristic antisymmetric O=U=O stretching mode at 940 cm -1 in the IRMPD spectrum of the dehydrated species, which therefore incorporates an UO 4+ core. Water elimination appeared to be favored by the direct apical U=OHO-N hydrogen bond that weakens the U=O bond and by the nearby location of a second hydroxyl proton [32,33]

Affinity capillary electrophoresis (ACE)

Uranyl complex formation equilibria with benzohydroxamic acid have been investigated at constant ionic strength (I = 0.1 M (H,Na)ClO4), pH (2.00(5) and 2.50(5)), and temperature (T = 298(1) K) by affinity capillary electrophoresis (ACE) in aqueous media. This speciation technique relies on the changes of the electrophoretic mobility of the detected species that is produced by the interaction with substrates present in the background electrolytes (BGE) [34][35][36][37]. Recently, ACE has been successfully used for measuring uranyl binding constants with both organic and inorganic ligands in aqueous solution [38][39][40]17,41]. Global stability constants mlh associated to equilibrium (1) are defined as molar concentration ratios according to Eq. ( 2), in which L -stands for the deprotonated ligand, while charges are omitted for sake of clarity.

m UO2 2+ + l L -+ h H +  [(UO2)m(L)lHh] (2m+h-l)+ ( 1
)
𝛽 𝑚𝑙ℎ = [(UO 2 ) 𝑚 (L) 𝑙 H ℎ ] [UO 2 ] 𝑚 [L] 𝑙 [H] ℎ (2)
A UV detector was used to monitor the migration of the uranium(VI) species as a function of the total BHAH concentration present in the BGE. The observed electrophoretic mobility (obs) for labile systems is related to the experimental migration time (t) by Eq. ( 3), in which Lt stands for the total capillary length (m), Ld for the distance between the capillary inlet and the detection window (m), U for the applied voltage (V), and teof for the migration time of the neutral marker (s). Noteworthy, teof (9 min 23 s) remained constant within  8 s over the entire concentration range, excluding any complications associated with viscosity effects [40,44].

𝜇 obs = 𝐿 t 𝐿 d 𝑈 ( 1 𝑡 - 1 𝑡 eof ) (3)
Variations of obs with the total amount of ligand introduced in the BGE (Fig. 2) can be modelled by expression (4), in which mlh corresponds to the molar fraction of any uranylcontaining species and mlh to the intrinsic electrophoretic mobility of the pure [(UO2)m(L)lHh] (2m+h-l)+ complex. It should be noted that neutral complexes, e.g.

[UO2(BHA)2(H2O)], do not contribute directly to the overall electrophoretic mobility (mlh = 0 m 2 V -1 s -1 ), but only indirectly as their occurrence impacts the molar fractions of the other charged species.

𝜇 obs = ∑ 𝛼 𝑚𝑙ℎ 𝜇 𝑚𝑙ℎ (4)
Under our acidic conditions, uranium(VI) prevails in the form of the pentaaquo uranyl dication.

According to the overall equilibrium ( 1 1:2 with 110 = 1.4 × 10 -8 m 2 V -1 s -1 taken as fixed value. The calculated curves for the 1:1 and 1:2 models are drawn as doted and dashed lines, respectively. Error bars correspond to the confidence interval at the 95% probability level of the average mobility calculated for three replicates ( 𝑡𝜎/√𝑛).

Since Eq. ( 4) is homogeneous to the expression of the NMR chemical shift for a given spin system present in several species in fast exchange, ACE data can be advantageously processed with the HypNMR program [45]. This software solves the mass-balance equations for all data points at each NLLS cycle and thus allows refining both the equilibrium constants and the intrinsic electrophoretic mobilities (mlh). Because [UO2(BHA)2(H2O)] is a neutral species, 120

was set at zero. Likewise, the protonation constant of BHA -was fixed to the value taken from the literature at the same temperature and ionic strength (log 011 = 8.80; selected data are collected in Table S1). Nevertheless, the refinement failed to converge when the 1l0 and 110

were allowed to vary simultaneously, as a consequence of the rather flat error hypersurface in the 110 direction. In other words, the system suffers from a mathematical indetermination, meaning that the product 110110 is well defined but each individual term cannot be precisely determined. This behavior is a direct consequence of the non-sigmoidal shape of both curves shown in Fig. 2, suggesting that the complex formation is far from being complete. To reach convergence, it was therefore necessary to make a reasonable guess of the 110 value and keep it constant. As a first approximation, the mobility of [UO2(BHA)(H2O)3] + was assumed to be close to the experimental value determined for the related complex [UO2(PIPO)(H2O)3] + (110 = 1.44(6) × 10 -8 m 2 V -1 s -1 ) [17], implicitly suggesting that the Stokes radii of both monovalent cations are similar. Considering the hydrophobicity of the phenyl substituent attached to the carbonyl group of BHAH and its larger size when compared to the cyclohexyl ring of PIPOH, it might be anticipated that the radius of [UO2(BHA)(H2O)3] + is somewhat larger than that of the model complex [UO2(PIPO)(H2O)3] + , and consequently 110 should be slightly lower. We therefore set 110 at 1.4 × 10 -8 m 2 V -1 s -1 , which can be considered as an upper limit. In turn, the well-defined plateau seen in Fig. 2 at low total BHAH concentrations enabled us to calculate a reliable estimate for the mobility of free uranyl. The best-fit value (100 = 3.295(3) × 10 -8 m 2 V -1 s -1 ) found for model 1:1 & 1:2 (vide infra) is in excellent accordance with the mean value determined in the absence of ligand at both pH's (100 = 3.287(8) × 10 -8 m 2 V -1 s -1 ).

