domain: sde phys sdu.ocean

text:
Irene Reinares Martínez

S Evan

F G Wienhold

J Brioude

E J Jensen

T D Thornberry

D Héron

B Verreyken

S Körner

H Vömel

J.-M Metzger

F Posny

Unprecedented Observations of a Nascent in Situ Cirrus in the Tropical Tropopause Layer

Homogeneous formation of tiny ice crystals (<1 µm) is due to a short vertical scale gravity wave at the tropopause • Such optically thin cirrus clouds would be missed by current aircraft instruments/remote sensing systems

Introduction

In situ cirrus formation in the tropical tropopause layer (TTL, Fueglistaler et al., 2009) occurs as rising air masses reach this extremely cold region [START_REF] Brewer | Evidence for a world circulation provided by the measurements of helium and water vapour distribution in the stratosphere[END_REF][START_REF] Jensen | Dehydration of the upper troposphere and lower stratosphere by subvisible cirrus clouds near the tropical tropopause[END_REF][START_REF] Jensen | Transport and freeze-drying in the tropical tropopause layer[END_REF]. Cirrus formation and sedimentation contribute to water vapor transport in the TTL, and its entrance value into the low stratosphere, where it has a non-negligible greenhouse effect (Forster & Shine, 2002;[START_REF] Solomon | Contributions of stratospheric water vapor to decadal changes in the rate of global warming[END_REF][START_REF] Dessler | Stratospheric water vapor feedback[END_REF]. However, the interaction between different-scale dynamical processes driving temperature anomalies in the TTL and the unknown composition of the TTL render the understanding of the cirrus formation very challenging.

Cirrus formation can occur either by homogeneous or heterogeneous ice nucleation mechanisms. For homogeneous freezing of aqueous aerosols, occurring at temperatures below -38 • C, theoretical models (e.g., [START_REF] Kärcher | A parameterization of cirrus cloud formation: Homogeneous freezing of supercooled aerosols[END_REF] predict that ice concentrations depend on the cooling rate, rather than on the concentration of aqueous solution droplets, which is rarely a limiting factor. Homogeneous nucleation generally requires conditions of supersaturation above 160% for TTL temperatures [START_REF] Koop | Water activity as the determinant for homogeneous ice nucleation in aqueous solutions[END_REF].

On the other hand, heterogeneous nucleation depends strongly on the population of ice forming nuclei (IFN, [START_REF] Cziczo | Clarifying the dominant sources and mechanisms of cirrus cloud formation[END_REF]Jensen et al., 2016b), which can be active at much lower supersaturations, but whose abundance is typically limited.

Observations of cirrus in the TTL show that ice concentrations are generally below 100 L -1 (e.g., [START_REF] Krämer | Ice supersaturations and cirrus cloud crystal numbers[END_REF][START_REF] Woods | Microphysical properties of tropical tropopause layer cirrus[END_REF]. In their recent climatology, [START_REF] Krämer | A microphysics guide to cirrus -Part 2: Climatologies of clouds and humidity from observations[END_REF] find median values of ice concentration between 10 and 100 L -1 .

These values are well below expected numbers for purely homogeneous nucleation. To explain these results, it has been suggested that the main mechanism for low-concentration cirrus formation is heterogeneous nucleation [START_REF] Cziczo | Clarifying the dominant sources and mechanisms of cirrus cloud formation[END_REF], which is constrained by IFN concentrations. Experimental results reported by [START_REF] Demott | Measurements of the concentration and composition of nuclei for cirrus formation[END_REF] show median values of heterogeneous IFN concentrations of ∼10 L -1 in the free troposphere. Similar measurements have not been made in the TTL, although lower concentrations are expected (Froyd et al., 2009). Several studies have shown, using a box model approach, that low ice concentrations can be generated by homogeneous nucleation if the cooling driving the supersaturation is replaced by warming within the duration of the nucleation event. Such cooling/warming can be attributed to fast changes of phase in high-frequency gravity waves [START_REF] Jensen | Ice nucleation and cloud microphysical properties in tropical tropopause layer cirrus[END_REF][START_REF] Spichtinger | Tropical tropopause ice clouds: a dynamic approach to the mystery of low crystal numbers[END_REF][START_REF] Dinh | Effect of gravity wave temperature fluctuations on homogeneous ice nucleation in the tropical tropopause layer[END_REF]. However, Jensen et al. (2016a) argued that the overall impact of the high-frequency waves is to increase ice concentrations produced by homogeneous freezing; the quenching of ice nucleation would only represent 11% of the events. In some infrequent cases, TTL cirrus have been observed with ice concentrations above several thousands per liter [START_REF] Jensen | Ice nucleation and dehydration in the tropical tropopause layer[END_REF][START_REF] Krämer | A microphysics guide to cirrus -Part 2: Climatologies of clouds and humidity from observations[END_REF], in which case homogeneous nucleation is the only plausible explanation.

High altitude cirrus are common over tropical oceans, and occur with highest frequency over the central and western Pacific, followed by the Indian Ocean [START_REF] Sassen | Global distribution of cirrus clouds from Cloudsat/Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO) measurements[END_REF][START_REF] Massie | HIRDLS and CALIPSO observations of tropical cirrus[END_REF]. Instrumented aircraft missions from programs like CRAVE (Costa Rica Aura Validation Experiment, e.g., [START_REF] Lawson | Aircraft measurements of microphysical properties of subvisible cirrus in the tropical tropopause layer[END_REF][START_REF] Jensen | Formation of large ( 100 µm) ice crystals near the tropical tropopause[END_REF], TWP-ICE (Tropical Warm Pool International Cloud Experiment, e.g., McFarquhar et al., 2007), ATTREX (Airborne Tropical TRopopause EXperiment, Jensen et al., 2017), POSIDON (Pacific Oxidants, Sulfur, Ice, Dehydration, and Convection Experiment, Jensen et al., 2018) and StratoClim [START_REF] Krämer | A microphysics guide to cirrus -Part 2: Climatologies of clouds and humidity from observations[END_REF] have provided valuable in situ observations of TTL cirrus, but have been so far mainly restricted to the Pacific Ocean or the Asian monsoon system (in the case of StratoClim). However, little effort has been dedicated to the undersampled southwestern Indian Ocean, despite the TTL cirrus occurrence reaching up to 60% during austral summer [START_REF] Sassen | Global distribution of cirrus clouds from Cloudsat/Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO) measurements[END_REF].

The aim of this study is to document a singular case of in situ TTL cirrus forma- The COBALD backscatter instrument, developed at ETHZ (Swiss Federal Institute of Technology Zurich), uses two light-emitting diodes at two wavelengths centered at 455 and 940 nm (blue and infrared, respectively) to detect and characterize cloud and aerosol particles in the atmosphere (e.g., [START_REF] Brabec | Particle backscatter and relative humidity measured across cirrus clouds and comparison with microphysical cirrus modelling[END_REF][START_REF] Brunamonti | Balloon-borne measurements of temperature, water vapor, ozone and aerosol backscatter on the southern slopes of the Himalayas during StratoClim 2016-2017[END_REF].

The detector has a field of view of ±6 • , and the signal detected typically originates from a distance of 0.5 to 10 m from the sonde. Beyond 10 m, the signal becomes negligible.

For each of the two COBALD wavelengths, the COBALD raw signal is converted to backscatter ratio (BSR), which is defined as:

BSR = β tot β mol = β mol + β part β mol = 1 + P BSR (1)
where P BSR is the particle backscatter ratio; β tot is the measured backscatter coefficient, expressed as the sum of the molecular (β mol ) and particle (β part ) contributions.

The particle backscatter coefficient includes aerosols, which are mainly sulfates in the TTL or lower stratosphere, and ice crystals (β ice ). The molecular backscatter coefficient is computed according to [START_REF] Bucholtz | Rayleigh-scattering calculations for the terrestrial atmosphere[END_REF], for stratospheric conditions, corresponding to 100 hPa and 203 K. The precision of the BSR resulting from the COBALD post-processing is around 1% in the TTL with an absolute accuracy of better than 5% [START_REF] Vernier | Increase in upper tropospheric and lower stratospheric aerosol levels and its potential connection with Asian pollution[END_REF].

