Cosmin Dragos Ciocan

Sebastian Voican

Laura Wrzosek

Cindy Hugot

Dominique Rainteau

Lydie Humbert

Dr Anne-Marie Cassard

Gabriel Perlemuter
email: gabriel.perlemuter@aphp.fr.20

Sebastian Cosmin

Voican

Prof Gabriel Perlemuter

Bile acid homeostasis and intestinal dysbiosis in alcoholic hepatitis

Keywords: Bile acid homeostasis and intestinal dysbiosis in alcoholic hepatitis Bile acid, microbiota and alcoholic hepatitis 2 16S sequencing, microbiota, UDCA, biotin, glutathione, Actinobacteria, 58 Bacteroidetes 59

2 statistical analysis, and wrote the manuscript. CSV designed the project and included 26 patients. LW designed the project and performed presequencing preparation 27 procedures. CH performed presequencing preparation procedures. DR and LH 28 performed bile acids analyses. AMC designed, performed and supervised 29 experiments and wrote the manuscript. GP designed the project, included patients, [START_REF] Chierico | Gut 651 microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients 652 unveiled by an integrated meta-omics-based approach[END_REF] supervised experiments and wrote the paper. All authors discussed the results, 31 commented and approved the final version of the manuscript. 32

Acknowledgments. The authors thank the NED group (Olivier Zemb, Béatrice 33 Gabinaud) and the GeT-PlaGe platform for their help with the sequencing data.

INTRODUCTION 60

Severe alcoholic hepatitis is a life-threatening complication seen in a subset of 61 patients with alcoholic liver disease with a mortality rate of up to 25% and few 62 therapeutic options [START_REF] Orrego | Prognosis of alcoholic cirrhosis in the 569 presence and absence of alcoholic hepatitis[END_REF][START_REF] Study | EASL clinical practical guidelines: 571 management of alcoholic liver disease[END_REF]. The causal role of the intestinal microbiota in the 63 development and individual susceptibility to alcoholic hepatitis has only recently been 64

shown [START_REF] Yan | Enteric dysbiosis 573 associated with a mouse model of alcoholic liver disease[END_REF][START_REF] Llopis | Intestinal 576 microbiota contributes to individual susceptibility to alcoholic liver disease[END_REF][START_REF] Cassard | Microbiota, a key player in alcoholic liver disease[END_REF][START_REF] Ciocan | 581 Characterization of intestinal microbiota in alcoholic patients with and without alcoholic 582 hepatitis or chronic alcoholic pancreatitis[END_REF]. It has also been suggested that faecal microbiota transplantation may 65 improve gut dysbiosis and clinical outcomes in patients with cortico-resistant severe 66 alcoholic hepatitis in a recent pilot study [START_REF] Philips | Healthy 584 Donor Fecal Microbiota Transplantation in Steroid-Ineligible Severe Alcoholic 585 Hepatitis: A Pilot Study[END_REF]. Nevertheless, the mechanisms related to 67 the role of the intestinal microbiota in alcoholic liver disease are not fully understood. 68

The relationship between bile acids and the intestinal microbiota is complex. Bile 69 acids have both direct antimicrobial effects on bacteria [START_REF] Begley | The interaction between bacteria and bile[END_REF], and indirect effects 70 through their signaling proprieties which allows them to induce antimicrobial peptides 71 production [START_REF] Inagaki | Regulation of 589 antibacterial defense in the small intestine by the nuclear bile acid receptor[END_REF]. Detergent properties of bile acids, needed for fat digestion, influence 72 the composition of the intestinal microbiota by acting on bacterial cell membranes [START_REF] Begley | The interaction between bacteria and bile[END_REF]. 73