Under such conditions, model 1:2 gave the poorest fit at any pH value and had to be discarded, while model 1:1 did not reproduce well all the data points, especially those measured at pH 2.50

for the highest ligand concentrations. The goodness-of-fit, assessed by the overall standard deviation returned by the software and by the visual inspection of the residues, significantly improved when the three species model 1:1 & 1:2 was considered. Best estimates of the overall stability constants defined by Eq. ( 2) were obtained after merging all data points recorded at pH 2.00 and 2.50. Refined values are log 110 = 7.43(2) and log 120 = 14.47(4). Accordingly, the stepwise stability constant of [UO2(BHA)2(H2O)], expressed as log K120 = log 120log 110, equals 7.04 (6).

Since the mobility of [UO2(BHA)(H2O)3] + is not known precisely, a sensitivity study was performed by assuming an uncertainty on 110 of ca. 25% or  0.4 × 10 -8 m 2 V -1 s -1 [46]. When 110 was set at the lower boundary value of 1.0 × 10 -8 m 2 V -1 s -1 , the returned equilibrium constants were lowered by 0.08 and 0.15 log units, respectively, as the refinement converged for log 110 = 7.35 [START_REF] Crançon | [END_REF] and log 120 = 14.32 (6), while the overall standard deviation of the fit increased by ca. 4.5%. For 110 = 1.8 × 10 -8 m 2 V -1 s -1 , a less likely situation as stressed above, the program returned higher l values (log 110 = 7.53(2), log 120 = 14.62(3)). As a matter of fact, the confidence interval on both stepwise binding constants has to be enlarged by about  0.1 log unit and the proposed values are log K110 = 7.4(1) and log K120 = 7.0(1). Accordingly, the molar fraction of [UO2(BHA)(H2O)3] + reaches 17 and 36% at pH 2.00 and 2.50, respectively, at the highest total ligand concentration used herein (50 mM). In turn, only 10%

of the total uranium occurs as the neutral bischelated complex at pH 2.50, which is within the threshold range for defining a minor species. At pH 2.00, 120 is less than 2%, in agreement with the experimental finding that [UO2(BHA)2(H2O)] does not contribute to the overall electrophoretic mobility. Based on that, the log K120 value should be considered as less reliable than log K110, and its uncertainty range is probably larger than  0.1. a Method used: absorption spectrophotometry (UV-vis), affinity capillary electrophoresis (ACE), glasselectrode potentiometry (Pot.), time-resolved laser-induced fluorescence spectroscopy (TRLFS).

b Extrapolation to I = 0 using the Davies equation (Eq. ( 5)).

Values of the stepwise equilibrium constants (Kmlh) determined in this work for the UO2 2+ /BHA -system are compared in Table 1 to those reported in the literature. Most data were determined by glass-electrode potentiometry, although some authors used UV-vis absorption spectrophotometry or time-resolved laser fluorescence spectroscopy (TRLFS), but ACE has never been applied until now. Examination of Table 1 reveals three potentiometric studies carried out in nitrate media at the same temperature (298.2 K) and ionic strength (I = 0.1 M) as our work, which propose remarkably consistent values for log K110 and log K120, averaging 7.44(4) and 6.72 (5), respectively [51][52][53]. Noteworthy, the log K110 value determined herein by ACE is in excellent accordance with those of references [51][52][53], while our log K120 is slightly higher by ca. 0.3 units.

In contrast, the equilibrium constants determined at 303.2 K by Dutt and Seshari [50] appear as outliers, as they exceed by more than 1.5 orders of magnitude the latter data. It can also be noted that log K120 = 8.91 almost equals log K110 = 9.03, which would suggest a cooperative uptake of the second monohydroxamato ligand, a quite unlikely situation for this class of ligands [55,56]. Indeed, the lower probability for the second compared to the first entering bidentate chelator to find an unoccupied binding position in the equatorial plane of a pentacoordinated uranium center decreases K120 with respect to K110. Based on that sole statistical effect, both constants should be in the 1/5 ratio, translating into a  difference ( = log K110log K120) of 0.70 units or Gstat = 4.0 kJ mol -1 [17]. In addition, the lower electrostatic interaction energy resulting from the overall charge reduction on the metal center upon progressive uptake of anionic ligands should further lower the binding affinity for the second hydroxamato anion and thus  values even larger than 0.70 are anticipated [17]. Based on that reasoning, the results of Dutt and Seshari [50] but also those of Glorius et al. obtained by TRLSF [48] can be considered as suspicious. The spectrophotometric measurements undertaken by the same authors [47] returned apparently more reasonable binding constants, although K110 and K120 are ca. 0.5 and 0.3 log units higher than our values, respectively. Nevertheless, charge reduction occurs upon successive chelation of UO2 2+ by BHA -. Thus, stability constants at infinite dilution (K 0 1l0) are predicted to be higher than those determined at the ionic strength conditions listed in Table 1. Extrapolation of K1l0 values to I = 0 can be achieved in first approximation by applying the Davies equation (Eq. ( 5) with z 2 = -4 and -2 for l = 1 and 2, respectively) for estimating the activity coefficients of the charged species [START_REF] Grenthe | TDB-2 Guidelines for the extrapolation to zero ionic strength[END_REF]. By doing so, Glorius et al.

seemingly calculated K 0 1l0 values lower than those measured at I = 0.1 M both by absorption and emission spectrophotometry [47]. If it is assumed that this obvious inconsistency is due to the accidental reversing of K1l0 and K 0 1l0 by the authors in both of their publications, then a good agreement can be found for their values determined by spectrophotometry [47] with those reported herein or in references [51][52][53].