The color index (CI) is defined as the ratio of the particle backscatter ratios at the infrared and blue wavelengths:

CI = P BSR 940 P BSR 455 (2)
which is independent of number concentration but depends on the size of the particles.

The CFH, developed by [START_REF] Vömel | Accuracy of tropospheric and stratospheric water vapor measurements by the cryogenic frost point hygrometer: Instrumental details and observations[END_REF], provides accurate water vapor measurements from the surface to the middle stratosphere. The instrument measures the frost point temperature, which combined with temperature measurements from the iMet-1-RSB allows water vapor mixing ratio and supersaturation (S i ) to be calculated using a saturation vapour pressure formula such as [START_REF] Murphy | Review of the vapour pressures of ice and supercooled water for atmospheric applications[END_REF] -see their equation (7)-. CFH water vapor mixing ratio uncertainties are 5% in the tropical troposphere [START_REF] Vömel | Accuracy of tropospheric and stratospheric water vapor measurements by the cryogenic frost point hygrometer: Instrumental details and observations[END_REF]) and 2-3% in the stratosphere [START_REF] Vömel | An update on the uncertainties of water vapor measurements using cryogenic frost point hygrometers[END_REF]. At the one per second telemetry data rate, the CFH and COBALD have a high vertical resolution of 2-3 m with a mean balloon ascent rate of 2-3 m s -1 on 11 January 2019. We use the pressure measured by Intermet iMet-1-RSB as the main vertical coordinate for all instruments.

All variables are binned in pressure intervals of 1 hPa (corresponding to a vertical resolution of ∼25 m in the TTL) to reduce measurement noise [START_REF] Brunamonti | Balloon-borne measurements of temperature, water vapor, ozone and aerosol backscatter on the southern slopes of the Himalayas during StratoClim 2016-2017[END_REF].

FLEXPART Lagrangian model

The origin of air masses sampled at the Maïdo Observatory is assessed using the FLEXible PARTicle (FLEXPART) Lagrangian Particle Dispersion Model [START_REF] Stohl | Technical note: The lagrangian particle dispersion model FLEXPART version 6.2[END_REF]. This transport model is run backward in time and is driven using European Centre for Medium Range Weather Forecasts -Integrated Forecast System (ECMWF-IFS) analysis (at 00, 12 UTC) and hourly forecast fields. To compute the FLEXPART trajectories, the ECMWF meteorological fields are retrieved at 0.50 • and 0.15 • and on full model levels (137 vertical model levels with a top at 0.01 hPa). Further details on the model set-up are described in [START_REF] Evan | A real-time global half-hourly pixel-resolution infrared dataset and its applications[END_REF]. Here, 100,000 air parcels were distributed randomly within boxes with a depth of 500 m and 0.10 • ×0.10 • longitude-latitude bins centered on the balloon at the cold-point tropopause when the cirrus was observed.

Microphysical and optical modeling

To estimate the microphysical properties of the cirrus from the COBALD backscatter ratios, we model the backscatter signal in a similar way as in the Zürich Optical and Microphysical Model [START_REF] Brabec | Particle backscatter and relative humidity measured across cirrus clouds and comparison with microphysical cirrus modelling[END_REF][START_REF] Cirisan | Balloon-borne match measurements of midlatitude cirrus clouds[END_REF]. A lognormal distribution of spherical ice crystals population is assumed, following [START_REF] Deshler | Thirty years of in situ stratospheric aerosol size distribution measurements from Laramie, Wyoming (41 o N), using balloon-borne instruments[END_REF]:

n(ln r) = N 0 √ 2π ln σ exp   -ln 2 r r M 2 ln 2 σ   (3)
where r M is the mode radius, σ is the geometric standard deviation parameterizing the distribution width and N 0 is the total number concentration. This size distribution possesses three degrees of freedom, r M , σ and N 0 , thus it cannot be fully constrained by the two COBALD measurements (BSR 455 and BSR 940 ). Assuming the crystals sphericity is a fair approximation under TTL conditions [START_REF] Woods | Microphysical properties of tropical tropopause layer cirrus[END_REF].

The backscatter coefficient from this distribution is evaluated as:

β ice = Q sc (x)πr 2 n(ln r)d ln r (4)
where Q sc (x) is the efficiency of the backward scattering to the ±6 • COBALD field of view for a single ice crystal, depending on the size parameter x = 2πr/λ. It is multiplied by the ice crystal geometric cross section (πr 2 for a sphere) and summed over the size distribution.

The optical calculus is based on Mie code tabulations to simulate backscatter coefficients. For ice particles, the refractive index is set to 1.31. For each wavelength, the backscatter efficiency Q sc is computed for varying r in intervals of d ln r = 0.0023. Then, by setting N 0 = 1 cm -3 , equation ( 4) provides the backscatter coefficient for a size distribution of one particle per cubic centimeter with a mode radius r M and width σ. The P BSR is computed by dividing β ice by the molecular backscatter coefficient, taken again from [START_REF] Bucholtz | Rayleigh-scattering calculations for the terrestrial atmosphere[END_REF]. For mode radii below 1 µm (in the blue) and below 2 µm (in the infrared), the backscatter coefficient weighted by the distribution (i.e., calculated as in equation ( 4) but without the cross section term) is found to linearly track r M . Up to these mode radius limits, the P BSR scales with the particle volume, relating it directly to the ice water content. The ratio of the infrared to the blue P BSR results in the CI, see equation (2), as function of the mode radius for each size distribution width. Without additional constraints on the particle distribution, it is difficult to identify uniquely r M from a given CI even for a single σ. This limitation is inherent in the computations of the backscatter coefficients using Mie calculations. With additional constraints, however, provided by a priori knowledge of the size distributions or microphysical considerations, a mode radius can be inferred by comparing the measured CI with the simulated CI for a prescribed σ. This finally allows deduction of the ice number concentration and the ice water content (IWC) as follows.

For each wavelength and prescribed r M and σ, the total particle number concentration N 0 is found by scaling the ice particle concentration leading to P BSR = 1 to the observed backscatter. That scaling factor is provided by the ratio of molecular backscatter coefficient from [START_REF] Bucholtz | Rayleigh-scattering calculations for the terrestrial atmosphere[END_REF] to the result from equation ( 4) with N 0 = 1 cm -3 (as mentioned above). With this scaled N 0 the ice water content (IWC) is computed as:

IW C = 4π 3 r 3 M N 0 ρ ice (5)
using ρ ice = 0.917 × 10 6 g m -3 .

3 Results and Discussion event that would contribute to the observed cooling/supersaturation. A mean temperature profile for January 2019 was computed using 14 radiosonde temperature profiles measured at the Maïdo Observatory from 7 to 19 January 2019. This allows the computation of the temperature anomaly (Figure 1). The characteristics of the temperature and vertical speed perturbations, most likely due to a GW event, are analyzed in Section 3.4.

Origin of the sampled air masses

Using FLEXPART, a retroplume consisting of 100,000 air parcels is released at 16.5 km altitude and 19:40 UTC, corresponding to the cirrus observation during the ascent.

The trajectories, simulated back to 00 UTC 11 January 2019, are shown on Figure 2. Almost all the backtrajectories had their origin between 15 and 17 km. The absence of deep convective clouds along the trajectories further implies that they had not experienced deep convection, which would be consistent with in situ cirrus formation.

Air masses had been advected from the south-southeast region of Réunion Island with a high moisture content, according to the ECMWF analyses (not shown). They remained within the TTL over the southwestern Indian Ocean for the full previous day (not shown).

