R Navarro-González

J Stern

B Sutter

D Archer

A Mcadam

H B Franz

C P Mckay

P Coll

M Cabane

D W Ming

F Raulin

A E Brunner

D Glavin

J L Eigenbrode

J H Jones

C Freissinet

L Leshin

M Wong

S Atreya

J J Wray

A Steele

A Buch

B D Prats

C Szopa

D Coscia

S Teinturier

P Conrad

P Mahaffy

F J Martín-Torres

M.-P Zorzano-Mier

J P Grotzinger

POSSIBLE DETECTION OF NITRATES ON MARS BY THE SAMPLE ANALYSIS AT MARS (SAM) INSTRUMENT

Introduction: Planetary models suggest that nitrogen was abundant in the early Martian atmosphere as dinitrogen (N 2 ). However, it has been lost by sputtering and photochemical loss to space [1,2], impact erosion [3], and chemical oxidation to nitrates [4]. Nitrates, produced early in Mars' history, are later decomposed back into N 2 by the current impact flux [5], making possible a nitrogen cycle on Mars. It is estimated that a layer of about 3 m of pure NaNO 3 should be distributed globally on Mars [5]. Nitrates are a fundamental source for nitrogen to terrestrial microorganisms. Therefore, the detection of soil nitrates is important to assess habitability in the Martian environment. The only previous mission that was designed to search for soil nitrates was the Phoenix mission but was unable to detect evolved N-containing species by TEGA and the MECA WCL [6]. Nitrates have been tentatively identified in the Nakhla meteorite [7]. The purpose of this work is to determine if nitrates were detected in first solid sample (Rocknest) in Gale Crater examined by the SAM instrument.

Materials and Methods: Samples collected from Rocknest, located in Gale Crater, which consists of an inactive, sandy wind drift mantled with dust, were analyzed by the SAM instrument. Prior to sample analysis, a blank was run using an empty quartz cup to characterize the background of the SAM instrument. The quartz cup was sealed inside the pyrolysis oven and heated to ~840°C at a rate of 35°C/min under a He carrier gas flow rate of 1.5 cm 3 /min and at an oven pressure of ~30 mbar. A small fraction of the gas released from the cup was measured directly by electron impact quadrupole mass spectrometry (QMS mass range 2-535 Da, resolution 0.1 Da). Then four samples of Rocknest bedform materials < 150 μm diameter size fraction (~20 mg) were examined by SAM. A thermal analyzer (Netzsch STA 449 F1 Jupiter Simultaneous TG/DSC) coupled to a mass spectrometer (Netzsch QMS 403 C Aeolos) was used in the laboratory to heat samples upto 1200°C at a rate of 20°C/min under a He or N 2 carrier gas flow rate of 20 cm 3 /min and at an oven pressure of 1000 mbar. Several mixtures of perchlorates, nitrates, and ammonium salts were analyzed and results utilized to assist in SAM data interpretation.

Results and Discussion: Nitrates generally decompose into nitric oxide (NO) and O 2 at temperatures above 560C and up to 900C [8]. Figure 1 shows the evolution of mass/charge (m/z) 30 as a function of temperature in a blank and the four portions of a scooped sample (Scoop#5) collected at the Rocknest site. There appear to be at least three peaks associated with the release of m/z 30: the first centered at 280C, the second at 380C, and the third at 460C. M/z 30 was identified as NO using the NIST database, which reports the ratio of (m/z 14)/(m/z 30) in NO as 0.075. Other plausible contributions to m/z 30 include an isotopologue of CO, 12 C 18 O. Surprinsingly the evolution of m/z 30 in the Rocknest samples is well below the expected thermal decomposition of nitrates. However, the lowest temperature peaks (~200° -300°C) have the best spectral match for NO based on (m/z 14)/(m/z 30) ratios, and diverge from the CO 2 trace (as represented by m/z 45). The higher temperature peaks (>300° C) track with m/z 45 and may represent some contribution of 12 C 18 O to m/z 30.

Temperature ( o C)

200 400 600 800

Mass/charge 30 (cps)

2648.pdf 44th Lunar and Planetary Science Conference (2013)

Since perchlorates have been detected on Mars [6], we have investigated their interaction effect on the thermal decomposition of nitrates. Thermal decomposition of metal (M) perchlorates follows two reaction paths: Reaction 1 for alkali metals (M 1 ) or group 1 (Na, K, etc): M 1 ClO 4 → M 1 Cl + 2O 2 ; and, reaction 2 for alkaline earth metals (M 2 ) or group 2 (Be → Ba except for Ca and Mg): 2M 2 (ClO 4 ) 2 → 2M 2 O + 2Cl 2 + 7O 2 ; however, for Mg-perchlorates reactions 1 and 2 are equally important. Therefore, we conducted experiments on the effect of Na-perchlorate on the decomposition of Mg-, Ca-, Na-, and K-nitrates and found no effect. The decomposition of nitrates with Mgperchlorate is enhanced as more perchlorate is added to the mixture (Figure 2). This effect is probably due to the reaction of HCl with nitrates derived from hydrated Mg-perchlorate decomposition [e.g., 9] according to the following reactions: (1) M 1 NO 3 + HCl → M 1 Cl + HNO 3 ; and (2) M 2 (NO 3 ) 2 + 2HCl → M 2 Cl 2 + 2HNO 3 . HCl arises by the reaction of Cl 2 with water vapor, and HNO 3 thermally decomposes into NO, water, and O 2 . We investigated the thermal stability of Mg-, Ca-, Na-, and K-nitrates by monitoring NO released during thermal analysis which spans from 330C to 600C (Figure 3), and from 360C to 650C (data not shown), in the presence of Mg-and Ca-perchlorate, respectively. These values may be consistent with the second and third evolved NO peaks but cannot explain the evolution of NO at 280C in Rocknest materials. A possible explanation that we are exploring is the presence of chlorate and chlorite species in the soil. To date we have conducted experiments with Na-chlorate and chlorite. While the release of O 2 as a function of temperature is decreased, the temperature of NO evolution is significantly increased, probably because HCl is not produced. It is possible that Mg-or Ca-chlorates or chlorites can explain the observed temperature of evolution of NO in the martian soil. Experiments are currenlty underway to examine Ca-and Mgchlorate/chlorite interactions with nitrates.