log 𝐾 1𝑙0 0 = log 𝐾 1𝑙0 -0.509∆𝑧 2 ( √𝐼 1 + √𝐼 -0.3𝐼) (5)
The binding affinity of BHA -for UO2 2+ found herein is significantly lower than that reported at the same temperature and ionic strength for acetohydroxamate (AHA -, log K110 = 8.22, log K120 = 7.08) [58], in agreement with the higher basicity of the latter ligand (log K011 = 9.30(4) [56,58] vs. 8.80 at I = 0.1 M). In turn, the secondary hydroxamate NMA -possesses a markedly lower basicity (log K011 = 8.68(3), I = 0.1 M, T = 298.2 K [17]) than AHA -, owing to the stabilization of the positive charge appearing on the nitrogen atom in the iminium canonical form, but is only very slightly more acidic (by ca. 0.1 log unit) than BHA -. Nevertheless, NMA - forms a moderately stronger 1:1 complex with uranyl (log K110 = 7.76(1)) than BHA -under similar conditions, while the reverse situation was found in the case of the 1:2 species (log K120 = 6.14(1) for NMA -) [17]. Very recently, we have investigated the speciation of the UO2 2+ in the presence of the six-membered cyclic hydroxamate PIPO -by combining potentiometric, UV-vis, ACE, and Raman equilibrium measurements [17]. This chelator assumes by construction a blocked cis orientation of both donor atoms, unlike the open-chain hydroxamates, which are prone to occur in solution as a mixture of both cis and trans rotamers (Fig. 1) [START_REF] Brandès | [END_REF]59,60]. The cis conformation of PIPO -confers a higher basicity (log K011 = 8.85(2))

and affinity towards UO2 2+ (log K110 = 8.58 [START_REF]The extraction of uranium in France: Data and key figures[END_REF], log K120 = 6.92(1), I = 0.1 M, T = 298.2 K) as compared to NMA -, the stability gain amounting 0.8 log units or 4.6 kJ mol -1 per bound ligand.

Interestingly, BHA -, which exhibits acid-base properties very similar to those of PIPO -and which prevails in water in its cis form [59], forms significantly weaker ML (log K110 = 7.44(4)

vs. 8.58 [START_REF]The extraction of uranium in France: Data and key figures[END_REF]) and ML2 (log K120 = 6.72(5) vs. 6.92(1)) complexes with uranyl, highlighting the advantage of a sterically constrained and predisposed structure over an acyclic one. A better solvation by the water molecules of the aliphatic PIPO -complexes might also contribute to enhance their stability.

Conclusions

This study has focused on BHAH, a hydroxamic acid carrying an aromatic carbonyl substituent.

Whereas this structural feature has virtually no influence on the pKa of BHAH by comparison with aliphatic analogues (NMAH and PIPOH), the binding properties of BHA -towards the uranyl dication are affected to a significant extent, since they can be viewed as intermediate between those of NMA -and PIPO -. The affinity of UO2 2+ for a first BHA -ligand is very similar to that for NMA -, whereas the second BHA -chelate is captured with the same affinity as PIPO - . Overall, however, BHA -behaves as a standard hydroxamate as far as the complexation of UO2 2+ in aqueous solution is concerned. The situation is more contrasted in the gas phase. The collision-induced U=O bond cleavage phenomenon observed in the case of the [UO2(NMA)(NMAH)2] + species, and reported in an earlier work, could be duplicated with the PIPOH ligand, but not with BHAH. In the latter case, the more classical release of a BHA - chelate was observed instead. Whereas gas phase studies are a direct entry into bond energetics, fundamental solution studies allow us to understand and describe phenomena taking place not only in vitro, but also in vivo. The demonstration that BHA -can complex uranyl as efficiently as aliphatic hydroxamic siderophores makes this chelate a pertinent model of humic substances, which have been recognized as a class of natural compounds involved in the mobilization of hard Lewis acids such as UO2 2+ or Fe 3+ .

Treatment of a slight excess ( 2 . 5 -

 25 3 equiv.) of hydroxamic acid dissolved in water with [UO2(NO3)2(H2O)2]4H2O immediately affords an orange solution, suggesting the formation of complexes that concomitantly lowers the pH to about 2.2. Upon neutralization with an aqueous base solution (~0.1 M N(CH3)4OH or KOH), a red-orange precipitate corresponding to the neutral bischelated complexes [UO2(L)2(H2O)]xH2O (x = 1 for L = BHA -, x = 0 for L = PIPO - ) could be isolated in good yield (60-90 %). Both synthesized compounds gave satisfactory elemental analysis and were further characterized by TGA (Fig. S1-S2), IR, Raman, and UVvis diffuse reflectance spectroscopy in the solid state, and by HR-ESI-MS spectroscopy in solution. Details can be found in the experimental section included in the Supplementary Information.

  spectrum. Electrospray ionization generates a variety of solvated monochelated species at m/z = 484.095 [UO2(BHA)(DMSO)] + , 502.105 [UO2(BHA)(DMSO)(H2O)] + , 516.121 [UO2(BHA)(DMSO)(CH3OH)] + , 562.109 [UO2(BHA)(DMSO)2] + , in addition to the bischelated sodium adduct [UO2(BHA)2(DMSO) + Na] + at m/z = 643.124, supporting the identity of the isolated complex. MS/MS collision induced dissociation (CID) experiments performed on all of the aforementioned mass-selected cations showed the progressive loss of the various solvent molecules with increasing collision energy (Fig. S4). These results are in marked contrast with the gas-phase behavior of [UO2(PIPO)2(H2O)]. Indeed, the MS spectrum of a pure methanolic solution of this compound shows the formation of different monocationic complexes of general [UO2(PIPO)(L)n] + formula, with L = PIPOH, H2O, or CH3OH, in addition to some sodium adducts (see the experimental section in the SI). To get further insight, some mass-selected cations were subjected to CID experiments. MS/MS fragmentation of [UO2(PIPO)(PIPOH)] + (m/z = 499.161) affords the [UO2(PIPO)] + cation (m/z = 384.098) together with its water adduct (m/z = 402.104). CID of the [UO2(PIPO)(PIPOH)2] + cation (m/z = 614.225) gives rise to a new signal at m/z = 499.156 associated to the loss of an intact molecule of bound PIPOH ligand (Fig. S5). The thereby generated [UO2(PIPO)(PIPOH)] + cation can further react in the collision chamber with a molecule of water, producing the [UO2(PIPO)(PIPOH)(H2O)] + ion detected at m/z = 517.167. More intriguingly, CID of [UO2(PIPO)(PIPOH)2] + generates another peak at m/z = 596.209.