Microphysical properties of the observed TTL cirrus

The CI for ice and for the background sulfate aerosols is displayed in Figure 3 These values are typical of stratospheric sulfates [START_REF] Pinnick | Stratospheric aerosol measurements III: Optical model calculations[END_REF][START_REF] Rosen | Backscattersonde: a new instrument for atmospheric aerosol research[END_REF]. The radius dependence of the CI is remarkably different between ice and sulfate particles. For ice, the particular dependence on σ and r M between 0.5 and 2 µm is used to constrain parameters of the size distribution. At the ascent, the CI from COBALD measurements was 7.3 (Figure 1). The CI for ice, which is estimated from subtracting the background sulfates signal, is 6.9 and can be as low/high as 6.5/7.4 when accounting for the BSR uncertainty. The observed backscatter cannot be produced with particles smaller than 0.25 µm in size. Therefore, the upper CI measurement limit implies σ to be less than 1.8 (the width for the sulfate aerosols) and a mode radius of at least 0.5 µm. From the observed CI of 16 at the descent, the same analysis yields a CI for ice of 15.4 and a possible range of values from 14.4 to 16.5. These ranges of CI values are shown as blue lines in Figure 3. A single solution for the mode radius does not exist from the obtained values of CI, even when the width of the distribution is prescribed, due to the oscillations of the simulated CI. The dip that manifests for ice slightly above 0.5 µm for a width of 1.6 sharpens and moves toward larger size as the distribution width reduces. This change of the mode that constitutes the COBALD signal typically occurs during the initial ice particles growth phase.

This unique feature in CI significantly constrains the particle radii. For σ = 1.3-1.6 the mode radius can be constrained to 0.5-1.1 µm during ascent and to 1.1-1.6 µm during descent.

The ice number concentration and IWC are deduced from the obtained mode radius for a lognormal distribution with σ = 1.6. They are estimated to be 520 L -1 and 0.43 µg m -3 during the ascent and 42 L -1 and 1.3 µg m -3 during the descent. The uncertainties of the estimated N 0 and IW C highly depend on the P BSR contribution to the BSR. In this case, the uncertainties range between 10 and 20%. The ice number concentration is reduced by a factor of more than 10 between the ascent and the descent.

Heterogeneity of microphysical properties encountered inside cirrus clouds can be explained by small-scale GWs [START_REF] Jensen | Ice nucleation and dehydration in the tropical tropopause layer[END_REF]. The climatology from [START_REF] Krämer | A microphysics guide to cirrus -Part 2: Climatologies of clouds and humidity from observations[END_REF] shows (in their Figure 8) that ice concentrations (for radius above 3 µm) in young cirrus are most frequently around 100 L -1 . Although cirrus with ice concentrations above 10,000 L -1 have already been observed [START_REF] Krämer | Ice supersaturations and cirrus cloud crystal numbers[END_REF][START_REF] Jensen | On the importance of small ice crystals in tropical anvil cirrus[END_REF][START_REF] Krämer | A microphysics guide to cirrus -Part 2: Climatologies of clouds and humidity from observations[END_REF], only less than 10% of the observed TTL ice concentrations exceed 1,000 L -1 [START_REF] Krämer | A microphysics guide to cirrus -Part 2: Climatologies of clouds and humidity from observations[END_REF]. The estimated IWC values have been previously reported in the same temperature range from in situ retrievals, but lie in the lower edge of the observed range [START_REF] Krämer | A microphysics guide to cirrus clouds -Part 1: Cirrus types[END_REF][START_REF] Krämer | A microphysics guide to cirrus -Part 2: Climatologies of clouds and humidity from observations[END_REF]. During ATTREX 2014 over the western Pacific, direct measurements of IWC were limited to 1 µg m -3 . The cloud probes used could only detect ice crystals with sizes above 1 µm (FCDP probe) or 10 µm (2D-S probe, Thornberry et al. ( 2017)). Twenty-seconds average of the FCDP data from ATTREX and POSIDON campaigns allows the sampling of larger volumes of air, leading to IWC below 0.01 µg m -3 . This indicates that while our measurements are sensible, the values found are very rare. The optical depth of the cirrus layer (τ cirrus ) is further computed. The uncertainty is much higher in the blue wavelength (90%) compared to the infrared (4-5%), as for the shorter wavelength the molecular Rayleigh scattering is 20 times stronger and the marginal particle scattering on top is much more affected by the observed error. In the infrared, the values of τ cirrus are roughly 1-2×10 -4 (see supporting information S1 and Figure S1 for details). With these values of the optical depth, the observed cirrus is beyond satellite detection capabilities. For example, for current CALIPSO (Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observation) V4 sensitivity, the optical depth is barely below 0.003 for night detection (Melody Avery, personal communication).

Analysis of the CI indicate the ice crystals are larger during the descent. Furthermore, the high supersaturation observed by the CFH (1.5) indicates that the environment could support the nucleation and initial growth of ice crystals. A growth calculation is performed to verify the consistency of the ice crystal size change. Neglecting the time dependence of S i and T , the growth rate of the ice crystals is estimated following [START_REF] Wallace | Atmospheric science: an introductory survey[END_REF]:

dm dt = 4πC(S i -1) f (T ) (6)
with

f (T ) = L s R v T -1 L s κT + R v T e i (T )D (7)
where m is the mass of the ice crystal, κ = 2.4 × 10 -2 J m -1 s -1 K -1 the thermal conductivity of air at 0 • C (independent of pressure), D = 2 × 10 -5 m 2 s -1 the diffusion coefficient of water vapor in air and L s = 2.85 × 10 6 J kg -1 the gas constant for water vapor. e i (T ) is computed using [START_REF] Murphy | Review of the vapour pressures of ice and supercooled water for atmospheric applications[END_REF] equation ( 7).

For a spherical particle, C equals the radius of the crystal. Using dm/dt = 4πρ ice r 2 dr/dt with ρ ice = 0.917 × 10 6 g m -3 equation ( 6) yields:

r(T ) = 2 ρ ice (S i -1) f (T ) t + r 2 0 ( 8
)
where S i = 1.5, T = 192.6 K, t = 93 min and r 0 = 0.6 µm (the estimate from COBALD in the ascent). This computation yields a radius of 6 µm. This value is on the same order of magnitude than COBALD estimates for the descent (1.1-1.6 µm, see Figure 3).

This value is on the same order of magnitude than COBALD estimates for the descent.

Moreover, given that the supersaturation was kept constant for the growth rate calculation, the radius of 6 µm would be an upper limit. The radius obtained from the growth rate calculation is consistent with the hypothesis that the crystals size increased between the COBALD observations at ascent and descent. Given the difference in the two methods used, the agreement in the radius found is reasonable. In reality, the COBALD sampled cirrus regions separated by 88 km (73 km when accounting for the advection). Some matching technique, like the one developed by [START_REF] Cirisan | Balloon-borne match measurements of midlatitude cirrus clouds[END_REF], would be needed to sample the same cirrus twice. Given the ubiquitous small-scale structure in TTL cirrus, it is challenging to relate the cloud properties at two different times and locations.

Possible mechanisms of cirrus formation

Wavelet analysis using S-transforms [START_REF] Stockwell | Localization of the complex spectrum: the S transform[END_REF] is applied to the temperature anomaly (T ) and the ascent rate (w) to deduce the vertical structure of the gravity wave event that most likely triggered the perturbations. This analysis yields a vertical wavelength of 1.5 km for a wave localized near the cold-point tropopause, for both T and w (see supporting information S2 and Figures S2 to S4 for details on the spectral analysis). The value of 1.5 km agrees with the vertical structure of GWs previously characterized over the tropics with the use of radiosondes. Using 6-hourly radiosonde data, [START_REF] Tsuda | Radiosonde observations of equatorial atmosphere dynamics over Indonesia: 2. Characteristics of gravity waves[END_REF] reported GWs with vertical wavelengths ranging from 1 to 4 km in the TTL, with a mean value of 2.5 km. Similarly, using 12-hourly radiosonde data in the equatorial western Pacific, [START_REF] Kim | Direct impacts of waves on tropical cold point tropopause temperature[END_REF] found gravity waves with vertical wavelenghts shorter than 2 km (value averaged over a 3-month period, cf. their Figure 5). The amplitude of the perturbations at the cold-point tropopause are estimated to be 1 K and 0.5 m s -1 in temperature and vertical speed, respectively. The amplitude of the temperature perturbation is consistent with [START_REF] Kim | A new wave scheme for trajectory simulations of stratospheric water vapor[END_REF]. They found that only 13% of the tropical temperature anomalies due to waves with less than a 3day period at 17 km altitude had amplitudes larger than 2 K. In addition, [START_REF] Kim | Direct impacts of waves on tropical cold point tropopause temperature[END_REF] determined that those waves with periods between 1 and 3 days accounted for a mean decrease of the CPT of ∼0.6 K. Theoretical or box model predictions of ice concentration at comparable temperature generally lead to higher ice concentrations (up to 10,000 L -1 ) compared to our results (∼500 L -1 ) for the same vertical wind speed (e.g., [START_REF] Kärcher | A parameterization of cirrus cloud formation: Homogeneous freezing of supercooled aerosols[END_REF][START_REF] Krämer | A microphysics guide to cirrus clouds -Part 1: Cirrus types[END_REF]. For our young cirrus case this difference cannot be explained by sedimentation or the entrainment of surrounding air (which might become important in longer-lived cirrus). Probably, the cirrus observed had not reached the peak ice concentration that would be produced in a persistent updraft because of the truncation of ice nucleation by the phase change of the gravity wave [START_REF] Jensen | Ice nucleation and cloud microphysical properties in tropical tropopause layer cirrus[END_REF][START_REF] Spichtinger | Tropical tropopause ice clouds: a dynamic approach to the mystery of low crystal numbers[END_REF][START_REF] Dinh | Effect of gravity wave temperature fluctuations on homogeneous ice nucleation in the tropical tropopause layer[END_REF].