Another possibility to explain the evolution of the first peak is the presence of NH 4 + in the soil [10], which undergoes combustion in the presence of perchlorates leading to the formation of NO. Figure 4 shows the evolution of NO by different ammonium salts. Ammoniun nitrate would probably explain the behavior observed for the first peak. However, we are exploring the possibility that the first peak could also arise from terrestrial contamination from MTBSTFA known to be present in the system [11,12].

Further work is underway to fully understand the release of NO in the Rocknest samples.

3 Figure 1 .

 31 Figure 1. Release of m/z 30 versus temperature from the blank and Rocknest samples as measured by SAM.

2 Figure 2 .

 22 Figure 2. Effect of magnesium perchlorate on the thermal stability of Na-nitrate in laboratory experiments.

2 Figure 3 . 4 Figure 4 .

 2344 Figure 3. Thermal stability of different (10%) nitrates in the precence of (90%) magnesium perchlorate in laboratory experiments.

44th Lunar and Planetary Science Conference (2013)


A Buch

C Szopa

C Freissinet

D P Glavin

P Coll

M Cabane

J Eigenbrode

K Miller

M Martin

R E Summons

D Archer

A Brunner

P Conrad

S Teinturier

D Coscia

J Dworkin

J Grotzinger

P Ma- Haffy

C Mckay

D Ming

R Navarro-González

IMPACT OF THE SAMPLE PREPARATION ON THE ORGANIC COMPOUNDS DETECTED ON MARS AT JK AND CB

Introduction:

The wet chemistry experiment on the Sample Analysis at Mars (SAM) experiment in the Curiosity rover of the Mars Science Laboratory mission allows organic compounds to be extracted from solid samples in such a way to improve their detection either by increasing the release of chemical species from solid sample matrices, or by changing their chemical structure to make compounds analyzable by gas chromatography mass spectrometry (GCMS). The latter is of particular importance if polar compounds are present. SAM, includes two different wet chemistry experiments: MTBSTFA derivatization [1][2] and TMAH thermochemolysis [3][4]. Here we report on the nature of the MTBSTFA derivatization experiment on SAM, the detection of MTBSTFA products in the SAM evolved gas analysis and GCMS experiments, and the implications of this detection.

Sample Processing and SAM Protocol:

The soil or powdered rock sample is heated up to ~1000C in one of the two pyrolysis ovens in SAM. The gases released from the sample, within a predefined temperature range, are pre-concentrated in a SAM hydrocarbon trap and then transferred to a GC injection trap for a final injection into the GC. Adsorbants used in the SAM and GC hydrocarbon trap are Tenax® (TA) silica beads and Carbosieve G. The GC injection trap contains Tenax GR. Trapped volatiles are released from these traps by heating to ~300 °C. Special cells containing the derivatization reagent can be utilized if needed. The chemical extraction and derivatization of polar molecular compounds is achieved with the MTBSTFA (N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide) and DMF (dimethylformamide) silylation reaction in order to transform refractory polar compounds into more volatile molecules that can be analyzed and detected by GCMS. The advantage of using MTBSTFA as the derivatization reagent is that it is a single-step reaction; it is less susceptible to hydrolysis compared with other common derivatization reagents and it does not require separation of the derivatives prior to GC separation analysis. In addition, MTBSTFA is sensitive to a wide range of organic compounds with acidic hydrogen atoms. The reaction yields obtained using MTBSTFA are typically in the 90-100% range. Finally, MTBSTFA has the major advantage to react in the gaseous phase and is therefore compatible with laboratory pyrolysis systems like the one used in SAM to treat the solid samples.

SAM solid sample experiment results:

Martian solid samples (Rocknest (RN), John Klein (JK), Cumberland (CB) have been analyzed by evolved gas analysis (EGA) and GC using thermal conductivity (TCD) and MS detection [5][6]. The samples were heated up to approximately 840 °C at a rate of 35°C/min under He flow. For GC analyses, the majority of the gas released was trapped on a hydrocarbon trap over a specific temperature range. Adsorbed volatiles on the GC injection trap (IT) were then released into the GC column (CLP-MXT 30m x 0.25mm x 0.25µm) by rapidly heating the IT to 300°C. Helium is used (0.9 bar inlet column pressure) as the carrier gas. The column temperature program started at 50 °C, and increased up to 220 °C with a ramp rate of 10 °C/min.

The first results obtained from Rocknest showed the presence of MTBSTFA by-products in the system. MTBSTFA was detected in the GC-TCD-QMS analysis blank meaning that MTBSTFA is part of the background signal under its gaseous phase and is derived from at least one of the seven MTBSTFA/DMF derivatization cups in SAM [START_REF] Freissinet | LPPSC[END_REF]. Since MTBSTFA is able to react in the gaseous phase, the detection of the hydrolysis products of MTBSTFA implies that some MTBSTFA reactions with all the labile compounds present in the Martian soil, in the sampling system, and/or inside the SAM instrument occurred during the experiment.

Several peaks have been identified by GCMS analysis of JK and CB as part of SAM background, some of them below the nmol level. Identification of these peaks reveal the presence of several aromatic, chlorinated hydrocarbons (Table 1) and silylated compounds such as water. The most interesting of these compounds are listed in Table 1. The question of the endogenous or exogenous origins of these compounds has to be asked. To answer it we have investigated several possibilities to produce these chlorinated hy-

2886.pdf 45th Lunar and Planetary Science Conference (2014)

drocarbons during the SAM experiment. They could come from : i) sample itself ii) MTBSTFA reaction iii)Tenax® degradation, and iv)Perchlorate oxidation.