  Fig.S7reproduces experimental electrophoregrams recorded for a total uranyl concentration of 0.1 mM at both considered pH values. Each trace shows a single signal for which the characteristic migration time taken at the peak maximum (t) increases with the total BHAH content. Such a behavior is typically observed for labile systems and reflects fast ligand exchange processes at the separation time scale (4 min) between the free and bound uranyl species[35,42,43]. At pH 2.50, uranyl migration is slowed down much more than at pH 2.00 for an identical total ligand concentration, suggesting complex formation to a greater extend. Chelation under strongly acidic conditions was further supported by a bathochromic shift of the absorption band, as the free uranyl peak detected at 200 nm progressively vanishes. Hence, the monitoring wavelength, initially set at 200 nm ([BHAH]tot = 0-2 mM), was changed first to 230 nm ([BHAH]tot = 5-20 mM), and finally to 250 nm for the last run ([BHAH]tot = 50 mM).

  ), progressive chelation of [UO2(H2O)5] 2+ by the hydroxamate anionic ligand lowers the overall charge and simultaneously increases the hydrodynamic radius. Both effects are responsible for reducing the apparent electrophoretic mobility (Fig. 2), as reflected by longer migration times. Nonlinear least-squares fit (NLLS) of the experimental obs values obtained at different total ligand concentrations, was performed for three chemical models including the formation of either the sole monochelate [UO2(BHA)(H2O)3] + (110, model 1:1), the sole neutral bischelate [UO2(BHA)2(H2O)] (120, model 1:2), or both complexes (110 and 120, model 1:1 & 1:2).

Fig. 2

 2 Fig. 2 Variations of the observed electrophoretic mobility of uranyl as a function of the total BHAH concentration at pH = 2.00(5) (a) and 2.50(5) (b). I = 0.1 M (H,Na)ClO4, T = 298(1) K, [U(VI)]tot = 0.1 mM. The solid lines correspond to the best fit obtained for the model 1:1 &

Table 1

 1 Literature survey of the stability constants for the uranyl/benzohydroxamate (BHA -)

	system
	log K110	log K120	I (M)	Medium	T (K)	Method a	Ref
	7.51(5)	7.1(1)	0 b	NaClO4	295.2	UV-vis	[47]
	7.5(1)	8.7(5)	0 b	NaClO4	298.2	TRLFS	[48]
	7.8(2)	7.2(2)	0 b	(H,Na)ClO4 298(1)	ACE	This work
	8.72	8.05	Variable KClO4	298.2	Pot.	[49]
	7.96(5)	7.3(1)	0.1	NaClO4	295.2	UV-vis	[47]
	7.9(1)	9.0(5)	0.1	NaClO4	298.2	TRLFS	[48]
	7.4(1)	7.0(1)	0.1	(H,Na)ClO4 298(1)	ACE	This work
	9.03	8.91	0.1	NaClO4	303.2	Pot.	[50]
	7.42(1)	6.77(1)	0.1	NaNO3	298.2	Pot.	[51]
	7.42(3)	6.70(1)	0.1	NaNO3	298.2	Pot.	[52]
	7.49(3)	6.68(8)	0.1	KNO3	298.2	Pot.	[53]
	7.7		1.0	NaClO4	293.2	UV-vis	[54]

Acknowledgements

The Centre National de la Recherche Scientifique (CNRS), the Conseil Régional de Bourgogne (CRB, program PARI II CDEA), the European Regional Development Fund (FEDER), the program "Défi NEEDS Environnement" (project PiRATE), and the Agence Nationale de la Recherche (ANR project PLUTON, grant N° ANR-17-CE08-0053) are gratefully acknowledged for their financial support. M.H. and P.J. thank the Chinese government and the CRB, respectively, for their PhD fellowships.


Desprez Charlotte
email: charlotte-desprez@orange.fr

Melchior Chloé

Wuestenberghs Fabien

Jacques Jérémie

Anne-Marie Leroi

Guillaume Gourcerol

Charlotte Desprez

Pyloric distensibility measurement predicts symptomatic response to intrapyloric botulinum toxin injection

Background and Aims:

Recent studies reported that pyloric distensibility was altered in 30% to 50% of patients with gastroparesis and this was correlated with gastric emptying and symptom severity.

The aim was to assess whether pyloric distensibility measurement was predictive of symptomatic response after intrapyloric botulinum toxin injection.

Method:

Pyloric distensibility was measured using EndoFLIP system before intrapyloric botulinum toxin injection. Altered pyloric distensibility was defined as distensibility below 10mm2/mmHg. Total symptomatic score (TSS), dyspeptic symptoms, Gastrointestinal Quality of Life Index (GIQLI), and gastric emptying were investigated prospectively before and 3 months after botulinum toxin injection.

Results:

Nineteen of 35 patients had altered pyloric distensibility. In those patients, TSS decreased at 3 months from 13.5 to 10.5 (p<0.01), whereas it remained unchanged in patients with normal pyloric distensibility (p=0.7). Gastric fullness (from 3.5 to 2.5; p=0.03) and bloating (from 3.0 to 2.0; p=0.01) were the only symptoms to be improved in patients with altered pyloric distensibility, whereas none of them were improved in patients with normal pyloric distensibility. GIQLI score increased from 59.5 to 76.5 in patients with altered pyloric distensibility (p=0.02), whereas there was no statistical difference (p=0.43) in patients with

normal pyloric distensibility. In patients with altered pyloric distensibility, gastric emptying half time was 223 minutes before and 190 minutes 3 months after injection (p=0.02), whereas it remained unchanged in patients with normal pyloric distensibility (p=0.6).