These vertical wind speed and temperature anomalies at the cold-point tropopause are most likely produced by a convectively generated gravity wave, although different convective sources could have generated the wave. For example, large-scale organized convection over Madagascar was very active a few hours before the observation (Figure 2)

and also small-scale oceanic storms developped to the west of Réunion Island during the evening (not shown). To determine the source of the gravity wave, a full ray-tracing analysis of the wave is required (e.g., [START_REF] Evan | Model study of intermediatescale tropical inertia-gravity waves and comparison to TWP-ICE campaign observations[END_REF]. To perform such an analysis, the complete spectral characteristics (horizontal/vertical wavelengths, intrinsic frequency)

of the wave would be needed, but this is not possible with a single observation.

Summary and Conclusion

The first observational study of a nascent TTL cirrus over the southwestern Indian Ocean is presented. On 11 January 2019, a balloon was launched from Réunion Island.

The retrieved data (backscatter ratio from COBALD, water vapor content and supersaturation from CFH) allow the characterization of the cirrus and environmental formation conditions. The cirrus is observed at the cold-point tropopause, at ∼16.5 km altitude. Backtrajectories from the FLEXPART Lagrangian model and the distribution of deep convection from Meteosat8 show that the air masses sampled by the instruments at the tropopause had remained over the ocean in the TTL and had not experienced any convection during the previous day.

The color indexes for ice and sulfate aerosols are simulated with an optical model for a set of lognormal (spherical) particle size distributions. The COBALD measurements for ice are compared with the simulations to constrain the parameters of the ice crystals distribution. Overeall, ice crystal radii are estimated to be smaller than 1 µm during the ascent. For a radius of 0.6 µm, the ice water content is 0.43 µg m -3 . The observed supersaturation (1.5) and estimated ice concentration (520 L -1 ) are consistent with homogeneous ice formation if the temperature tendency changes during the nucleation event, which can truncate the process and limit the ice concentration (e.g., [START_REF] Spichtinger | Tropical tropopause ice clouds: a dynamic approach to the mystery of low crystal numbers[END_REF][START_REF] Dinh | Effect of gravity wave temperature fluctuations on homogeneous ice nucleation in the tropical tropopause layer[END_REF]. Spectral analysis of concomitant observed temperature and vertical wind speed anomalies observed indicates that the nucleation event was most likely triggered by a gravity wave event of 1 K and 0.5 m s -1 amplitudes, respectively. The vertical wavelength is estimated to be 1.5 km for both perturbations. Such short vertical scale GW event and subsequent cooling/cirrus formation would be difficult to represent in most current numerical weather prediction and climate models with coarse vertical resolution in the TTL/lower stratosphere.

This study presents unique and rare observations of a relatively low concentration, very optically thin cirrus (τ cirrus ∼10 -4 ). It might result from the quenching of the nucleation event by a high-frequency wave. Such cirrus are difficult to measure remotely or in situ by current instrumented aircraft. Thus, the occurrence frequency of these thin cirrus could be greatly underestimated in remote sensing climatologies. This emphasizes the need for a long-term monitoring system of the TTL composition, with this kind of CFH-COBALD joint measurements performed here, especially in tropical oceanic regions with scarce TTL in situ measurements [START_REF] Müller | The need for accurate long-term measurements of water vapor in the upper troposphere and lower stratosphere with global coverage[END_REF]. Adding an ozone sonde would also be valuable to better identify air mass origins. To further assess the mechanisms controling this specific cirrus case numerical modeling is needed.

  tion around Réunion Island, in the southwestern Indian Ocean. A set of balloon-borne observations, Lagrangian trajectories and microphysical and optical calculations are presented and used to characterize the microphysical properties and the environment of the observed cirrus. The measurements presented in this study were taken in the framework of the CONCIRTO (CONvection CIRrus tropical Tropopause layer over the indian Ocean) project. This project aims to further our understanding of deep convection and cirrus clouds and how they affect the TTL over the Indian Ocean. It funded coincidental highresolution balloon-borne in situ measurements of water vapor, ozone and aerosol/ice particles in austral summer 2019 (January-March). In this study, balloon-borne observations of a nascent in situ cirrus encountered on 11 January 2019 are analyzed and the results are interpreted in terms of the different cirrus formation mechanisms. 2 Data and Methods 2.1 Balloon-borne in situ observations In order to characterize the nascent TTL cirrus cloud and its environment, a COBALD (Compact Optical Backscatter and AerosoL Detector) and CFH (Cryogenic Frost Point Hygrometer) instrument were flown on the same payload on 11 January 2019. Data from the COBALD and CFH were telemetered to the ground by an Intermet iMet-1-RSB meteorological radiosonde, which additionally provided measurements of ambient pressure, temperature and wind speed and direction. The sonde was launched at 17:54 UTC from the Maïdo observatory (21.1 • S,55.4 • E), located on Réunion Island, at 2160 m above sea level.

3. 1

 1 Observed cirrus eventThe balloon was launched at 17:54 UTC and reached the cold-point temperature (CPT) of 192.6 K at approximately 16.5 km altitude at 19:40 UTC. The vertical profiles of temperature, ascent rate and saturation ratio with respect to ice (S i ) retrieved

Figure 1 .

 1 Figure 1. Vertical profiles measured during the sounding at Maïdo observatory on 11 January 2019 at 17:54 UTC. Left: profiles of temperature (black), temperature perturbation (blue) and ascent rate (yellow). Right: profiles of saturation ratio with respect to ice (pink), backscatter ratios at 455 and 940 nm for the ascent (dark blue and red, respectively), and for the descent (cyan and brown, respectively) and color index at ascent/desent (light/dark green). The black dashed line corresponds to the cold point tropopause at ∼16.5 km altitude.

Figure 2 .

 2 Figure 2. Backward trajectories from the FLEXPART model. They were initialized for the 16.25-16.75 km layer on 11 January 2019, 19:40 UTC around cirrus observation at the ascent of the balloon at the cold-point tropopause. The particle positions (pink dots) and Meteosat8 brightness temperature are shown on 11 January 2019 at 13:00 UTC, meaning that ∼7 hours before the observation the particles were located in the 15-17 km altitude range.

  as function of the mode radius. To simulate the CI for ice, three different size distribution widths are used (σ = 1.3-1.6) and a refractive index of 1.31. The simulated CI for sulfate aerosols is shown for a single width (σ = 1.8) and refractive indexes of 1.45 and 1.51, which correspond to different H 2 SO 4 and H 2 O proportions of the aerosols composition.

Figure 3 .