However, the diversity of the bile acids pool and enterohepatic circulation are 74 dependent on the intestinal microbiota. Indeed, primary bile acids (cholic acid, CA, 75 and chenodeoxycholic acid, CDCA) are synthesized in the liver, but secondary bile 76 acids are produced in the digestive tract. The complex pool of bile acids is then 77 reabsorbed in the portal circulation via a large panel of transporters. In addition, bile 78 acids are signaling molecules involved in regulating hepatic metabolism, 79 inflammation, and their own synthesis through the activation of various nuclear 80 receptors, such as the farnesoid X receptor (FXR) [START_REF] Fickert | Biliary bile acids in hepatobiliary injury -What is the link?[END_REF]. 81 Chronic alcohol consumption is associated with an impaired bile acids homeostasis 82 [START_REF] Trinchet | Serum bile acids and 594 cholestasis in alcoholic hepatitis. Relationship with usual liver tests and histological 595 features[END_REF][START_REF] Kakiyama | Colonic 597 inflammation and secondary bile acids in alcoholic cirrhosis[END_REF][START_REF] Bajaj | Continued Alcohol 600 Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis[END_REF](14). The level of plasma bile acids positively correlates with the histological 83 severity of AH [START_REF] Trinchet | Serum bile acids and 594 cholestasis in alcoholic hepatitis. Relationship with usual liver tests and histological 595 features[END_REF] and is predictive of poor patient survival [START_REF] Nissenbaum | Prognostic significance of 606 cholestatic alcoholic hepatitis. VA Cooperative Study Group #119[END_REF]. Moreover, FXR-84 5 specific agonists attenuate chronic alcohol-induced liver injury and steatosis in 85 experimental alcoholic liver disease models [START_REF] Lívero | The FXR agonist 6ECDCA reduces hepatic steatosis and oxidative stress induced by 610 ethanol and low-protein diet in mice[END_REF][START_REF] Wu | Activation of farnesoid X receptor 612 attenuates hepatic injury in a murine model of alcoholic liver disease[END_REF]. Conversely, FXR-deficient mice 86 develop more severe liver injury [START_REF] Wu | Activation of farnesoid X receptor 612 attenuates hepatic injury in a murine model of alcoholic liver disease[END_REF]. Overall, these results suggest that bile acids-87 dependent hepatotoxicity may be due, in part, to impaired FXR signaling. 88

We have shown in a recent work that patients with severe alcoholic hepatitis have a 89 specific dysbiosis that renders their liver more susceptible to alcohol-induced injury 90 (4). This sensitivity was transmissible from patients to mice by intestinal microbiota 91 transplant. In these humanized mice, the bile acids pool was impaired in the feces. 92

Moreover we also showed that alcoholic patients that develop severe alcoholic 93 hepatitis have a different microbial composition as compared to patients that develop 94 other types of complications such as alcoholic pancreatitis [START_REF] Ciocan | 581 Characterization of intestinal microbiota in alcoholic patients with and without alcoholic 582 hepatitis or chronic alcoholic pancreatitis[END_REF]. 95 However, while both the bile acids and intestinal microbiota profiles were reported in 96 alcoholic liver disease, these studies focused either only on the bile acids profile 97 [START_REF] Trinchet | Serum bile acids and 594 cholestasis in alcoholic hepatitis. Relationship with usual liver tests and histological 595 features[END_REF][START_REF] Aranha | Bile 615 acid levels are increased in the liver of patients with steatohepatitis[END_REF] or intestinal microbiota profile (4) and never on both, in the same cohort of 98 patients or didn't included patients with severe alcoholic hepatitis [START_REF] Kakiyama | Colonic 597 inflammation and secondary bile acids in alcoholic cirrhosis[END_REF][START_REF] Bajaj | Continued Alcohol 600 Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis[END_REF]. In order to 99 study the relationships between intestinal microbiota modifications and bile acids 100 metabolism, we investigated herein the interplay between bile acids and intestinal 101 microbiota in well phenotyped patients at different stages of alcoholic liver disease by 102 assessing and comparing plasma and faecal bile acids profiles and intestinal 103 microbiota composition and functions in currently drinking alcoholic patients 104 according to the severity of liver lesions. 105 6 METHODS 106

Study subjects 107

All patients included in this prospective study were admitted to the Hepato-108

Gastroenterology and Nutrition Department of Antoine-Béclère University Hospital, 109

Clamart, France, for the management of excessive drinking. 110 Alcoholic patients were eligible for inclusion if they were between 17 and 75 years old 111 and had been consuming at least 50 g of alcohol/day and were negative for hepatitis 112 B surface antigens and hepatitis C. The exclusion criteria were gastrointestinal 113 bleeding, bacterial infection, hepatocellular carcinoma, any other carcinoma, other 114 associated severe diseases, the presence of anti-HIV antibodies, antibiotic intake in 115 the last three months, probiotic drugs use, refusal to undergo a liver biopsy if required 116 (abnormal liver function), use of any hepatoprotective treatment (UDCA, TUDCA). A 117 standardized questionnaire was used to collect information about alcohol 118 consumption [START_REF] Williams | Variations in quantity-frequency 618 measures of alcohol consumption from a general population survey[END_REF] and patients' families were also interviewed, when possible. 119

General demographic and clinical characteristics were recorded for all patients at 120 inclusion. The study was carried out in accordance with the Helsinki Declaration and 121 was approved by the Ile de France VII ethics committee (Bicêtre Hospital, 94270 le 122 Kremlin-Bicêtre, France). All patients provided written informed consent for 123 participation in the study. 124