Conclusion:

Pyloric distensibility measurement before intrapyloric botulinum toxin injection predicted symptomatic and quality of life response 3 months after injection in patients with gastroparesis.

Key words: Gastroparesis, dyspepsia, botulinum toxin, pylorus, distensibility

INTRODUCTION:

Gastroparesis is defined as objectively delayed gastric emptying in the absence of mechanical obstruction, leading to dyspeptic symptoms [START_REF] Camilleri | Clinical Guideline: Management of Gastroparesis[END_REF] . Its prevalence in the United States general population was estimated at 24.2 per 100,000 persons in 2007 [START_REF] Jung | The incidence, prevalence, and outcomes of patients with gastroparesis in Olmsted County, Minnesota, from 1996 to 2006[END_REF] , revealing medical issues in long-term care with an increase in morbidity and mortality in those patients. In the most severe cases, gastroparesis can alter quality of life and nutritional status, with an increasing number of hospitalizations [START_REF] Bharucha | Epidemiology and natural history of gastroparesis[END_REF] . In addition to idiopathic gastroparesis, which is the most frequent form, the main causes of gastroparesis are diabetes and surgery [START_REF] Soykan | Demography, clinical characteristics, psychological and abuse profiles, treatment, and long-term follow-up of patients with gastroparesis[END_REF] .

Pathophysiology of gastroparesis is complex, with possible contribution of several mechanisms 5-6-7 leading to gastroduodenal motor abnormalities [START_REF] Camilleri | Relationship between impaired gastric emptying and abnormal gastrointestinal motility[END_REF] , including pylorospasm [START_REF] Mearin | Pyloric dysfunction in diabetics with recurrent nausea and vomiting[END_REF] . The study of Mearin et al [START_REF] Gourcerol | Impaired fasting pyloric compliance in gastroparesis and the therapeutic response to pyloric dilatation[END_REF] is the first one to our knowledge, to have assessed the implication of impaired pyloric contraction in gastroparesic patients. It revealed that the occurrence and duration of pylorospasm were increased in patients with diabetic gastroparesis compared with healthy controls. A previous prospective study using EndoFLIP system to assess pyloric characteristics showed that fasting pyloric distensibility could be altered in a subgroup of 10 out of 26 patients with gastroparesis [START_REF] Gourcerol | Impaired fasting pyloric compliance in gastroparesis and the therapeutic response to pyloric dilatation[END_REF] . Moreover, there was an association in this study between altered pyloric distensibility and delayed gastric emptying. Altered pyloric distensibility was also correlated with more severe dyspeptic symptoms and altered quality of life. In contrast, there was no association with pyloric pressure. These results were confirmed in other populations of patients with gastroparesis or dyspepsia [START_REF] Malik | Assessing pyloric sphincter pathophysiology using EndoFLIP in patients with gastroparesis[END_REF][START_REF] Snape | Evaluation of the pylorus with concurrent intraluminal pressure and EndoFLIP in patients with nausea and vomiting[END_REF] .

Botulinum toxin (BT) is a protein natively produced by the bacterium Clostridium botulinum, which blocks neuromuscular conduction. The first case of intrapyloric injection of BT was reported in 1998 [START_REF] Sharma | Pyloric intrasphincteric botulinum toxin (Botox) improved symptoms and gastric emptying in a patient with diabetic gastroparesis (abstr)[END_REF] . The positive findings of open-label case series [START_REF] Ezzeddine | Pyloric injection of botulinum toxin for treatment of diabetic gastroparesis[END_REF] and a retrospective review [START_REF] Bromer | Endoscopic pyloric injection of botulinum toxin A for the treatment of refractory gastroparesis[END_REF] were not confirmed in the two randomized placebo-controlled trials 16- 17 . A retrospective analysis identified the existence of a potentially definable subgroup of patients for whom intrapyloric BT injection might be beneficial [START_REF] Coleski | Factors associated with symptom response to pyloric injection of botulinum toxin in a large series of gastroparesis patients[END_REF] . However, there is a lack of direct measurement of pyloric motility to identify the best candidates for such treatment in randomized controlled-placebo studies. Therefore, we hypothesized that altered fasting pyloric compliance could predict positive response to intrapyloric BT injection among patients with gastroparesis.

The objective of this study was to assess whether pyloric distensibility measurement was predictive of symptomatic response after intrapyloric botulinum toxin injection.

MATERIAL AND METHODS:

Approval:

The study received the agreement of the local ethical review board (Comité d'Ethique de la Recherche Non-Interventionnelle, CERNI, No E2018-68). All patients had given written informed consent before the investigation.

Patients:

Patients with diagnosis of gastroparesis refractory to medical therapy (involving resistance to all prokinetics at maximal posology available in France like oral metoclopramide 40 mg per day, oral domperidone 40 mg per day, and oral erythromycin 750 mg per day 1 ) were consecutively included in the study. Organic gastrointestinal obstruction was ruled out using routine biological blood tests and upper gastro-intestinal endoscopy with biopsies. Patients who had previous endoscopic treatment for gastroparesis were excluded from the study.

Improvement at 3 months on symptom scores, quality of life, and gastric emptying were used as evaluation of treatment.

Clinical evaluation:

Dyspeptic symptoms (vomiting, nausea, epigastric pain, bloating, fullness, and satiety) were recorded before and at 3 months after intrapyloric botulinum toxin injection using specific symptomatic scales. Each item was graded using a 5-point likert scale from 0 (absent) to 4 (most-severe symptoms). Total symptomatic score (TSS) was defined as the aggregation of individual symptom scores. Quality of life was measured using Gastrointestinal Quality of Life Index (GIQLI) score ranging from 0 (worst quality of life) to 144 (best quality of life) [START_REF] Eypasch | Gastrointestinal Quality of Life Index: development, validation and application of a new instrument[END_REF] .