 3 Figure 3. Color index versus mode radius of the ice crystals (green) and sulfate aerosols (pink). For ice, the refractive index (n) is set to 1.31, and three different widths (σ = 1.3, 1.4 and 1.6) of the lognormal size distribution are used. For the sulfate aerosols, two values of the refractive index (n = 1.45, 1.51) are used to simulate the color index for σ = 1.8. The blue horizontal lines indicate the CI uncertainty range for the ascent (lower lines) and descent (upper lines) observations.

  . M. d. F., & Shine, K. P. (2002). Assessing the climate impact of trends in stratospheric water vapor. Geophysical Research Letters, 29 (6), 10-1-10-4. doi: 10.1029/2001GL013909 Froyd, K. D., Murphy, D. M., Sanford, T. J., Thomson, D. S., Wilson, J. C.., Wienhold, F. G., Hoyle, C. R., Immler, F., & Peter, T. (2011). Modeling the ascent of sounding balloons: derivation of the vertical air motion. Atmospheric Measurement Techniques, 4 (10), 2235-2253. doi: 10.5194/amt-4-2235

Acknowledgments

The CONCIRTO project was funded by the Agence Nationale de la Recherche (ANR-17-CE01-0005-01). We thank OPAR (Observatoire de Physique de l'Atmosphère à La Réunion, including Maïdo Observatory). It is part of OSU-R (Observatoire des Sciences de l'Univers à La Réunion), which is funded by Université de la Réunion, CNRS-INSU,

Météo-France and the French research infrastructure ACTRIS-France (Aerosols, Clouds and Trace Gases Research Infrastructure).

Data from the sounding is available at https://lacy.univ-reunion.fr/activites/programmesde-recherche/anr-concirto/donnees. Meteosat8 data are accessible at https://disc.gsfc.nasa.gov/datasets/ GPM MERGIR 1/summary. The FLEXPART code is available at https://www.flexpart.eu/ and the ECMWF data can be accessed at https://apps.ecmwf.int/datasets/.

The authors thank the two anonymous reviewers for their useful comments.


Mu-Yi Chen

Xavier Pannecoucke

Philippe Jubault

Tatiana Besset

Pd-Catalyzed Selective Chlorination of Acrylamides at Room Temperature

ABSTRACT:

In this manuscript, the transition metal catalyzed chlorination of alkenes was reported. In the presence of the commercially available and inexpensive N-chlorosuccinimide and without additive, the Pd-catalyzed chlorination of acrylamides by C-H bond activation was developed at room temperature under air. Under these mild reaction conditions, the versatility of the methodology was demonstrated as an array of acrylamides was functionalized to selectively provide the corresponding difficult-to-synthesize chlorinated olefins as a single Z stereoisomer. Mechanistic studies were conducted to get insights into the reaction mechanism and post-functionalization reactions further demonstrated the synthetic utility of the approach towards the access to high value-added chlorinated compounds.

Chlorinated molecules are widely present in compounds of interest such as pharmaceuticals, agrochemicals and natural products. 1 Especially, halogenation of aromatic derivatives is at the forefront of innovation since they have widespread applications in organic chemistry. Therefore, the development of novel methods to access them has attracted a strong interest from the scientific community. [START_REF] Petrone | Modern Transition-Metal-Catalyzed Carbon-Halogen Bond Formation[END_REF] Among them, a special attention was paid to the transition metal catalyzed halogenation of aromatic derivatives by C-H bond activation with I, Br and Cl atoms. [START_REF] Patra | Group 9 Transition Metal-Catalyzed C-H Halogenations[END_REF] Indeed, transition metal catalyzed C-H bond functionalizations have completely reshaped the field of organic chemistry. [START_REF] Lyons | Palladium-Catalyzed Ligand-Directed C-H Functionalization Reactions[END_REF] This straightforward tool for molecular synthesis further opened the chemical space, enabling applications to various fields. [START_REF] Wang | Late-Stage Peptide Diversification by Position-Selective C-H Activation[END_REF] Especially, the transition metal catalyzed directed functionalization of the more challenging vinylic derivatives by C-H bond activation was well studied over the years, offering a straightforward and selective access to the difficult-to-synthesize Z isomer. [START_REF] Besset | Recent Progress in Direct Introduction of Fluorinated Groups on Alkenes and Alkynes by Means of C-H Bond Functionalization[END_REF] In sharp contrast, the halogenation of vinylic derivatives by C-H bond activation [START_REF] Besset | Recent Progress in Direct Introduction of Fluorinated Groups on Alkenes and Alkynes by Means of C-H Bond Functionalization[END_REF] still remains restricted to a handful of examples and requires to be further investigated as it offers a selective access to the challenging and ubiquitous Z olefinic moiety. The quest to efficient and selective tools towards the difficult-to-synthesize Z-olefins is of high importance, since this scaffold might be found in several bioactive compounds such as Selinexor®.

In the realm of transition metal catalyzed halogenation of olefins by C-H activation, bromination and iodination reactions were mainly studied using various transition metal catalysts and led to major contributions. In 2013, Glorius reported the Rh(III)catalyzed bromination and iodination of acrylamides [START_REF] Kuhl | Rh(III)-Catalyzed Halogenation of Vinylic C-H Bonds: Rapid and General Access to Z-Halo Acrylamides[END_REF] and one year later they depicted the iodination of acrylamides 8 via a Cp*Co(III) catalysis. In 2019, Carreira described the Pd-catalyzed iodination of unactivated olefins using picolamide as a directing group (Scheme 1). [START_REF] Schreib | Palladium-Catalyzed Regioselective C-H Iodination of Unactivated Alkenes[END_REF] However, the direct and regioselective synthesis of Z-chlorinated acrylic acid derivatives is still elusive despite the clear interest of the corresponding vinyl chloride compounds. 1 Therefore, the elaboration of a new synthetic approach towards these chlorinated molecules represents a significant challenge in modern organic chemistry and is still an unmet goal. Up to date, the existing methods to synthesize Z--chloroacrylic acid derivatives generally relied on a Vilsmeier-Haack reaction, [START_REF] Krishnaraj | Synthesis of 2-Hydroxy-5,6-Diarylnicotinonitriles and 2-Chloro-5,6-Diarylnicotinonitriles From[END_REF] a Wittig reaction, 11 a chloropalladation/Heck reaction sequence, 12 chlorination of propagyl alcohols, [START_REF] Trost | Vanadium-Catalyzed Synthesis of Geometrically Defined Acyclic Tri-and Tetrasubstituted Olefins From Propargyl Alcohols[END_REF] as well as hydrochlorination, 14 chloroacylation 15 and chlorocarbonylation 16 reactions of alkynes, among others. A lack of E/Z selectivity or the need to have the proper alkynes are the main synthetic limitations of these methods that are needed to be overcome. In addition, to meet the continuous demand for more sustainable transformations, the development of C-H activation reactions under mild conditions (no additives, room temperature) is still highly desirable. Keeping these considerations in mind and pursuing our current interest towards the functionalization of vinylic derivatives by C-H bond activation, 17 we report herein the unprecedented Pd(II)-catalyzed chlorination of various -and -substituted acrylamide derivatives at room temperature.

Scheme 1. State of the art and present work.

At the outset of the study, the Pd-catalyzed chlorination of the -phenylacrylamide 1a was studied (Table 1). In the presence of a stoichiometric amount of NCS and using PdCl2 as a catalyst, 1a was smoothly converted into the corresponding chlorinated product 2a in 85% yield under an air atmosphere in 8 h (Table 1, entry 1). The reaction turned out to be completely diastereoselective as a single Z-isomer was obtained as ascertained by 2D NMR experiments. 18 The replacement of PdCl2 by other copper-based catalysts (Table 1, entries 2 and 3) led to no expected product 2a, and only the product resulting from the chlorination at the C5 position of the 8-aminoquinoline part was observed. The catalyst loading was also important to selectively get the functionalization of the olefin. Indeed, when 5 mol% of the catalyst was used, the reaction furnished 2a in only 25% yield, along with the chlorinated product on the aminoquinoline part (Table 1, entry 4). A control experiment was conducted and in the absence of catalyst, 2a was not obtained and the same sidereaction was prominent ( a Isolated yields. All reactions were performed on a 0.2 mmol scale. b The product was obtained along with the product resulting from the chlorination at the C5 position of the 8-aminoquinoline part in 31% yield.