Patients were classified into four groups: 125

• patients with alcoholic cirrhosis and severe alcoholic hepatitis (Cir_sAH, n = 126 17). Severe alcoholic hepatitis was suspected in patients with a Maddrey 127 score > 32 and was confirmed by a liver biopsy (histological score for AH ≥ 6 128 with neutrophilic infiltration) [START_REF] Llopis | Intestinal 576 microbiota contributes to individual susceptibility to alcoholic liver disease[END_REF][START_REF]Alcoholic liver disease: morphological manifestations. Review by an international 621 group[END_REF]. 129 7

• patients with alcoholic cirrhosis, but without severe alcoholic hepatitis 130 (Cir_noAH, n = 17). As the impact of non-severe alcoholic hepatitis was limited 131 on the parameters that we studied in non-cirrhotic patients, we pooled the 132 patients with mild alcoholic hepatitis with patients with no alcoholic hepatitis in 133 the cirrhotic patients group. 134

• patients without alcoholic cirrhosis or alcoholic hepatitis (noCir_noAH, n = 61), 135

• patients without alcoholic cirrhosis, but with alcoholic hepatitis (noCir_AH, n = 136 13). Alcoholic hepatitis was defined by aspartate aminotransferase>50, 137 aspartate aminotransferase/alanine aminotransferase > 1.5, and both values < 138 400 IU/L [START_REF]Alcoholic liver disease: morphological manifestations. Review by an international 621 group[END_REF][START_REF] Crabb | Standard 623 Definitions and Common Data Elements for Clinical Trials in Patients With Alcoholic 624 Hepatitis: Recommendation From the NIAAA Alcoholic Hepatitis Consortia[END_REF] or, if a liver biopsy was available (12/13 patients), an AH 139 score between 3 and 5 (with neutrophilic infiltration) or ≥ 6 (with neutrophilic 140 infiltration and a Maddrey score <32). 141

Diagnosis of cirrhosis was made based on clinical examination, laboratory test, 142

imaging and endoscopy studies or by a liver biopsy, when available. As patients with 143 cirrhosis have a different intestinal microbiota profile than those without, and most 144 patients with severe alcoholic hepatitis exhibit histological evidence of micronodular 145 cirrhosis, we did not perform a global comparison between the four groups but 146 separately compared the patients with and without cirrhosis. 147

Biochemical assays 148

Bile acids measurements in plasma of 55 patients and feces of 73 patients were 149 performed using high-performance liquid chromatography-tandem mass 150 spectrometry as previously described [START_REF] Humbert | Bile acid 627 profiling in human biological samples: comparison of extraction procedures and 628 application to normal and cholestatic patients[END_REF]. Serum fibroblast growth factor-19 (FGF-151 19) was measured for 55 patients using a sandwich ELISA kit (R&D Systems) 152 according to the manufacturer's instructions. 153

Analysis of the intestinal microbiota by 16S ribosomal RNA sequencing 154 8

Faecal samples were available for 96 patients. The composition of the faecal 155 microbiota was analyzed by high-throughput sequencing with Illumina MiSeq 156 technology, targeting the 16S ribosomal DNA V3-V4 region in the paired-end mode 157

(2 x 300 base pairs) (GenoToul, Toulouse), as previously described [START_REF] Ferrere | Fecal 631 microbiota manipulation prevents dysbiosis and alcohol-induced liver injury in mice[END_REF]. Data were 158 processed with the quantitative insights into microbial ecology (QIIME v1.9.0) 159 pipeline, using its default parameters. Closed reference operation taxonomic 160 mapping was performed using the Greengenes database (v13.8, 97% sequence 161 similarity). 162

The mean number of quality-controlled reads was 26535±7840 (mean ± SD) per 163 sample (minimum count: 9833, maximum count: 56968). After rarefaction at 9,000 164 reads per sample, bacterial alpha diversity was estimated on the basis of the 165 Shannon's index. OTUs with a prevalence < 5% were removed from the analysis. 166

Functional composition of the intestinal metagenome was predicted using 167

Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 168

(PICRUSt) [START_REF] Langille | Predictive functional profiling of microbial communities using 16S rRNA marker gene 635 sequences[END_REF]. This is a computational approach that accurately predicts the 169 abundance of gene families in the microbiota and thus provides information about the 170 functional composition of the microbial community. Linear discriminant analysis (LDA) 171 effect size (LEfSe) analysis was performed to identify the taxa and functions 172 displaying the largest differences in abundance in the microbiota between groups 173 [START_REF] Segata | Metagenomic 637 biomarker discovery and explanation[END_REF]. Only taxa and functions with an LDA score > 2 and a significance of < 0.05, as 174 determined by Wilcoxon signed-rank tests, are shown. LEfSe and PICRUSt were 175 accessed online (http://huttenhower.sph.harvard.edu/galaxy/). 176