The GIQLI score is specific for digestive disease and has also been validated in French [START_REF] Slim | First validation of the French version of the Gastrointestinal Quality of Life Index (GIQLI)][END_REF] . All questionnaires were answered prospectively by patients during their hospitalization before intrapyloric BT injection and at 3-month evaluation.

C-Octanoic acid breath-test:

All patients had gastric emptying measurement before and 3 months after intrapyloric BT injection, and were off prokinetics for at least 1 week. After 12 hours of fasting, [START_REF] Sharma | Pyloric intrasphincteric botulinum toxin (Botox) improved symptoms and gastric emptying in a patient with diabetic gastroparesis (abstr)[END_REF] C-octanoic breath acid breath test was performed according to a previously published method 21 22 . The breath test started between 8:00 AM and 9:00 AM with ingestion of a 250-kcal test meal (19% protein, 43% carbohydrate, 38% fat) consisting of white bread (50 g), butter (17 g), and an egg dosed with 91 mg of [START_REF] Sharma | Pyloric intrasphincteric botulinum toxin (Botox) improved symptoms and gastric emptying in a patient with diabetic gastroparesis (abstr)[END_REF] C-octanoic acid (Euriso-Top, Saint Aubin, France). Breath samples were collected immediately before meal ingestion and every 15 minutes after the meal for up to 8 hours. Breath samples were investigated for the presence of [START_REF] Sharma | Pyloric intrasphincteric botulinum toxin (Botox) improved symptoms and gastric emptying in a patient with diabetic gastroparesis (abstr)[END_REF] CO 2 by isotope-selective non-dispersive infrared spectrometry (IRIS; Wagner/Analysen Technik, Bremen, Germany). The solid half-time rate (T 1/2 ) for gastric emptying was calculated by linear regression analysis using the IRIS software GE2.DEM.

Pyloric distensibility:

No chronic intake of medication known to affect gastrointestinal function was noted and patients were investigated off prokinetic agents. Pyloric distensibility was investigated after 12-hour fasting using EndoFLIP probe (Crospon Inc) before intrapyloric botulinum toxin injection. The pyloric measurements were performed with the patient under general anesthesia (propofol). An EndoFLIP catheter (EF-325N; Crospon Ltd, Galway, Ireland) was inserted through the single channel of the endoscope. Visual control of the EndoFLIP catheter through the pylorus and the distinctive shape of the pylorus represented as a narrow segment with decreased cross-sectional area (CSA) were necessary to validate correct positioning. The 8cm balloon was filled with different volumes (10, 20, 30, 40, and 50 mL) and for each set volume, at least 3 recordings were made. Retained values needed stable pyloric pressure and CSA for at least 5 seconds, in order to avoid variations in pylorus volume and/or pressure during peristaltic contractions. Moreover, measurement of antral pressure was realized with the deflated bag before and after intrapyloric probe positioning. In our study, pyloric pressure was expressed relative to antral pressure as previously described [START_REF] Gourcerol | Impaired fasting pyloric compliance in gastroparesis and the therapeutic response to pyloric dilatation[END_REF] . The mean value of 3 measures of the smallest CSA and the corresponding pressure were taken for compliance analysis for each subject. Distensibility was computed using minimum CSA divided by the pressure at each balloon fill volume. A cut-off of 10 mm 2 /mm Hg at 40 mL of inflation for normality of distensibility was determined in a previous study 10 in healthy subjects. We used this threshold value of 10 mm 2 /mm Hg to classify patients into 2 groups on the basis of normal (>10 mm 2 /mm Hg) or decreased (<10 mm 2 /mm Hg) pyloric distensibility.

Intrapyloric botulinum toxin injection:

Immediately after investigation of pyloric distensibility by EndoFLIP, intrapyloric botulinum toxin injection was performed during upper GI endoscopy with the patient under general anesthesia. A 0.5-mm sclerotherapy needle was introduced through the biopsy channel of the endoscope. Two hundred units of botulinum toxin (Dysport; Ipsen/Biotech, France) were diluted into 4 mL of saline solution and then injected per 1 mL dose in each quadrant of the pylorus.

Data analysis:

Intra-individual analysis used Wilcoxon signed ranked test for continuous variable (2-tailed). A p<0.05 was considered as statistically significant. Correlation studies used the Spearman test. Results were expressed as the median and the 25 th and 75 th percentile. Data were analyzed using GraphPad Prism version 5.03 for Windows (GraphPad Software Inc, San Diego, California, USA, www.graphpad.com).

RESULTS:

Patients:

Thirty-five patients with gastroparesis (12 men), median age 55.1 years (range 25-80), were included between January 2016 and June 2018. The main etiologies of gastroparesis among patients were diabetes (11/35; 31.4%) and surgery (6/35; 17.1%) with 6 Nissen fundoplications. 51% (18/35) of patients had idiopathic gastroparesis. Using the threshold of 10 mm 2 /mm Hg at 40 mL of inflation, we found that 19 of 35 (54.2%) patients had altered pyloric distensibility whereas 16 of 35 patients had normal pyloric distensibility (Figure 1).

Characteristics of patients are summarized in Table 1 and no difference was found between the 2 groups.

Impact of pyloric distensibility on TSS and dyspeptic symptoms at 3-month evaluation:

Twenty-eight of 35 patients answered questionnaires and were included in statistical analysis (reasons for missing data are summarized in Supplementary Figure 1). In patients with altered pyloric distensibility, TSS decreased at 3 months from 13.5 (range 11.0-16.0) to 10.5 (range 8.5-13.0; p<0.01), whereas it remained relatively unchanged in patients with normal pyloric distensibility from 12.0 (range 9.0-14.5) to 13.0 (range 8.8-16.0; p=0.7; Figure 2). Regarding dyspeptic symptoms, gastric fullness, and bloating were improved in patients with altered pyloric distensibility, with a decrease in scores respectively from 3.5 (range 2.3-4.0) to 2.5 (range 1.8-3.0; p=0.03) and from 3.0 (range 2.0-3.8) to 2.0 (range 1.8-3.0; p=0.01). None of the dyspeptic symptoms were improved in patients with normal pyloric distensibility. Results of dyspeptic symptoms are more detailed in Table 2. Moreover, similar results were found when the threshold for pyloric distensibility was set at 8 and 9 mm 2 /mm Hg (data not shown).