With the best reaction conditions in hand, an access to tri-substituted acrylamides was achieved via the Pd-catalyzed chlorination of an array of -arylsubstituted acrylamides (Scheme 2, 2a-2q). When 1a was used as the substrate, the reaction was efficiently 20-fold time scaled up, offering an access to about 1g of 2a (0.99 g, 80%) without erosion of the yield (85% on 0.2 mmol scale). The reaction turned out to be highly regio-and diastereo-selective to the expected product and only the mono-chlorination of the olefin part was observed. Several acrylamides bearing an arene at the -position substituted by an electrondonating (1b-e) group or halogens (1f-i) at the para position were chlorinated. The reaction was also tolerant to the trifluoromethyl group (2k). Meta and ortho substituted -aryl acrylamides (2j-2o) were chlorinated and the substitution pattern on the arenes did not have any impact on the efficiency of the transformation (similar yields for compounds 2b, 2j, 2m). Note that the structure of 2n was further confirmed by X-ray analysis (CCDC 2016282). 20 Acrylamides with disubstituted arenes (1p and 1q) were also suitable substrates in this transformation.

The methodology was successfully extended to the chlorination of the methacrylamide 1r leading to the corresponding product 2r in 53% yield.

Scheme 2. Scope of the chlorination reaction. Pleasingly, the approach was successfully applied to the functionalization of the -substituted acrylamides, the stereoselective access to tetrasubstituted acyclic olefin being still a synthetic challenge. , -Substituted acrylamides such as the dimethyl acrylamide (1s) and the cyclohex-1enecarboxamide (1t) were smoothly converted into the fully decorated olefins 2s and 2t. Even the amide derived from the 5-methoxy-8-aminoquinoline 1u was smoothly functionalized in a longer time.

To further illustrate the modularity of the chlorinated amides, they were easily converted into other classes of compounds. The directing group was cleaved under various reaction conditions (Scheme 3). When 2a and 2u were engaged under acidic or oxidative conditions, the corresponding ester 3 and the primary amide 4 were obtained in 55% and 77% yields, respectively. Scheme 3. Deprotection of the directing group. Taking benefit from the versatility brought by the introduction of a chlorine atom on the olefinic derivative as a "synthetic transformable handle" for post-functionalization, we investigated further structural modifications by the transformation of the carbon-chlorine bond into other carbon-functional groups (Scheme 4). Reaction of 2a with morpholine led to the corresponding product 5 as the single Eisomer, ascertained by NMR. 18 Subsequent thiolation of 2a provided an inseparable Z/E mixture of the thiolated compound (6a/6b) in 75% yield, the Zisomer being the major one. 21 Moreover, taking benefit from the presence of an iodine atom on the aromatic ring of 2f, a Suzuki reaction was performed leading to the corresponding compound 7, the chlorinated olefin part remaining intact at the end of the reaction. To gain more insight into the mechanism of the reaction, several experiments were conducted. 18 First, when scrambling experiments were performed, a H/D exchange was observed, suggesting that the C-H bond activation step is reversible. Then, 1i and the isotopically labeled olefin [D]-1i were engaged in parallel reactions and a kinetic isotopic effect (KIE) of 2.1 was measured. These results indicated that the rate-determining step is most likely the palladacycle formation. Experiments conducted with 1a in the presence of TEMPO, BHT and 1,4-dinitrobenezene as radical scavengers showed no significant effect on the outcome of the reaction, the reaction was just slowed down (remaining unreacted 1a at the end of the reaction), explaining the slightly lower yields. 18 Therefore, a radical process might be ruled out. Based on these considerations, the following plausible mechanism was suggested: at first, coordination of the Pd(II) catalyst with the bidentate directing group of 1a followed that the reversible formation of the palladacycle II. This latter underwent an oxidative addition followed by a reductive elimination to furnish the expected product 2a and to regenerate the catalyst (Scheme 5). Note that a redox neutral Pd(II)based mechanism might not be excluded. [START_REF] Haines | Mechanistic Details of Pd(II)-Catalyzed C-H Iodination with Molecular I2: Oxidative Addition vs Electrophilic Cleavage[END_REF] Scheme 5. Suggested mechanism.

In summary, we developed a new methodology allowing the direct chlorination of acrylamide derivatives under Pd catalysis by C-H bond activation. This approach offered an access to tri-and tetrasubstituted olefins in a complete stereoselective manner towards the Z-isomer (20 examples, up to 89% yield). This original transformation occurred at room temperature and was suitable with a broad variety of acrylamides. Indeed, -substituted and disubstituted acrylamide derivatives were efficiently functionalized. The salient features of this chlorination transformation are the lack of additives (oxidant, ligand, acid or base…), the air and moisture tolerance, a total control of the regio-and diasteroselectivity, the very mild reaction conditions (room temperature) and an easy scale-up. Moreover, access to value-added scaffolds, which hinged on the use of the chlorine atom as a linchpin, demonstrated further the key importance of chlorinated compounds and the need to develop methodologies to reach them. By tackling an unmet synthetic goal, this new approach considerably extended the portfolio of chlorinated molecules and paved the way towards original synthetic routes to unsaturated chlorinated compounds.

i:

  TsOH (3 equiv.), MeOH (0.2 M), 100 °C, 7 days, Ar. ii: CAN (3 equiv.), MeCN/H2O (5:1, 0.3 M), 25 °C, 16 h, Ar.

Table 1 .

 1 Table 1, entry 5). Increasing the reaction time (Table 1, entry 6) or running the reaction under inert atmosphere (Table1, entry 7) did not have any significant impact on the outcome of the reaction. Finally, using non-distilled DMF or a mixture of DMF/H2O (9:1), the reaction afforded 2a albeit in somehow lower yields (Table1, entries 8 and 9), showcasing hence the robustness of the transformation. Note that attempts to extend the reaction to the bromination of 1a was unsuccessful.19 Reaction of 1a with NCS: optimization studies.

	entry	variation from standard	yields
		conditions	(%) a
	1	None	85
	2	CuI instead of PdCl2	0
	3	CuCl2 instead of PdCl2	0
	4	5 mol% of PdCl2 instead of 10	25 b
		mol%
	5	No catalyst	0
	6	16 h instead of 8 h	82
	7	Ar atmosphere instead of air 83
	8	No distilled DMF instead of	75
		distilled one
	9	DMF/H2O (9:1) instead of	48
		DMF

ACKNOWLEDGMENT

This work was partially supported by Normandie Université (NU), the Région Normandie, the Centre National de la Recherche Scientifique (CNRS), Université de Rouen Normandie (URN), INSA Rouen Normandie, Labex SynOrg (ANR-11-LABX-0029), the French National Research Agency (ANR-17-CE07-0038-01) and Innovation Chimie Carnot (I2C). T.B. thanks the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. 758710). M.-Y.C. thanks the French National Research Agency for a doctoral fellowship (ANR-17-CE07-0038-01). We are thankful to Prof. Hassan Oulyadi for his help for NMR analysis.


François Mifsud

Sébastien Czernichow

Claire Carette

Rachel Levy

Philippe Ravaud

Luc Cynober

Nathalie Neveux

Claire Rives-Lange
email: claire.rives-lange@aphp.fr

Claire Rives

Behaviour of plasma citrulline after bariatric surgery in the BARIASPERM cohort

Keywords: Bariatric surgery, gastric bypass, sleeve gastrectomy, citrulline, functional enterocyte mass

Background: Plasma citrulline is currently used in clinical practice as a marker of small bowel functional mass. Behaviour of plasma citrulline after bariatric surgery and its link with post-operative outcome are still poorly understood.

Objective: Primary objective was to compare plasma citrulline 12 months after two types of bariatric surgery with pre-operative concentrations. Secondary objectives were to search for correlation between plasma citrulline variation and body weight and fat mass loss.