SAM experiment lab:

A first set of laboratory tests were done to evaluate the thermal stability of Tenax® GR and the possible products released. followed by tests with Tenax® that was exposed to MTBSTFA during the thermal test. The Tenax® sample was successively submitted to thermal treatments from 200°C to 600°C with 50°C steps. Each temperature was held for 5 minutes, then the liner was cooled down to 50°C during the rest of the run. Operating conditions are summarized in Figure 1. 2). Indeed, Tenax® is an organic polymer and the detected compounds are coming from the thermal degradation of this polymer. The structure of the Tenax® polymer (Figure 4) explains why aromatic compounds are detected during its thermal degradation. The main compound detected (fig. 4) is the terphenyl which is a part of the monomer of Tenax®. Other reactive compounds such as phenol or hydroxy-biphenyl are also directly generated from this monomer degradation.

Table 1.

Compounds generated by Tenax® degradation in lab experiment compared to compounds detected on Mars (J. Klein and Cumberland).

Compounds Lab T (°C)

SAM blank At higher temperatures, the number of released compounds increases and the size of the released molecules decreases. The temperature range studied far exceeds the maximum temperature reached by the SAM trap which stays below 300°C Under these condiditons only a few organic compounds should be released such as benzene. But the continuous use of the GC trap could generate a long term ageing of Tenax® resulting in the potential release of additional compounds. Among these compounds some of them could be reactive and give other by product when contacted with MTBSTFA or perchlorate.

SAM sample (JK and CB) H2O

Tenax® degradation results in presence of MTBSTFA:With MTBSTFA a part of the background signal, it could possibly interact either with martian organics or compounds produced by the thermal degradation of the trap. This is why we have studied the influence of the MTBSTFA on the compounds released from the Tenax®. During the thermal degradation of Tenax®, we have added a few microliters of MTBSTFA (0.5µL of MTBSTFA for 10 g of Tenax®).

Results of adding MTBSTFA show that no derivatization reaction occurred on compounds with labile H. We have shown that phenol was one of the compounds released by Tenax® when temperature reached 550°C. But contrary to the results of the SAM experiment (table 1), after MTBSTFA addition, no TBDMS-phenol was detected at the retention time of 36 min against 16.7 min for phenol. The explanation is certainly the shorter time of contact since the laboratory derivatization reactions requires 15 min at 75°C.

Figure 1 .

 1 Figure 1. Operating conditions Tenax® degradation results: Thermal degradation of Tenax® starts at 400°C. Detected compounds are mainly aromatic compounds such as benzene, biphenyl, or phenol (Figure 3 and Table2). Indeed, Tenax® is an organic polymer and the detected compounds are coming from the thermal degradation of this polymer. The structure of the Tenax® polymer (Figure4) explains why aromatic compounds are detected during its thermal degradation. The main compound detected (fig.4) is the terphenyl which is a part of the monomer of Tenax®. Other reactive compounds such as phenol or hydroxy-biphenyl are also directly generated from this monomer degradation.

Conclusion:

We have confirm the presence of MTBSTFA by-products in the EGA and GC-TCD-MS background signals.

Major part of aromatic detected compounds on mars could be generate from the degradation of the Tenax® trap or from the MTBSTFA reaction. the possible range of products that might be produced by the SAM background and the Tenax trap are being elucidated by laboratory studies and it will help clarify which of the compoiunds observed by SAM are indigeneous.

SAM-GC team acknowledges support from the French Space Agency (CNES), French National Programme of Planetology (PNP), National French Council (CNRS), Pierre Simon Laplace Institute 2886.


A Buch

V Pinnick

Cyril Szopa

R Danell

F H W Van Amerom

Daniel Glavin

Caroline Freissinet

Patrice Coll

Fabien Stalport

Noël Grand

Coupling Moma Gc-Ms

la diffusion de documents scientifiques

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction:

The goals of the joint ESA-Roscosmos Exo-Mars-2018 rover mission is to search for "signs of life" molecules (large, non-volatile organic or biological molecules that suggest existing or prior biosynthetic activity) on the Martian surface and near subsurface and to distinguish between the endogenous and exogenous sources of Martian organics. The Mars Organic Molecule Analyzer (MOMA) aboard the ExoMars rover will be a key analytical tool in providing chemical (molecular) information from the solid samples, with particular focus on the characterization of organic content. Central to MOMA instrumentation is a gas chromatograph-mass spectrometer (GC-MS) which provides the unique ability to characterize a broad range of compounds allowing chemical analyses of volatile and non-volatile species. Both instruments have been tested separately first and have been coupled in order to test the efficiency of the future MOMA GC-MS instrument.

Instrumental Suite:

The French brassboard GC coupled has been coupled to the US brassboard ion trap mass spectrometer in a flight-like configuration (fig 1) for the coupling campaign. The MOMA GC setup is based on the Sample Analysis at Mars (SAM) heritage design with a He reservoir and 4 separate analytical modules including traps, columns and TCDs. Solid samples are sealed and heated in this setup using the manual tapping station (fig. 1), design and built at MPS in Germany, for GC-MS analysis. Then the effluent is ionized by an electron impact ionization source in the MS chamber and analyzed by the linear ion trap mass spectrometer (fig. 1).

MOMA GCMS Results:

Qualitative Ananlysis: Volatile and non volatile compounds have been injected in the MOMA instrumental suite. Both of these compounds family have been detected by the TCD and by the MS. By using the TCD signal and the retention time we were able to identify each of these injected compounds. MS signal and single mass spectra, by comparison with the NIST librarie, give us an unambiguous confirmation of these identifications (fig 2). The TCD trace (a) and the total ion chromatogram (TIC) from the MS (b) for a standard gas-phase mixture of 100 ppm butane, pentane, hexane and benzene in He. The mass spectra (c-e) arise from an average of 10 spectra averaged from the TIC peaks. Quantitative Ananlysis: The sensitivity of the end-toend test of the GC-MS interface depends heavily on the ionization efficiency of the analyte, the quality of the thermal control along the sample path, and the split manifold design. The MOMA requirement for sensitivity in the GC-MS mode for organic molecules is 1 pmol. In this test, sensitivity was determined for the GC TCD and MS response to a dilution series containing IPA, hexane and benzene deposited onto silica beads in the MOMA oven. For the MS, peak areas were integrated in the total ion chromatogram as well as in the resulting mass spectra. Again, IPA was not detected in the MS because the characteristic peaks fell below the low mass cutoff for the instrument. Generally, the MS was found to be 5-10 times more sensitive than the GC TCD for hexane and benzene respectively.