Statistical analysis 177

The results are expressed as the means ± SD for normally distributed data or median 178 The demographic and laboratory data are summarized in Table 1. There was no 201 difference in age, sex, body mass index (BMI), or duration of alcohol intake between 202 the groups. As expected, patients with noCir_AH had higher aspartate 203 aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, gamma-204 glutamyl transferase (GGT), and C-reactive protein levels than noCir_noAH patients. 205

[
Cir_sAH patients had lower alcohol consumption than the Cir_noAH patients and 206 higher total bilirubin and C-reactive protein levels, a higher MELD score, and lower 207 albumin levels. 208

Intestinal microbiota profiles 209

We first studied interindividual bacterial diversity (beta diversity). Cir_sAH patients 210 had a different intestinal microbiota structure (proportion of bacteria) than Cir_noAH 211 patients (weighted UNIFRAC distances, R = 0.09, p = 0.04) (Figure 1A). There was 212 no difference in the overall bacterial composition of the intestinal microbiota between 213 these two groups (unweighted UNIFRAC distances, R = 0.05, p = 0.1) (Figure 1B). 214

This result suggests that the two groups have an intestinal microbiota with similar 215 bacterial species, but with different relative abundances. There was no difference in 216 the beta diversity between noCir_noAH and noCir_AH patients (data not shown Christensenellaceae families (Firmicutes phylum) and a higher relative abundance of 229 Lactobacillus and Lactococcus (Firmicutes phylum) than Cir_noAH patients (Figure 230 1C and Supplemental Table 1). 231

Although there was no difference in the overall composition of the intestinal 232 microbiota between noCir_AH and noCir_noAH patients, LEFsE analysis showed 233 that noCir_AH patients had a higher abundance of Dorea (Firmicutes phylum), 234 Wolbachia (Proteobacteria phylum) and Rothia (Actinobacteria phylum) than 235 noCir_noAH patients (Figure 1D). These results suggest that a specific dysbiosis is 236 associated with hepatic inflammation in AH in both patients with and without cirrhosis 237 and independently of alcohol consumption. 238

Functional Intestinal Metagenome Prediction in Alcoholic Hepatitis: 239

The dysbiosis identified in Cir_sAH patients prompted us to also examine the 240 metabolic pathways associated with this specific intestinal microbiota. The intestinal 241 12 microbiota of the Cir_sAH group had a higher proportion of metabolic pathways 242 containing gene functions, such as glutathione metabolism, membrane transport 243 (phosphotransferase system), and nucleotide metabolism than that of the Cir_noAH 244 group. The intestinal microbiota of Cir_sAH patients also had a lower proportion of 245 genes for energy metabolism (methane metabolism and carbon fixation pathways in 246 prokaryotes), amino acid metabolism (arginine, proline and histidine metabolism), 247 lipid metabolism (lipid biosynthesis proteins), glycan biosynthesis and metabolism, 248 metabolism of cofactors and vitamins (biotin metabolism), metabolism of terpenoïds 249 and polyketides (polyketide sugar unit biosynthesis), biosynthesis of other secondary 250 metabolites (streptomycin biosynthesis), and of the transcription machinery than that 251 of Cir_noAH patients (Figure 1E). There was no difference between the two groups in 252 the secondary bile acids biosynthesis pathway. 253

These results indicate that the dysbiosis observed in patients with severe alcoholic 254 hepatitis is also associated with a shift in the bacterial metabolic pathways. 255

Bile acids 256

Bile acids can shape the intestinal microbiota, and in turn, the intestinal microbiota 257 alters the bile acids pool. Thus, we studied plasma bile acids and faecal bile acids 258 profiles and their relationship with the dysbiosis observed in alcoholic patients with 259 and without alcoholic cirrhosis. 260

Plasma bile acid profile 261

We first studied the plasma bile acids profile associated with liver inflammation in 262 alcoholic patients. Cir_sAH patients had a different plasma bile acids profile than 263

Cir_noAH patients, as shown by PCA (Figure 2A) and on a heatmap (Supplementary 264 figure 1A). Cir_sAH patients had higher levels of total bile acids, total primary bile 265 13 acids, total conjugated bile acids (total glyco-and tauroconjugated bile acids), 266 primary glyco-and tauroconjugated bile acids, total CA (glycocholate (GCA) and 267 taurocholate (TCA)), total CDCA (glycochenodeoxycholate (GCDCA) and 268 taurochenodeoxycholate (TCDCA)), and total UDCA (tauroursodeoxycholate 269 (TUDCA)) than Cir_noAH patients (Figures 2 B-D and Supplementary Table 2). 270