Design: This is an ancillary study of the BARIASPERM study. Forty-six adult men (mean age 38.9 ± 7.9 years) who underwent gastric bypass (GB, n=20) or sleeve gastrectomy (SG, n=26) were included in this prospective study. Plasma citrulline was measured at baseline, 6 months and 12 months after surgery, as well as total body weight and fat mass measured by Dual x-ray absorptiometry (DEXA).

Results: Plasma citrulline increased significantly 12 months after surgery, both after gastric bypass and sleeve gastrectomy (respectively 30.2% [18.3-42.2] and 17.8% [5.8-29.7]). The increase was significantly higher after GB than after SG (p=0.02) while total body weight and fat mass loss were not significantly different between GB and SG. The increase in plasma citrulline levels tended to be positively correlated with both weight and fat mass loss however the association did not reach statistical significance (p=0.07 and p=0.06 respectively).

Conclusion:

These results confirm the increase in plasma citrulline after GB published in two previous small studies. Citrulline also significantly increased after SG, and in spite of similar weight loss obtained with both surgery types, citrulline increase was higher after GB than SG. This suggests different modifications of intestinal functional mass after these two anatomically different techniques.

Introduction

Plasma citrulline is now currently used in clinical practice as a non-invasive marker of functional enterocyte mass in severe small bowel diseases such as short bowel syndrome (1), villous atrophy [START_REF] Crenn | Plasma citrulline: A marker of enterocyte mass in villous atrophy-associated small bowel disease[END_REF][START_REF] Papadia | Plasma citrulline as a quantitative biomarker of HIV-associated villous atrophy in a tropical enteropathy population[END_REF], radiation enteritis [START_REF] Lutgens | Plasma citrulline concentration: a surrogate end point for radiation-induced mucosal atrophy of the small bowel. A feasibility study in 23 patients[END_REF][START_REF] Lutgens | Biomarkers for radiation-induced small bowel epithelial damage: an emerging role for plasma Citrulline[END_REF] and other alterations of small bowel absorptive function(see ref. [START_REF] Crenn | La citrullinémie : un biomarqueur de la fonctionnalité intestinale[END_REF] for a recent review on this topic). Citrulline is a non protein amino acid involved as an intermediary product in the urea cycle [START_REF] Rabier | Metabolism of citrulline in man[END_REF][START_REF] Van De Poll | Intestinal and hepatic metabolism of glutamine and citrulline in humans[END_REF]. Plasma citrulline is mainly produced in small bowel enterocytes through the glutamate pathway and is considered not to depend on regimen [START_REF] Curis | Citrulline and the gut[END_REF]. Fasting venous concentration of plasma citrulline varies between 20 and 60 µmol/L in healthy subjects [START_REF] Crenn | Citrulline as a biomarker of intestinal failure due to enterocyte mass reduction[END_REF]. In patients with intestinal failure, it has been shown to correlate with the remnant functional enterocyte mass, independently of the nutritional and inflammatory status [START_REF] Papadia | Plasma citrulline concentration: a reliable marker of small bowel absorptive capacity independent of intestinal inflammation[END_REF]. Citrulline concentration increases in patients with renal failure as a consequence of its renal metabolism [START_REF] Van De Poll | Intestinal and hepatic metabolism of glutamine and citrulline in humans[END_REF][START_REF] Papadia | Plasma citrulline concentration: a reliable marker of small bowel absorptive capacity independent of intestinal inflammation[END_REF]. As citrulline can increase from a glomerular filtration rate below 50 ml/min, it is commonly slightly elevated in patients over 70 years of age [START_REF] Crenn | Citrulline as a biomarker of intestinal failure due to enterocyte mass reduction[END_REF]. In the field of obesity, it has been shown in a cohort of 70 severely obese patients that citrulline concentration is increased in patients with type 2 diabetes versus non-diabetic patients [START_REF] Verdam | Small intestinal alterations in severely obese hyperglycemic subjects[END_REF]. Bariatric surgery can take the form of a restrictive (SG) or malabsorptive technique (which combines reduced stomach volume and intestinal bypass) (GB). Behaviour of plasma citrulline after bariatric surgery is still unclear. A preliminary study in 20 patients first showed that citrulline decreased by 20% but stayed in the normal range three months after a Roux-en-Y gastric bypass [START_REF] Thibault | Assessment of plasma citrulline, iron, vitamine D and nutritionnal status in the short-term follow-up of Roux-en-Y gastric bypass for morbidly obese patients[END_REF]. This was confirmed in a transversal study in 13 patients who had malabsorptive bariatric surgery in the past with citrulline levels comparable to controls after a mean follow-up of 43 months [START_REF] Costa | Citrullinemia stimulation test in the evaluation of the intestinal function[END_REF]. In a more recent series of 30 patients undergoing RYGB, citrulline concentrations significantly increased over time after surgery, and citrulline was higher in patients who had successfully lost weight compared to patients in whom RYGB had failed [START_REF] De Hollanda | Gastrointestinal hormones and weight loss response after Roux-en-Y gastric bypass[END_REF]. Studying the behaviour of citrulline after different types of bariatric surgeries could therefore allow a better understanding of the links between intestinal functional modifications and generated weight loss. We investigated plasma citrulline changes at 6 and 12 months in 46 patients from the BARIASPERM cohort undergoing either Roux-en-Y GB or SG. Our main outcome was the variation of plasma citrulline at 12 months after surgery according to the two surgery types. The secondary goals were to study the relation between citrullinemia behaviour and the surgical outcome (weight loss and fat mass loss).

Subjects and methods

The present work is an ancillary study of the BARIASPERM cohort (16) which included 52 male patients from 5 different hospital centers undergoing either GB or SG. The 46 patients who have completed the one year follow-up were included in the present study.

Twenty patients underwent a GP (19 Roux-en-Y and 1 Omega bypass) and 26 underwent a SG, according to a joint decision between the surgeon, the reference physician and the patient.

In patients operated by Roux-en-Y GB, length of alimentary limb ranged from 1.2 to 1.5 m (mean length 1.4 m, standard deviation 0.15 m).

Plasma citrulline was measured in these subjects, before surgery, and during follow-up at 6 months and 12 months after surgery.

Anthropometric variables were measured according to a standardized protocol. Fat mass was measured by bioelectrical impedance analysis (SECA, Belgium). Blood samples were drawn before surgery, 6 months, and 12 months after surgery. Plasma citrulline was measured in all patients in post-absorptive state (after 8 hours of fasting) by ion exchange chromatography with post-column ninhidrine derivatization using an Aminotac Jeol apparatus (Jeol, Tokyo, Japan). Plasma glucose was measured routinely.

Ethics

Written informed consent was signed by all participants prior to enrollment. The study was approved by the local ethic committee (CPP2012-08-01), and was performed in accordance with the guidelines proposed in the Declaration of Helsinki (2000) of the World Medical Association.

Outcome

The primary outcome was the variation of plasma citrulline concentration 12 months after surgery. We also assessed the impact of surgery type, total body weight loss, fat mass loss and of the presence of diabetes prior to surgery on citrulline variation.

Statistical analysis

Association between baseline citrulline and clinical covariates was tested with linear regression analysis for continuous covariates and Student t-test for categorical covariates. The evolution of citrulline during follow up was modeled by a linear mixed effects model using the R library nlme, the latter model was used to test the possible impact of surgery type and other covariates on citrulline evolution after surgery compared to baseline. The association between post-operative citrulline changes and weight/fat mass loss was studied by linear regression analysis. All tests were conducted with a level of significance of 0.05.

Results

Participants mean age and BMI were 38.9 (±7.9) years old and 44.1 (±5.5) kg/m 2 respectively as shown in Table 1. 58% of patients had known diabetes mellitus before surgery. Mean plasma citrulline before surgery was 27.7 µmol/L (±7.9). Four patients had a plasma citrulline below 20 µmol/L before surgery (Figure 2A). Baseline citrullinemia did not differ according to age at inclusion and baseline BMI (Figure 2B and2C). Diabetic patients did not have a significantly different plasma citrulline at baseline than non-diabetic patients (p=0.058). Mean plasma citrulline was not different at baseline between the two surgery type groups (Figure 3A).