2907.pdf 45th Lunar and Planetary Science Conference (2014)

Conclusion:

The Mars Organic Molecule Analyzer is a key analytical instrument aboard the ExoMars Rover set to launch in 2018. The results described here demonstrate the current status of the end-to-end performance of the gas chromatography-mass spectrometry mode of operation. Both prototypes individually meet the performance requirements, but this work particularly demonstrates the capabilities of the critical GC-MS interface. Future work will focus on improving detection limits to provide margin to ensure the requirements will be met, as well as analyzing higher molecular weight compounds including derivitized amino acids and thermochemolized compounds.

Figure 1 :

 1 Figure 1: picture of the manual taping station (a) where sample has been introduced before that it has been traped, injected in the gas chromatograph (b) and detected in the MS ion trap (c).

Figure 2 :

 2 Figure 2: The TCD trace (a) and the total ion chromatogram (TIC) from the MS (b) for a standard gas-phase mixture of 100 ppm butane, pentane, hexane and benzene in He. The mass spectra arise from an average of 10 spectra averaged from the TIC peaks.

Table 1 :

 1 TCD and MS sensitivity

		TCD Sensitivity	MS Sensitivity
	Compounds	LOD (pmol)	Peak integrated	LOD (pmol)
	Isopropanol	14	ND	ND
			m/z=57	3
	Hexane	32	m/z=86	22
			TIC	0.8
	Benzene	24	m/z=78 TIC	5 5

Ecole Centrale Paris, LGPM, Grande voie des vignes, 92295 Châtenay-Malabry, France,

NASA Goddard Space Flight Center, 8800 Greenbelt Rd, Greenbelt, MD 20771, USA,

LATMOS, Univ. Pierre et Marie Curie, Univ. Versailles Saint-Quentin & CNRS, 75005 Paris, France, 5 Danell Consulting, Inc., Winterville, NC 28590

, 4 LISA, Univ. Paris-Est Créteil, Univ. Denis Diderot & CNRS, 94000 Créteil, France


Audrey Huguet

Marianne Latournerie

Pauline Houssel Debry

Caroline Jezequel

Ludivine Legros

Michel Rayar

Karim Boudjema

Dominique Guyader

Edouard Bardou Jacquet

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

The psoas muscle transversal diameter predicts mortality in patients with cirrhosis on waiting-list for liver transplantation: a retrospective cohort study

Keywords: ADPM, axial diameter of psoas muscle APTI, axial psoas thickness index AUC, area under the curve BMI, Body mass index BIA, bioimpedance analysis CI, confidence interval CT, computed tomography DEXA, dual-energy X-ray absorptiometry INR, International Normalized Ratio MAMC, midarm muscle circumference MELD, muscle mass, malnutrition, computed tomography scan, liver failure

Transversal psoas thickness index (TPTI) is measured on an umbilicus-targeted CT.

TPTI was independently associated with mortality.

TPTI is easy, quick, cheap, and highly reproducible, reliable by a non-expert operator.

TPTI could be a marker of muscle mass and function.