Because of the higher total bile acids levels in Cir_sAH patients, we also studied the 271 relative proportion of each bile acids (bile acids concentration/total bile acids 272 concentration) between the two groups. Cir_sAH patients had a higher relative 273 proportion of total primary bile acids, total CDCA (TCDCA), total primary conjugated 274 bile acids, primary tauroconjugated bile acids, and TUDCA than Cir_noAH patients. 275

They also had a lower relative proportion of total sulphoconjugated bile acids, total 276 secondary bile acids, total secondary conjugated bile acids, secondary 277

glycoconjugated

Cir_noAH patients (Figures 2 B, C, E). 283

We then used PLS-DA to find the plasma bile acids that best discriminate between 284 the two groups. The model showed a significant distinction (R2 = 0.6, Q2 = 0.4, 285 prediction accuracy during training: p = 0.001, 1000 permutations) between the 286 Cir_sAH and Cir_noAH groups (Figure 3A). The average AUROC confirmed that the 287 model was able to discriminate Cir_noAH from Cir_sAH patients (0.955, 95% CI: 288 0.763-1, Supplementary Figure 2A). TUDCA was the most discriminant bile acids 289 14 between the two groups according to the PLS-DA (variable importance in projection, 290 VIP = 2.1, Figure 3B). 291

Comparison of the bile acids profile of noncirrhotic patients showed higher levels of 292 TUDCA in noCir_AH patients than noCir_noAH patients. Moreover, noCir_AH 293 patients had higher proportions of TUDCA and total conjugated bile acids and a 294 lower proportion of DCA than noCir_AH patients (data not show). However, these 295 changes were no longer significant after correction for multiple comparisons. 296

These results indicate an increase in the pool of bile acids in Cir_sAH patients 297 Moreover, the increase in TUDCA levels, a bile acids produced exclusively by the 298 intestinal microbiota from CDCA, and that of primary bile acids s are consistent with a 299 shift in bile acids transformation in the gut. 300

Faecal bile acids 301

We further studied the bile acids profile in Cir_sAH patients. Cir_sAH patients had a 302 different faecal bile acids profile than Cir_noAH patients (Figure 4A and 303 Supplementary Figure 1B). Cir_sAH patients had lower total faecal bile acids, total 304 unconjugated bile acids, total glycoconjugated bile acids, total secondary bile acids, 305 secondary unconjugated bile acids, secondary glyco-and tauroconjugated bile acids, 306 total LCA (LCA), and total DCA (DCA, GDCA, TDCA, deoxycholate 3-sulfate: 307 DCA3s) than Cir_noAH patients (Figures 4B-D and Supplementary Table 3). We also 308 examined the relative amount of each bile acids (faecal bile acids/total faecal bile 309 acids). Cir_sAH patients had a higher percentage of total primary bile acids, total 310 CDCA (CDCA, TCDCA), primary unconjugated bile acids, and CA than Cir_noAH 311 patients and a lower percentage of total secondary bile acids, total secondary 312 15 unconjugated bile acids, secondary glycoconjugated bile acids, secondary 313 tauroconjugated bile acids, and total DCA (DCA, GDCA, TDCA) (Figures 4 B,C,E). 314 PLS-DA showed a significant distinction (R2 = 0.8, Q2 = 0.6, prediction accuracy 315 during training: p = 0.001, 1000 permutations) between the Cir_sAH and Cir_noAH 316 groups (Figure 5A). The average AUROC confirmed that the model was able to 317 discriminate Cir_noAH from Cir_sAH patients (0.977, 95% CI: 0.769-1) 318 (Supplementary Figure 2B). Secondary glycoconjugated bile acids and GDCA were 319 the most discriminant bile acids between the two groups according to the PLS-DA 320 (variable importance in projection, VIP = 2.1 and 2.1, respectively, Figure 5B). 321

Comparison of the faecal bile acids profiles between noncirrhotic patients showed the 322 observed changes (higher taurolithocholate, TLCA, and GLCA ratios in noCir_AH 323 than noCir_noAH patients) to no longer be significant after correction for multiple 324 comparisons (data not show), as observed in plasma. 325

These results confirm the decrease in bile acids excretion and impaired bile acids 326 transformation in the gut by the intestinal microbiota of Cir_sAH patients relative to 327 that of Cir_noAH patients. 328