Plasma citrulline levels significantly increased both in patients operated by SG (p<0.01) and by GB (p<0.001) (Figure 2A). Indeed when tested in the linear mixed effect model, the increase in citrulline at 12 months was significantly higher after GB than after SG (p=0.02) (Figure 3B).

Both bariatric surgery types resulted in a massive decrease in weight and fat mass at 6 and 12 months, with no significant difference (Table 2). The increase in plasma citrulline levels with time tended to be correlated with both weight loss and fat mass loss (Figure 4A and4B), however the association did not reach statistical significance (p=0.07 and p=0.06 respectively for correlation with weight loss and fat mass).

Patients with diabetes did not have a significantly different increase in citrulline levels 12 months after surgery (Supplementary figure 2). Similarly no difference was found according to age, weight or fat mass at baseline (data not shown).

Discussion

This is the largest study to date that measures variations of plasma citrulline 6 and 12 months after bariatric surgery. In addition, to our knowledge, it is the first study to compare the evolution of citrulline in the two main types of bariatric surgery i.e. gastric bypass and sleeve gastrectomy. Plasma concentrations of citrulline at baseline were comparable with the general population. However four subjects had a plasma citrulline lower than 20 µmol/L which might suggest unknown intestinal failure but could be simply explained by un-compliance to the fasting period before blood sampling, as plasma citrulline concentration has been shown to decrease after food intake [START_REF] Crenn | Citrulline as a biomarker of intestinal failure due to enterocyte mass reduction[END_REF]. Indeed clinical data of these four patients did not suggest any reason to any decrease functional enterocyte mass. By contrary to one previous report, we did not find a significant increase in baseline concentrations of citrulline in patients with diabetes compared to non-diabetic ones [START_REF] Verdam | Small intestinal alterations in severely obese hyperglycemic subjects[END_REF]. Interestingly we confirmed the significant increase in citrulline levels with time 12 months after gastric bypass and for the very first time, we shown that the increase is observed in both GB and SG. The mechanism underlying this increase in plasma citrulline is still unclear and suggests modifications of the small bowel function after bariatric surgery. In these two surgery types, the totality of the jejunum is left in place but the montages are very different. In particular, it is worth noting that an upstream intervention such as sleeve gastrectomy could affect downstream physiology. This has been previously described in gastric surgery and could be explained by a downstream effect on amino absorption in the gut [START_REF] Ziegler | Pharmacokinetic assessment of an oligopeptide-based enteral formula in abdominal surgery patients[END_REF]. The significantly higher increase of citrullinemia after GB compared with SG, despite comparable weight and fat mass loss, could suggest that functional intestinal modifications induced by each of these two very different types of surgery are different. Malabsorption induced by GB may result in greater compensatory growth of the enterocyte mass. Indeed Cavin et al. [START_REF] Cavin | Malabsorption and intestinal adaptation after one anastomosis gastric bypass compared with Roux-en-Y gastric bypass in rats[END_REF] have demonstrated intestinal adaptation after two types of gastric bypass with intestinal hyperplasia, increased jejunum diameter, higher villi, and deeper crypts than in sham non operated rats. We could also hypothesize that malabsorption could play an additional role in compensatory action compared to restriction alone. Furthermore, it was healthy small bowel and therefore with an ability to compensate for the intact enterocyte mass. During the first year after bariatric surgery, the catabolism and weight loss are at maximal rate but we can suppose that the signal for this compensatory growth of enterocyte mass is also at maximal rate. The impact of this intestinal adaptation after surgery on long term weight regain remains to be investigated.

Although the effect did not reach statistical significance due probably to the relatively small sample size, we showed a positive correlation between the post-operative increase in citrulline and both weight loss and fat mass loss. We also looked at whether the change in the level of inflammation or insulin after surgery bariatric could influence our results and it was not the case. Even if causality cannot be formally proven from these data, it is possible that the functional modifications underlying the increase in citrulline are associated with the effectiveness of bariatric surgery. It is thus tempting to suggest that citrullinemia could be used as a potential biomarker of the surgical outcome but its interpretation in clinical practice remains to clarify. In addition, large fat mass is associated with low grade inflammation [START_REF] Hotamisligil | Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance[END_REF][START_REF] Trayhurn | Adipokines: inflammation and the pleiotropic role of white adipose tissue[END_REF] which in turn leads to a decrease in lean mass [START_REF] Beyer | Chronic low-grade inflammation and age-related sarcopenia[END_REF]. This organ cross-talk is mediated by adipokines but also by pro-inflammatory cytokines synthetized and released by immune cells.

Actually, in immune cells citrulline is both the product and the precursor of nitric oxide [START_REF] Rath | Metabolism via Arginase or Nitric Oxide Synthase: Two Competing Arginine Pathways in Macrophages[END_REF][START_REF] Wijnands | Arginine and citrulline and the immune response in sepsis[END_REF][START_REF] Papadia | Citrulline in health and disease. Review on human studies[END_REF] a major player of non-specific immunity. Thus, bariatric surgery, by reducing systemic inflammation, decreases the use of citrulline by immune cells thus causing an increase in its plasma levels. The study of citrulline metabolism in gut-associated lymphoid tissue (GALT) before and after bariatric surgery should be therefore of major interest to get mechanistic data.

There are several limitations to our study. First, the sample size is limited and caused likely a lack of statistical power. The BARIASPERM cohort included only male patients, the generalization of these results to women is therefore unsure especially because the intestinal synthesis of citrulline is affected by the genre [START_REF] Levillain | Amino acid handling in uremic rats: citrulline, a reliable marker of renal insufficiency and proximal tubular dysfunction[END_REF]. Follow-up was limited to 12 months after surgery and we did not recorded markers of renal function, which is known to impact the plasma concentration of citrulline [START_REF] Pironi | Plasma citrulline after small bowel transplantation: effect of time from transplantation, acute cellular rejection, and renal failure[END_REF].

In conclusion this study strengthens the idea of an increase in plasma citrulline 12 months after gastric bypass and sleeve gastrectomy. We found that gastric bypass was followed by a significantly higher citrulline increase than sleeve gastrectomy, despite comparable weight loss, suggesting distinct modifications of intestinal function mass after these two anatomically different techniques.

Tables

Table 1 . Characteristics of the 46 study participants at baseline Characteristics at inclusion

 1

		Surgery group Gastric By-pass Sleeve Gastrectomy (n=20) (n=26)	Both groups (n=46)
	Anthropometrics measures, mean (SD)
	Age, years	41.7 (6.9)	36.8 (8.0)	38.9 (7.9)
	Weight, kg [Range]	138.4 (18.2) [106-178]	141.8 (23.5) [106-192]	140.3 (21.2) [106-192]
	BMI, kg/m²	44.1 (4.9)	44.1 (6.0)	44.1 (5.5)
	Waist circumference, cm [Range]	140.1 (12.1) [117-158]	137.5 (15.0) [113-171] 138.6 (13.8) [113-171]
	Fat mass percentage (%)	41.4 (7.0)	40.6 (7.3)	41.0 (7.1)
	Fasting Laboratory values, mean (SD)
	Fasting glucose, mmol/L	6.0 (2.1)	6.1 (1.7)	6.1 (1.8)
	Known diabetes mellitus (n)	Yes=5, No=9	Yes :10, No : 7,	Yes :15, No :16, NA=15
		NA=6	NA=9
	Plasma citrulline, umol/L	29.5 (8.7)	26.1 (6.9)	27.7 (7.9)

The authors declare no relevant conflict of interest.

Authors contribution

F. Mifsud done the statisctical analysis and wrote de manuscript N. Neveux was responsible for citrulline analysis and corrected the manuscript.

L. Cynober had a significant contribution correcting the manuscript. C. Carette, S. Czernichow, C. Rives-Lange had correcting the manuscript Bariasperm Study Group: Eustache F, Coupaye M, Msika S, Cohen R, Catheline JM, Brugnon F, Slim K, Barsamian C, Chevallier JM, Bretault M, Bouillot JL.

The study was funded by a grant from Programme Hospitalier de Recherche Clinique -PHRC 2011 (Ministère de la Santé, N° AOM 11193).