INTRODUCTION

Chronic liver diseases are often complicated with malnutrition [START_REF] Schuppan | Liver Cirrhosis[END_REF], that impairs the prognosis [START_REF] Coltorti | Nutritional Status in Cirrhosis. Italian Multicentre Cooperative Project on Nutrition in Liver Cirrhosis[END_REF][START_REF] Shaw | Influence of Selected Patient Variables and Operative Blood Loss on Six-Month Survival after Liver Transplantation[END_REF]. Nevertheless, because of rapid fluid shifts and altered water compartmentalization due to ascites and oedema, standard methods of nutritional assessment (weight loss, body mass index, biological markers, i.e. albumin, transthyretin, or multicomposite scores) are not usable. Also, body composition assessment methods, such as bioimpedance analysis (BIA) or dual-energy Xray absorptiometry (DEXA) [START_REF] Pirlich | Bioelectrical Impedance Analysis Is a Useful Bedside Technique to Assess Malnutrition in Cirrhotic Patients With and Without Ascites[END_REF][START_REF] Riggio | Whole Body and Regional Body Composition Analysis by Dual-Energy X-Ray Absorptiometry in Cirrhotic Patients[END_REF] are not applicable to patients with liver cirrhosis (BIA) [START_REF] Schloerb | Bioelectrical Impedance in the Clinical Evaluation of Liver Disease[END_REF], or not routinely applicable in the daily clinical practice (DEXA) [START_REF] Pietrobelli | Dual-Energy X-ray absorptiometry: fat estimation errors due to variation in soft tissue hydration[END_REF]. Liver cirrhosis is associated with significant changes in body composition: the prevalence of muscle mass loss is estimated to be 20-60% [START_REF] Montano-Loza | Inclusion of Sarcopenia within MELD (MELD-Sarcopenia) and the Prediction of Mortality in Patients with Cirrhosis[END_REF][START_REF] Meza-Junco | Sarcopenia as a Pronostic Index of Nutritional Status in Concurrent Cirrhosis and Hepatocellular Carcinoma[END_REF]; muscle mass loss is an independent prognostic factor in cirrhosis [START_REF] Montano-Loza | Muscle Waisting Is Associated with Mortality in Patients with Cirrhosis[END_REF]. Mid-arm muscle circumference is an independent predictor of survival in patients with liver cirrhosis [START_REF] Alberino | Nutrition and Survival in Patients with Liver Cirrhosis[END_REF], but its interobserver variability limits its use in the daily practice. In the last decade, the development of cross sectional imaging techniques, such as computed tomography (CT) or magnetic resonance imaging (MRI) [START_REF] Shen | Total Body Skeletal Muscle and Adipose Tissue Volume: Estimation from a Single Abdominal Cross-Sectional Image[END_REF], is very promising to assess muscle mass. Developed from studies conducted in oncology [START_REF] Prado | Prevalence and Clinical Implications of Sarcopenic Obesity in Patients with Solids Tumors of the Respiratory and Gastrointestinal Tracts: A Population-Based Study[END_REF], disease prognosis could be assessed by the whole muscle cross sectional area measured on a third lumbar vertebra (L3)-targeted CT normalized by height. This L3 skeletal muscle index predicted mortality after liver transplantation [START_REF] Englesbe | Sarcopenia and Mortality after Liver Tranplantation[END_REF][START_REF] Hamaguchi | Impact of Quality as Well as Quantity of Skeletal Muscle on Outcomes after Liver Transplantation[END_REF][START_REF] Masuda | Sarcopenia Is a Prognostic Factor in Living Donor Liver Transplantation[END_REF], and recently in cirrhotic patients on waiting-list for liver transplantation [START_REF] Tandon | Severe Muscle Depletion in Patients on the Liver Transplant List: Its Prevalence and Independence Prognostic Value[END_REF][START_REF] Carey | A multicenter study to define sarcopenia in patients with end-stage liver disease[END_REF]. However this technique could be time-consuming. Therefore there is a need to develop simpler and easier methods to assess muscle mass, doable for all in the daily clinical practice. Psoas muscle measurement could appear as a reliable marker to assess cirrhosis prognosis. Recently, Golse et al suggested that psoas muscle area better predicts post-liver transplantation 1-year survival than L3 skeletal muscle index, but they did not assess the predictive role of transversal psoas muscle thickness [START_REF] Golse | A New Definition of Sarcopenia in Patients with Cirrhosis Undergoing Liver Transplantation[END_REF]. Durand et al showed that the measurement of the transversal psoas muscle thickness by CT images targeted on the umbilicus, standardized to height, was an independent predictive factor for mortality in cirrhotic patients with refractory ascites on waiting-list for liver transplantation [START_REF] Durand | Prognostic Value of Muscle Atrophy in Cirrhosis using Psoas Muscle Thickness on Computed Tomography[END_REF]. However these results were never confirmed by an independent study, nor in a whole population of cirrhotic patients on waiting-list for liver transplantation. Therefore, the main aim of this retrospective study was to determine whether the measurement of the transversal or axial diameter of the psoas muscle on an abdominal CT predicted mortality in patients with cirrhosis on waiting-list for liver transplantation. The secondary aims were to determine the feasibility and reliability of the measurements of transversal or axial psoas muscle diameters by a non-trained operator.

METHODS

Study design

A monocentric observational retrospective study was conducted in the Department of Liver Transplantation and the Hepatic diseases Unit of the Rennes University Hospital (CHU Rennes), Rennes, France. Our unit is a tertiary referral centre for liver transplantation in France.

Patients' recruitment

All patients with liver cirrhosis registered on the waiting-list for liver transplantation for isolated

Study endpoints

The primary endpoint was the occurrence of death on waiting-list for liver transplantation. As previously published [START_REF] Englesbe | Sarcopenia and Mortality after Liver Tranplantation[END_REF], patients removed from the transplantation waiting list because of the worsening of their liver cirrhosis were considered as deaths. The secondary endpoint was the reliability of the measurements of transversal (TDPM) and axial (ADPM) diameters of the psoas muscle by a non-trained operator.

Measurement of psoas muscle diameters

The measurements of the axial and transverse diameters of the right psoas were performed on one single CT scan image targeted on the umbilicus. We chose to assess psoas muscle diameters on umbilicus-targeted scan, because the study by Taguchi et al [START_REF] Selberg | Norms and Correlates of Bioimpedance Phase Angle in Healthy Human Subjects, Hospitalized Patients, and Patients with Liver Cirrhosis[END_REF] indicated that axial and/or transversal psoas thickness at the umbilicus level was more associated with mortality than at the L3 level, in patients with metastatic urothelial carcinoma; Durand et al used this method in cirrhotic patients; the recent study by Golse et al [START_REF] Golse | A New Definition of Sarcopenia in Patients with Cirrhosis Undergoing Liver Transplantation[END_REF] compared L3-targeted psoas muscle area to SMI, but they did not assess the predictive role of the axial or transversal diameters of the psoas muscle. The assessment was performed by one single non expert-operator (AH), a gastroenterologist fellow without any specific skill in radiology. For the first 50 patients, the measurements were checked by a radiologist specialized in Gastrointestinal Imaging, blinded of the first operator's measurement. The operators were blinded from the demographics and clinical data including mortality on liver transplantation list. The axial diameter of psoas muscle (ADPM) was determined as the longest antero-posterior diameter and expressed in millimetre (mm) (Figure 1). The transversal diameter of the psoas muscle (TDPM) was defined as the diameter perpendicular to the axial diameter (Figure 1). Axial psoas thickness index (APTI), was expressed as mm/m, and calculated as: ADPM (mm)/height (m). Transversal psoas thickness index (TPTI) was expressed as mm/m, and calculated as: TDPM (mm)/height (m).

Data collection

The list of patients fulfilling the eligibility criteria was obtained by extraction from the Cristal®

Statistical analysis

Variables are expressed as mean ± standard deviation (SD) or percentage. Means were compared with Student t or Wilcoxon test as appropriate. Univariate analysis was performed by Cox proportional hazards model with a significance level of P<0.05. Variables significant (P<0.2) in univariate analysis were included in the multivariate analysis. To select the optimal cut-off of psoas muscle index associated with the primary endpoint, receiver operating characteristic (ROC) curve and the Youden method were used. Survival rates of subgroups of patients were calculated with Kaplan Meier method, and compared by the log-rank test. Patients transplanted and removed of transplantation list because they had an improvement of the liver disease or for personal decision were censored. Interobserver agreement of the measurements of axial (ADPM) and transversal (TDPM) diameters of the psoas muscle were analysed by kappa correlation test.