Impact of pyloric distensibility on quality of life at 3-month evaluation:

Among patients with altered pyloric distensibility, quality of life was improved, with an increase in GIQLI score from 59.5 (range 38.0-82.0) to 76.5 (range 66.5-91.0; p=0.03), whereas it remained relatively unchanged in patients with normal pyloric distensibility (from 75.5 [range 52.0-87.0] to 67.0 [range 48.0-89.5; p=0.43; Figure 3]). Similar results were found when the threshold for pyloric distensibility was set at 8 and 9 mm 2 /mm Hg (data not shown).

Gastric emptying half-time:

Twenty-eight of 35 patients had [START_REF] Sharma | Pyloric intrasphincteric botulinum toxin (Botox) improved symptoms and gastric emptying in a patient with diabetic gastroparesis (abstr)[END_REF] C-Octanoic acid breath-test and were included in statistical analysis (reasons for missing data are summarized in Supplementary Figure 1). Gastric (range 149-243) minutes at 3 months after injection (p=0.6; Figure 4). Last, similar results were found when the threshold for pyloric distensibility was set at 8 and 9 mm 2 /mm Hg (data not shown).

DISCUSSION:

To our knowledge, the present study is the first to have assessed the efficacy of intrapyloric BT injection in patients with gastroparesis, based on the alteration of fasting pyloric distensibility. In our study, altered fasting pyloric compliance was associated with improvement in symptom scores (TSS, gastric fullness, and bloating) and quality of life, in contrary to gastroparesis associated with normal fasting pyloric compliance. Moreover, only decreased fasting pyloric compliance was associated with a decrease in gastric emptying. This is a first signal based on the pathophysiology of gastroparesis to better select patients for pyloric targeting therapeutics. Further confirmatory studies are needed to verify whether this marker is relevant to predict the outcome of gastroparesis treatment.

We chose fasting pyloric distensibility to assess the efficacy of BT injection in the present study. It has been shown that pyloric distensibility, but not pyloric pressure, was different between healthy volunteers and gastroparetic patients [START_REF] Gourcerol | Impaired fasting pyloric compliance in gastroparesis and the therapeutic response to pyloric dilatation[END_REF][START_REF] Malik | Assessing pyloric sphincter pathophysiology using EndoFLIP in patients with gastroparesis[END_REF] . Moreover, altered pyloric distensibility was also predictive of delayed GE, worse symptoms and quality of life [START_REF] Gourcerol | Impaired fasting pyloric compliance in gastroparesis and the therapeutic response to pyloric dilatation[END_REF][START_REF] Malik | Assessing pyloric sphincter pathophysiology using EndoFLIP in patients with gastroparesis[END_REF] , confirming our findings. In addition, even if a second pyloric distensibility measurement after BT injection was not reported in the present study, we found in a previous report that pyloric dilatation in 10 patients with gastroparesis and altered pyloric compliance increased pyloric compliance from 7.4 to 20.1 mm²/mm Hg [START_REF] Gourcerol | Impaired fasting pyloric compliance in gastroparesis and the therapeutic response to pyloric dilatation[END_REF] .

Regarding intrapyloric BT injection, the efficacy of this treatment remains controversial based on the analysis of the 2 randomized controlled-placebo trials available in the literature. Arts et al [START_REF] Arts | Clinical trial: a randomized-controlled crossover study of intrapyloric injection of botulinum toxin in gastroparesis[END_REF] did not find a difference in symptoms improvement after saline solution or botulinum toxin injection, suggesting an important placebo effect by endoscopic therapy. But in this study, the population was heterogeneous with mostly idiopathic gastroparesis and the number of subjects in this study was limited to 23 with insufficient statistical power reported by authors. Similarly, Friedenberg et al [START_REF] Friedenberg | Botulinum toxin A for the treatment of delayed gastric emptying[END_REF] did not find an improvement in symptom scores and quality of life. These similar findings could be partially explained by the overestimated effect of BT on the basis of open reports, but also by the absence of data on pyloric distensibility. It could be interesting to perform further randomized placebo-controlled studies using the EndoFLIP system to assess pyloric distensibility and consequently confirm the association between altered pyloric distensibility and response to BT injection. One of the limits of BT injection is the necessity of repeating injections every 6 to 9 months according to their efficacy among patients. Therefore, other therapeutic interventions specific to the pylorus have been proposed in recent years, with early results for many of them which are promising [START_REF] Ahuja | Pyloric Therapies for Gastroparesis[END_REF] . On the basis of our results, it could be interesting to reproduce pyloric measurements to assess the efficiency of these different interventions and if possible at a longer time than 3-month evaluation. A French prospective study 26 evaluated the efficacy of peroral endoscopic pyloromyotomy (G-POEM) with assessment of pylorus characteristics using EndoFLIP. They found a distensibility threshold <9.2 mm²/mm Hg before the G-POEM to discriminate responders and not responders patients; however, all patients were responders at 3 months in this study.