The relationship between intestinal microbiota and bile acids homeostasis 329

We assessed the correlation between the bile acids profiles with the bacteria species 330 identified in the feces, as the composition and quantity of the bile acids pool influence 331 the intestinal microbiota, and conversely, the metabolism of bile acids is dependent 332 on intestinal microbiota composition. Primary and secondary plasma bile acids levels 333 positively correlated with most of the taxa in Cir_noAH patients (Figure 6A) while 334 primary plasma bile acids negatively correlated with taxa from Bacteroidetes and 335 Firmicutes phyla in Cir_sAH patients (Figure 6B). Total UDCA, TUDCA, and 336 16 glycoursodeoxycholate (GUDCA) positively correlated with most of the taxa in 337 Cir_noAH patients while it was negatively correlated with taxa from the Cir_sAH 338 patients (Figure 6A and6B). 339 Faecal primary bile acids were mostly negatively correlated with most of the taxa in 340 Cir_noAH patients (Figure 6C) while in Cir_sAH patients, primary faecal bile acids 341 were negatively correlated and secondary faecal bile acids were positively correlated 342 with most of the taxa (Figure 6D) 343

These correlations suggest that the abundance of bacteria carrying the enzymes 344 needed for bile acids deconjugation and transformation into secondary bile acids and 345 UDCA is reduced in the intestinal microbiota of Cir_sAH patients. 346

FXR-FGF-19 in severe alcoholic hepatitis 347

As signaling molecules, bile acids activate ileal FXR and induce the production of 348 FGF19. Cir_sAH patients had higher plasma levels of FGF19 than Cir_noAH patients 349 (282 ± 431 vs. 55 ± 75 pg/mL, p = 0.03, Table 1). In patients without cirrhosis, 350 plasma levels of FGF19 were higher in patients with AH patients than those without, 351 but did not reach statistical significance (154 ± 368 vs. 69 ± 74 pg/mL, p = 0.5). 352 In this study, we characterized the intestinal microbiota, its functions, and its 358 relationship with bile acids homeostasis in well phenotyped alcoholic liver disease 359 patients, in order to overcome potential confounders such as alcoholic liver disease 360 stage and previous or concomitant treatments. Moreover, as alcohol induces a 361 specific dysbiosis in both animal models of alcoholic liver disease [START_REF] Llopis | Intestinal 576 microbiota contributes to individual susceptibility to alcoholic liver disease[END_REF][START_REF] Ferrere | Fecal 631 microbiota manipulation prevents dysbiosis and alcohol-induced liver injury in mice[END_REF]27) and 362 humans [START_REF] Kakiyama | Colonic 597 inflammation and secondary bile acids in alcoholic cirrhosis[END_REF][START_REF] Bajaj | Continued Alcohol 600 Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis[END_REF][START_REF] Mutlu | Colonic 644 microbiome is altered in alcoholism[END_REF], including higher levels of some members of Proteobacteria and 363 lower Bacteroidaceae, Lachnospiraceae, and Prevotellaceae, we only compared 364 patients with ongoing alcohol consumption. This allowed us to identify specific 365 changes related only to the liver disease, independently on the amount and duration 366 of alcohol consumption. 367 Alcoholic hepatitis did not modify the overall composition of the intestinal microbiota 368 in patients without cirrhosis. A similar result has been recently reported in a mouse 369 model of acute-on-chronic alcohol feeding [START_REF] Lowe | Alcohol-related changes in the intestinal microbiome influence neutrophil infiltration, 648 inflammation and steatosis in early alcoholic hepatitis in mice[END_REF]. However, we observed an increase 370 in the abundance of Dorea, Wolbachia and Rothia in noCir_AH patients. Among 371 patients with alcoholic cirrhosis, severe alcoholic hepatitis patients had higher 372 abundance of bacteria of the Actinobacteria phylum including the Actinomyces, 373 Rothia, and Bifidobacterium genus. The abundance of Lactobacillus (Firmicutes 374 phylum), Haemophylus (from the Pasteurellaceae family, Proteobacteria phylum), 375 and an unidentified member of the Enterobacteriaceae family (Proteobacteria 376 phylum) was also higher. Conversely, the abundance of bacteria of the Bacteroidetes 377 phylum was lower. Interestingly, these changes are consistent with data from other 378 studies that investigated the intestinal microbiota in alcoholic liver disease and in 379 other liver diseases such as NAFLD [START_REF] Mutlu | Colonic 644 microbiome is altered in alcoholism[END_REF][START_REF] Lowe | Alcohol-related changes in the intestinal microbiome influence neutrophil infiltration, 648 inflammation and steatosis in early alcoholic hepatitis in mice[END_REF][START_REF] Chierico | Gut 651 microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients 652 unveiled by an integrated meta-omics-based approach[END_REF][START_REF] Chen | Microbiota 655 Protects Mice Against Acute Alcohol-Induced Liver Injury[END_REF][START_REF] Chen | Characterization of fecal 658 microbial communities in patients with liver cirrhosis[END_REF], suggesting that these changes may be 380 related to cirrhosis and impaired liver function rather than to the cause of liver 381 18 disease. These results also confirms the increase seen in Bifidobacterium genus in 382 severe alcoholic hepatitis patients that we previousely reported in a smaller sample of 383 severe alcoholic hepatitis patients (4). Furthermore, by increasing the number of 384 patients included in the present study we also identifies new taxa associated with 385 severe alcoholic hepatitis as compared to our previous study. 386