Data were analysed using SPSS version 22 (SPSS Inc., Chicago, IL, USA). P value less than 0.05 was considered significant with a two-tailed test.

RESULTS

Patients' selection

The study flow chart is shown in Figure 2. Among the 175 eligible patients, 173 were enrolled into the study and included in the final analysis.

Patients' characteristics and outcomes

The characteristics of the 173 included patients at the time of their registration on liver transplantation list are detailed in Table 1. The most frequent aetiology of liver cirrhosis was alcoholic (71%). Most of cirrhoses were CHILD C (66%). The mean MELD score at registration was 21.2±8.1. Forty-seven percent of cirrhoses were complicated with refractory ascites. During the mean follow-up of 5.6±6.2 month, 143 patients were transplanted (82.7%) and 13 died (7.5%). The remaining 17 patients (9.8%) were removed from the waiting-list because of disease improvement or personal decision. The mean time length on waiting-list before accessing to liver transplantation was 5.3±6.4 month. The mean time length on waiting-list before death was 7.3±6.8 month.

Relation between psoas muscle thickness and mortality

The univariate analysis including the patients' characteristics at registration (gender, age, body mass index), disease variables (aetiology of cirrhosis, CHILD and MELD scores, ascites, hepatic encephalopathy, plasma bilirubin, albumin, prothrombin time, creatinine, and sodium), APTI and TPTI, is shown in Table 2. The CHILD score, the MELD score, refractory ascites, plasma creatinine and sodium, and TPTI, were significantly associated with death or exclusion of transplant list for worsening of the liver cirrhosis (Table 2). In the Cox multivariate analysis, only TPTI was independently and significantly (P<0.05) associated with mortality (Table 3).

TPTI was associated with mortality: AUC=0.66 [95% confidence interval, 0.51-0.80] (Figure 3A). According to the optimal cut-off for predicting the occurrence of death with the ROC curve, muscle mass loss was defined as TPTI<15.2 mm/m and normal muscle mass as TPTI ≥15.2 mm/m; then a low muscle mass was associated with higher mortality (log rank test: p<0.01) (Figure 3B). Thirty-three percent of cirrhotic patients had low muscle mass at registration on waiting-list for liver transplantation, and had an overall mortality of 14% (vs. 4% in the normal muscle mass group).

Feasibility and interobserver agreement of the measurements of transversal (TDPM) and axial (ADPM) diameters of the psoas muscle

TDPM and ADPM measurements were easily performed in 100% of patients by the non-trained operator (AH). There was an almost perfect interobserver agreement: kappa coefficient correlations between the two operators-blinded TDPM and ADPM measurements (n=50) were respectively: k=0.97, p<0.0001; k=0.94, p<0.0001. This very good agreement between two operators of opposite levels of radiologic expertise suggests the reliability of the measurements by a non-trained operator.

DISCUSSION

In this cohort of 173 patients with liver cirrhosis on waiting-list for liver transplantation, a low TDPM measured on an umbilicus-targeted CT scan image and normalized by height (i.e. TPTI) was independently associated with mortality. The increase of 1 mm/m of the TPTI was associated with a 13% decrease in death risk. This study confirms that the assessment of muscle mass from an abdominal CT scan is an accurate method to assess mortality in liver cirrhosis patients on waiting-list for liver transplantation. Our study clearly showed that the measurement of the transversal diameter of the right psoas muscle is an easy (i.e. no need for dedicated software such as for the measurement of L3 skeletal muscle index), quick, cheap (i.e. no additional costs as abdominal CT are routinely performed before registration on liver transplantation list), and highly reproducible method, and most of all, reliable by a non-expert operator, i.e. without any specific radiological competence. These make the TDPM the elective tool, accessible for all, to assess the liver cirrhosis prognosis for the daily clinical practice of Liver Disease, Radiology, and Liver Transplantation Departments.

Our study confirms the findings by Durand et al [START_REF] Durand | Prognostic Value of Muscle Atrophy in Cirrhosis using Psoas Muscle Thickness on Computed Tomography[END_REF], but in a whole population of cirrhotic patients waiting for liver transplantation. Indeed, Durand et al only studied TPTI impact on mortality on liver transplantation waiting list in a subgroup of liver cirrhosis patients with refractory ascites and MELD<25 [START_REF] Golse | A New Definition of Sarcopenia in Patients with Cirrhosis Undergoing Liver Transplantation[END_REF]. We found that the optimal TPTI cut-off to predict mortality was 15.22 mm/m with an AUC of 0.66. Comparable thresholds have been reported in the literature [START_REF] Masuda | Sarcopenia Is a Prognostic Factor in Living Donor Liver Transplantation[END_REF][START_REF] Durand | Prognostic Value of Muscle Atrophy in Cirrhosis using Psoas Muscle Thickness on Computed Tomography[END_REF]. The recent study by Golse al [START_REF] Golse | A New Definition of Sarcopenia in Patients with Cirrhosis Undergoing Liver Transplantation[END_REF] compared predictability for 1-year liver transplantation survival with different muscle indexes measured on a CT-scan cross section between L3 and the fourth lumbar vertebra: AUC values of right and left psoas muscle area, psoas muscles area normalized by height or body surface area, and the L3 skeletal muscle index, varied between 0.72 and 0.75. Psoas muscles area alone (below 1561 mm 2 in men, or below 1464 mm 2 in women) best predicted survival [START_REF] Golse | A New Definition of Sarcopenia in Patients with Cirrhosis Undergoing Liver Transplantation[END_REF]. This method looks like as less easy and more time-consuming that the measurement of TPTI based on the right psoas only. The predictive role of the transversal diameters of the psoas muscles was not assessed by Golse et al [START_REF] Golse | A New Definition of Sarcopenia in Patients with Cirrhosis Undergoing Liver Transplantation[END_REF]. More studies are needed in larger populations of cirrhotic patients to validate the optimal cut-off of TPTI or other muscle mass indexes, and most importantly the best indicator of muscle mass associated with long term prognosis. In all published studies, including the one presented here, the AUC values below 0.80 of the different muscle indexes suggested that the mortality prediction could be improved by associating muscle indexes prognostic with other variables associated with mortality such as refractory ascites or MELD, as previously suggested [START_REF] Montano-Loza | Inclusion of Sarcopenia within MELD (MELD-Sarcopenia) and the Prediction of Mortality in Patients with Cirrhosis[END_REF][START_REF] Durand | Prognostic Value of Muscle Atrophy in Cirrhosis using Psoas Muscle Thickness on Computed Tomography[END_REF]. Indeed a MELD-psoas score outperformed MELD score alone to predict mortality on liver transplant list in subgroups restricted to patients with refractory ascites [START_REF] Golse | A New Definition of Sarcopenia in Patients with Cirrhosis Undergoing Liver Transplantation[END_REF], and previous history of variceal haemorrhage [START_REF] Montano-Loza | Inclusion of Sarcopenia within MELD (MELD-Sarcopenia) and the Prediction of Mortality in Patients with Cirrhosis[END_REF]. Future studies should focus on determining whether the combination of several predictive factors, e.g. TPTI and MELD score, could improve the mortality prediction after liver transplantation.