The convenience and reproducibility of EndoFLIP to assess pyloric dysfunction have been also demonstrated using either the single channel therapeutic endoscope, with catheter easily inserted into the pylorus and adjusted under endoscopic and fluoroscopic control [START_REF] Malik | Assessing pyloric sphincter pathophysiology using EndoFLIP in patients with gastroparesis[END_REF] or transnasal insertion under local anesthesia [START_REF] Gourcerol | Impaired fasting pyloric compliance in gastroparesis and the therapeutic response to pyloric dilatation[END_REF] . We found a mean fasting pyloric distensibility of 10.04  1.67 at 40 mL in our cohort of patients with gastroparesis, with similar findings as previous studies, 10 11 which were performed with the patients under local or general anaesthesia. Only fasting pyloric compliance can be measured because of technical feasibility and the possibility of adverse events. This fasting measure is the only pyloric compliance measurement available in the literature to our knowledge. Nineteen out of 35 patients had decreased fasting pyloric distensibility in our study, using the cut-off of 10 mm 2 /mm Hg, as previously demonstrated [START_REF] Gourcerol | Impaired fasting pyloric compliance in gastroparesis and the therapeutic response to pyloric dilatation[END_REF] . This cut-off was fixed on the basis of a fasting pyloric distensibility measure in healthy volunteers in this study, using 90% percentile of results on fasting pyloric distensibility at 40 mL of inflation. Our results on symptom scores, quality of life and gastric emptying were similar when the threshold was set at 8 and 9 mm 2 /mm Hg at 40 mL inflation (data not shown). Moreover, we found a correlation between the results of pyloric distensibility with bag volume set at 40 mL and at 50 mL (95% CI, 0.7407-0.9340; p<0.0001).

A limitation of our study is the number of missing data (Supplementary Figure 1), with a high number of patients who declined to fill questionnaires (7/35) or who did not have the [START_REF] Sharma | Pyloric intrasphincteric botulinum toxin (Botox) improved symptoms and gastric emptying in a patient with diabetic gastroparesis (abstr)[END_REF] C-Octanoic acid breath-test (7/35). It has to be underlined that intrapyloric BT injections for refractory gastroparesis are not yet reimbursed and that our hospital accepted financial support in exceptional cases. However, even if a small number of patients were included, a significant difference was shown on both symptomatic response and gastric emptying, revealing efficient statistical power. In addition, in previous randomized controlled placebo studies, the number of patients included was similar . The BT injection has local neurolytic effects and its duration of response is probably limited by axonal regeneration of cholinergic neurons. Even if a 3-month evaluation can be considered as premature, there is often a decreased effect of BT after this time. Moreover, in the same randomized controlled trials, the evaluation was performed earlier after only 1 month of follow-up.

We did not use Gastroparesis Cardinal Score Index (GCSI) [START_REF] Revicki | Gastroparesis Cardinal Symptom Index (GCSI): development and validation of a patient reported assessment of severity of gastroparesis symptoms[END_REF] in our study because to our knowledge, this score has not yet been validated in French at the time we started the study. In addition, the TSS 28-29-10 is based on the same items as the GCSI, and only the likert scale and the nausea/vomiting subscale which is composed of nausea, retching, and vomiting differs from TSS. In fact, the literal translation of retching in French is similar to that of nausea. We did not use the Patient Assessment of Upper GastroIntestinal Disorders-Quality of Life (PAGI-QOL) questionnaire [START_REF] Loge C De La | Cross-cultural development and validation of a patient self-administered questionnaire to assess quality of life in upper gastrointestinal disorders: the PAGI-QOL[END_REF] . This questionnaire was developed to assess quality of life in upper gastrointestinal disorders, but the number of patients with gastroparesis was low (only 28) with none French patients in this subgroup. Each item in the PAGI-QOL is graduated in a 6-point scale, with difficulty of translation in French between them. On the contrary, the GIQLI score has been validated in French [START_REF] Slim | First validation of the French version of the Gastrointestinal Quality of Life Index (GIQLI)][END_REF] and uses a 5-point scale for each item, even if less specified for upper gastrointestinal disorders. Last, all questionnaires were answered prospectively by all patients at the time of evaluation.

CONCLUSION:

In the present study, we have shown that pyloric distensibility measurement before intrapyloric BT injection predicted symptomatic and quality of life response 3 months after intrapyloric BT injection in patients with gastroparesis. Further confirmatory studies are needed to verify whether this marker is relevant to predict outcome of gastroparesis treatment and better select patients for pyloric targeting therapeutics. Table 1. Patients characteristics before intrapyloric botulinum toxin injection. BT: botulinum toxin. GE: gastric emptying.

FIGURES LEGENDS:

Normal pyloric distensibility

Altered pyloric distensibility

Vomiting

  emptying half-time was decreased from 223 (range 157-282) minutes to 190 (range 151-242) minutes at 3 months after intrapyloric botulinum toxin injection (p=0.02) in patients with altered pyloric distensibility. In contrast, in patients with normal pyloric distensibility, gastric emptying half-time remained relatively unchanged from 214 (range 193-240) minutes to 210

Figure 1 .

 1 Figure 1. Representative recording of fasting pyloric distensibility in one patient with altered pyloric distensibility of 6.3 mm 2 /mm Hg (A) and in one patient with altered pyloric distensibility of 13.4 mm 2 /mm Hg (B). Measurements were made at 40 mL of

Figure 2 .

 2 Figure 2. Evolution of TSS at 3 months after intrapyloric botulinum toxin injection in

Figure 3 .

 3 Figure 3. Evolution of GIQLI at 3 months after intrapyloric botulinum toxin injection in

Figure 4 .

 4 Figure 4. Evolution of solid half-time rate (T 1/2 ) for gastric emptying at 3 months after

  mass index; TSS, Total symptomatic score; GIQLI, gastrointestinal quality of life index.

Table 2 .Figure S1 .

 2S1 Figure S1. Flowchart of patients who had intrapyloric botulinum toxin injection.

Acknowledgements: The authors are grateful to Nikki Sabourin-Gibbs, Rouen University Hospital, for editing the manuscript.

Conflicts of interest:

Guillaume Gourcerol received consulting fees from Allergan. The other authors have no conflict of interest to declare.