We further explored the role of the intestinal microbiota in Cir_sAH using PICRUSt to 387 predict the metagenomic profile of the intestinal microbiota. We observed a switch in 388 the functions of the intestinal microbiota in Cir_sAH patients, including a decrease in 389 the biotin metabolic pathway. Biotin is a member of the vitamin-B family of vitamins 390 and acts as a cofactor for several carboxylases in mitochondria. Exogenous biotin is 391 obtained from dietary sources or intestinal biotin-producing bacteria [START_REF] Zempleni | Biotin. Biofactors[END_REF]. Plasma 392 biotin levels in chronic alcohol patients are reduced due to inhibition of carrier-393 mediated biotin transport in the jejunum and colon [START_REF] Subramanya | Inhibition of 662 intestinal biotin absorption by chronic alcohol feeding: cellular and molecular 663 mechanisms[END_REF]. Thus, reduced production in 394 the gut and decreased absorption in alcoholic patients could lead to the dysfunction 395 of mitochondria, which could impair the hepatic response to inflammation in severe 396 alcoholic hepatitis. 397

We also observed altered glutathione metabolism in the intestinal microbiota of 398 Cir_sAH patients. Glutathione is a powerful antioxidant and patients with alcoholic 399 liver disease have low hepatic and plasma glutathione levels [START_REF] Lee | Abnormal 665 hepatic methionine and glutathione metabolism in patients with alcoholic hepatitis. 666[END_REF]. In this context, it 400 has been shown that the bile salt hydrolase (BSH) gene from Bifidobacterium longum 401 is co-transcribed with the gene encoding glutamine synthetase adenylyltransferase 402 (glnE), a component of the nitrogen regulation cascade [START_REF] Tanaka | Bile salt hydrolase of Bifidobacterium longum-668 biochemical and genetic characterization[END_REF]. Thus, the increase in 403 the abundance of Bifidobacterium in Cir_sAH patients could be responsible, at least 404 partially, for the increased proportion of primary unconjugated faecal bile acids, as a 405 19 result of BSH activity and increased glutathione metabolism of the intestinal 406 microbiota of these patients. 407

It has been suggested, in a previous work, that an increase in primary bile acids was 408 associated with the severity of histological lesions in AH [START_REF] Trinchet | Serum bile acids and 594 cholestasis in alcoholic hepatitis. Relationship with usual liver tests and histological 595 features[END_REF]. A similar result was 409 observed in our study. Of note, in the previous work [START_REF] Trinchet | Serum bile acids and 594 cholestasis in alcoholic hepatitis. Relationship with usual liver tests and histological 595 features[END_REF], the authors provided an 410 overall plasma bile acids profile in patients with AH ranging from mild AH to severe 411 alcoholic hepatitis and irrespective of the presence of cirrhosis, that was present in 412 75 % of their patients. As alcoholic cirrhosis is associated with an impaired bile acids 413 profile [START_REF] Kakiyama | Colonic 597 inflammation and secondary bile acids in alcoholic cirrhosis[END_REF][START_REF] Bajaj | Continued Alcohol 600 Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis[END_REF], their results might be biased by the mix of cirrhotic and non-cirrhotic 414 patients. Moreover, according to the method used for bile acids assay (HPLC), they 415 could only detect primary and secondary bile acids but neither their conjugated forms 416 nor UDCA. Other studies have investigated plasma bile acids, faecal bile acids and 417 intestinal microbiota in alcoholic patients [START_REF] Kakiyama | Colonic 597 inflammation and secondary bile acids in alcoholic cirrhosis[END_REF][START_REF] Bajaj | Continued Alcohol 600 Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis[END_REF]. They suggested, that alcohol intake 418 in both cirrhosis and non-cirrhotic patients is associated with a decrease in 419 conjugated CDCA in plasma [START_REF] Kakiyama | Colonic 597 inflammation and secondary bile acids in alcoholic cirrhosis[END_REF]. In our study, CDCA was increased in severe 420 alcoholic hepatitis, suggesting that this increase is due to liver inflammation (ie 421 severe alcoholic hepatitis) independently of the presence of cirrhosis of alcohol 422 intake. We also suggest that perturbation of intestinal microbiota is involved in the 423 specific modifications bile acids metabolism observed in patients with severe 424 alcoholic hepatitis. 425 More hydrophobic bile acids (CA, CDCA, DCA) rapidly induce apoptosis [START_REF] Attili | Bile acid-induced liver 671 toxicity: relation to the hydrophobic-hydrophilic balance of bile acids[END_REF], 426 whereas less hydrophobic bile acids (UDCA) are less toxic [START_REF] Hillaire | Effects of ursodeoxycholic acid 674 and chenodeoxycholic acid on human hepatocytes in primary culture[END_REF]. Moreover, total 427 plasma bile acids and primary plasma bile acids (CA and CDCA) levels have been 428