In our study, contrary to TPTI, MELD score alone was not associated (P<0.08) with mortality in the multivariable analysis. This was expected, because, since the MELD score is used to determine patients' priority to liver transplantation, the time to access to transplantation and the mortality on the waiting-list have been reduced. Therefore patients with higher MELD scores have a much shorter waiting time on transplantation list, thus better survival, than patients with lower MELD scores. However, the MELD score fails to identify patients with malnutrition or muscle mass loss, who, despite their poor prognosis related to malnutrition, are attributed a low MELD score, and therefore are not eligible for priority liver transplantation. In 2012, the French 'Agence de Biomédecine' (Bio-Medicine Agency) reported that 20% of liver cirrhosis with a low MELD score were registered on waiting-list for liver transplantation. These included patients with cirrhosis complications, such as refractory ascites, encephalopathy, hepatorenal syndrome, hepatopulmonary syndrome, or malnutrition. In these cases, except for malnutrition, the appeal to expert component allows deciding priority liver transplantation according to disease severity. Despite presumed muscle mass loss-related poor prognosis, malnutrition is not considered as a liver transplantation priority indication. Thanks to TPTI measurement, early identification of cirrhosis complicated with malnutrition, i.e. muscle mass loss, should allow triggering an early nutritional intervention with the aim to improve patients' outcome on waiting list for liver transplantation.

Anthropometric methods such as mid-arm muscle circumference should be abandoned because of their interobserver variability to assess muscle mass. Also in cirrhotic patients with ascites, bioimpedance analysis lacks of reliability [START_REF] Pirlich | Bioelectrical Impedance Analysis Is a Useful Bedside Technique to Assess Malnutrition in Cirrhotic Patients With and Without Ascites[END_REF][START_REF] Riggio | Whole Body and Regional Body Composition Analysis by Dual-Energy X-Ray Absorptiometry in Cirrhotic Patients[END_REF][START_REF] Schloerb | Bioelectrical Impedance in the Clinical Evaluation of Liver Disease[END_REF][START_REF] Pietrobelli | Dual-Energy X-ray absorptiometry: fat estimation errors due to variation in soft tissue hydration[END_REF]. However BIA-derived phase angle could be useful to assess cirrhotic patients' prognosis [START_REF] Selberg | Norms and Correlates of Bioimpedance Phase Angle in Healthy Human Subjects, Hospitalized Patients, and Patients with Liver Cirrhosis[END_REF][START_REF] Ruiz-Margáin | Malnutrition assessed through Phase Angle and its Relation to Prognosis in Patients with Compensated Liver Cirrhosis: a Prospective Cohort Study[END_REF][START_REF] Belarmino | Phase Angle obtained by Bioelectrical Impedance Analysis independently Predicts Mortality in Patients with Cirrhosis[END_REF]. Beside radiologic assessment of muscle mass, the clinical assessment of muscle function, e.g. by handgrip strength measurement, could be of interest because of its prognostic value [START_REF] Taguchi | Sarcopenia evaluated using the Skeletal Muscle Index is a significant prognostic factor for metastatic urothelial carcinoma[END_REF]. This remains to be demonstrated in liver cirrhosis patients on waiting-list for liver transplantation.

In our study, contrary to TPTI, APTI was not predictive of mortality. We have no clear explanation for this observation. This may be because the TDPM is more sensitive in case of catabolic situations associated to decreased protein synthesis such as malnutrition or reduced physical activity. The psoas muscle is involved in the ability of standing up, staying in the upright position, and walking. Therefore, we believe that TPTI could be a marker of muscle mass, as well as muscle function, both being related to prognosis [13-20;24-25]. This remains to be demonstrated in further studies.

In this retrospective study, the mechanisms underlying the relation between low psoas muscle transversal diameter and mortality were not explored. However we could hypothesize that increased mortality is due to a defect of immune response in relation with the decrease in protein reserves. This assumption is supported by our finding (data not shown) that infectious complications are the first cause of mortality (30% of deaths) on waiting-list for liver transplantation, and that nine out of the ten patients who died from infections had a low TDPM at registration on transplantation list.

. Study limitations. The retrospective and monocentric design of the study exposed to the risk of bias. Nevertheless the characteristics of the study population were very similar to those of similar studies [START_REF] Tandon | Severe Muscle Depletion in Patients on the Liver Transplant List: Its Prevalence and Independence Prognostic Value[END_REF][START_REF] Golse | A New Definition of Sarcopenia in Patients with Cirrhosis Undergoing Liver Transplantation[END_REF], and the proportion of missing data was less than 3%. Umbilicus-targeted CT scan could suffer from a lack of precision, particularly in the case of umbilical hernia, sacralisation of the L5 vertebrae, lumbar wedge fractures, and pronounced lordosis, frequent situations in the presence of ascites. However a lack of precision and a greater risk of error are also observed when analysing L3/L4-targeted images, in cases of degenerative bone diseases, i.e. vertebral osteoporosis, or lordosis related to ascites.

In conclusion, the transverse psoas muscle index measured by umbilicus-targeted CT scan is a predictive factor of mortality in cirrhotic patients on waiting-list for liver transplantation. This easy, cheap, reproducible, and reliable method is routinely feasible even by non-trained staff.

  cirrhosis from 01/01/2011 to 31/12/2014 were eligible. The date of 01/04/2011 was chosen because CT-scan images are included in the CHU Rennes computerized medical records only since this date. To be included, patients must have undergone an abdominal CT scan within the 3 months before registering on the waiting-list for liver transplantation. Exclusion criteria were hepatocellular carcinoma as the indication for liver transplantation, multi-organ transplant, and temporary contraindication to transplantation during the follow-up. The study protocol conformed to the ethical guidelines and the 1975 Declaration of Helsinki as reflected in a priori exemption by the appropriate institutional review committee.

  software, the computerized biomedicine agency database. The data were then collected by consultation of computerized files and included age, gender, body weight, height, aetiology of cirrhosis, year of cirrhosis diagnosis, presence of ascites (none, refractory), presence of hepatic encephalopathy, serum albumin, serum bilirubin, prothrombin time, serum sodium level, and serum creatinine. Clinical and laboratory data were obtained at the registration on liver transplantation list. Child Pugh and Model for End-Stage Liver Disease (MELD) scores were calculated.

Table 1 - 1 #

 11 Prospective randomized controlled trials should assess whether a dedicated nutritional intervention combining nutrition support and physical rehabilitation in liver cirrhosis patients with low muscle mass could improve the clinical outcome of liver cirrhosis patients before and after liver transplantation. designed the study, and carried out the collection of data. CZ, LL, and MR carried out the 316 collection of data. KB and DG carried out the collection of data, and drafted the manuscript. 317 Liver cirrhosis patients' baseline characteristics, at the time of their inscription on liver transplantation list (n=173). creatinine ( mol/l) -mean ± SD 0 85.5 ± 50.3 Plasma sodium (mmol/l) -mean ± SD 0 135 ± 5 APTI (mm/m) -mean ± SD 0 22.6 ± 3.6 TPTI (mm/m) -mean ± SD 0 17.0 ± 4.Others included biliary cirrhosis, autoimmune hepatitis, sclerosing cholangitis, hemochromatosis. APTI, Axial Psoas Thickness Index; CI, confidence interval; HR, hazard ratio; MELD, Model for End-Stage Liver Disease; SD, standard deviations; TPTI, Transversal Psoas Thickness Index.

#Table 3 -Figure 1 -

 31 Figures legends

Figure 2 -

 2 Figure 2 -Study flow chart.

Figure 3 -

 3 Figure 3 -Predictive value of Transversal Psoas Thickness Index (TPTI) on mortality of liver

  Figure 1

Figure 3 A

 3 Figure 3

Table 2 -Univariate analysis of variables associated with mortality on waiting-list for liver transplantation according to univariate Cox analysis (n=173).

 2 Mortality was defined as the occurrence of death on waiting-list for liver transplantation; as previously published[START_REF] Englesbe | Sarcopenia and Mortality after Liver Tranplantation[END_REF], patients removed from the transplantation waiting list because of the worsening of their liver cirrhosis were considered as deaths.

		Alive	Dead	HR [95% CI] P-value
		n=160	n=13
	Male / female -n	126 (78) /	9 (69) /	1.38 [0.77 -	0.28
	(%)	34 (22)	4 (31)	2.49]
	Age (year) -mean ±	54.4 ±	58.5 ± 6.0 1.06 [0.98 -	0.13
	SD	10.0		1.14]
	Body mass index	26.0 ± 4.6 27.7 ± 5.2 1.08 [0.96 -	0.19
	-mean ± SD			1.22]
	Etiology of cirrhosis
	-n (%)
	Alcohol	114 (71.3)	9 (69)	Reference	0.97
	Metabolic	12 (7.5) 3	1 (8)0	1.12 [0.14 -	0.49
	HBV	(1.9)	2 (15)	8.96]	0.93
	HCV	11 (6.9)	1 (8)	0.00 [0.00 -	0.98
	Others #	20 (12.5)		inf]	0.78
				1.24 [0.26 -
				5.81]
				0.53 [0.07 -
				4.24]
	Duration of disease	48 ± 57	39 ± 41	0.99 [0.98 -	0.36
	-mean ± SD			1.00]
	Child-Pugh score	10 ± 2.2	10.6 ± 1.1	1.45 [1.08 -	0.014
	-mean ± SD			1.96]
	A -n (%) B-n (%)	17 (11) 39 (24)	0 (0) 2 (15)	-	-
	C-n (%)	104 (65)	11 (85)	--	--
	Severe/ Refractory	49	8 (61.5%) 4.34 [1.31 -	0.016
	ascites	(30.6%)		14.30]
	Plasma bilirubin	134.1 ±	68.4 ±	1.00 [0.99 -	>0.99
	( mol/l) -mean ±	159.0	39.8	1.01]
	SD
	Plasma albumin (g/l)	31.5 ± 6.1 30.6 ± 6.4 0.95 [0.86 -	0.29
	-mean ± SD			1.04]
	Prothrombin time	44.3 ±	44.5 ±	0.97 [0.93 -	0.11
	(%) -mean ± SD	19.2	10.2	1.01]
	MELD score -mean 21.2 ± 8.3 19.9 ± 3.3 1.12 [1.01 -	0.033

Acknowledgements:

We thank Dr Koch, radiologist in Rennes University Hospital (CHU Rennes), Rennes, France, for his precious help for CT-scan analysis.

Conflict of interest statement: none. The authors have no conflict of interest to declare.

Statement of authorship:

AH, ML, EBJ and RT conceived and designed the study, carried out the collection of data, interpreted the data, performed the statistical analyses, and drafted the manuscript. PHD B