shown to positively correlated with the AH severity and steatosis [START_REF] Trinchet | Serum bile acids and 594 cholestasis in alcoholic hepatitis. Relationship with usual liver tests and histological 595 features[END_REF][START_REF] Aranha | Bile 615 acid levels are increased in the liver of patients with steatohepatitis[END_REF][START_REF] Puri | The presence 677 and severity of nonalcoholic steatohepatitis is associated with specific changes in 678 circulating bile acids[END_REF]. Several 429 mechanisms may explain the increase of the bile acids pool, including upregulation of 430 20 cholesterol 7α-hydroxylase (Cyp7A1) induced by both chronic and acute alcohol 431 consumption (40) and/or a decrease in bile acids excretion in the bile and 432 subsequent release in the plasma in the context of AH. Moreover, it has also been 433

suggested that the intestinal microbiota can contribute to biliary inflammation [START_REF] Schrumpf | 683 The gut microbiota contributes to a mouse model of spontaneous bile duct inflammation[END_REF], 434 which could impair bile acids circulation. These hypotheses are supported by the 435 increased levels of primary and conjugated plasma bile acids in the Cir_sAH patients 436 and decrease in the proportion of secondary plasma bile acids and total faecal bile 437 acids. We can thus hypothesize that the excess plasma bile acids do not reach the 438 gut where they could be deconjugated and transformed into secondary bile acids. 439 Moreover, conjugation of either taurine or glycine to bile acids decreases their 440 hydrophobicity and thus their toxicity. There was also a trend towards switching the 441 plasma bile acids pool from gylcoconjugated forms towards tauroconjugated forms, 442 which are less toxic. 443 CDCA is the most potent FXR agonist capable of inducing FGF19 expression [START_REF] Zweers | 686 Prolonged fibroblast growth factor 19 response in patients with primary sclerosing 687 cholangitis after an oral chenodeoxycholic acid challenge[END_REF]. 444

Here, plasma FGF-19 levels were higher in Cir_sAH than Cir_noAH patients. FGF-19 445 is produced in the ileum by FXR activation. Faecal bile acids activates FXR and acts 446 in a negative feedback loop by blocking CYP7A1 and bile acid synthesis (classical 447 pathway). FGF-19 is absent from primary, non-activated hepatocytes, but bile acids -448 activated hepatic FXR can induce FGF19 secretion in vitro [START_REF] Song | Bile acids activate fibroblast growth 690 factor 19 signaling in human hepatocytes to inhibit cholesterol 7α-hydroxylase gene 691 expression[END_REF] and in vivo in patients 449 with cholestasis (44) by an autocrine/paracrine mechanism, independently of SHP 450 [START_REF] Song | Bile acids activate fibroblast growth 690 factor 19 signaling in human hepatocytes to inhibit cholesterol 7α-hydroxylase gene 691 expression[END_REF][START_REF] Wunsch | Expression of hepatic Fibroblast Growth Factor 19 is enhanced in Primary Biliary 694 Cirrhosis and correlates with severity of the disease[END_REF]. However, a recent study found that FGF19 levels were significantly elevated 451 in patients with alcoholic hepatitis while serum 7-alpha-hydroxy-4-cholesten-3-one 452 (C4) levels, a bile acids synthesis marker for de novo synthesis was decreased 453 suggesting a prominent role of cholestasis (14). Moreover, the authors showed that in 454 alcoholic hepatitis FGF-19 originates in cholangiocytes and ductular cells from 455 21 smaller ductules (progenitor cells). Here, FGF-19 positively correlated with the MELD 456 score. This is consistent with other studies that reported a correlation between FGF-457 19 levels and liver disease severity (14,44) and suggests that the increase in FGF-19 458 levels observed in our study has a double origin, hepatic (due to increased plasma 459 bile acids , dominated by CDCA) and intestinal. Indeed, there was a shift of the faecal 460 bile acids pool in the gut toward species with a higher affinity for FXR, as shown by 461 the increase in the proportion of CDCA, although there was an overall decrease in 462 total faecal bile acids. Thus, high levels of FGF-19 could increase CDCA synthesis by 463 directing bile acids synthesis from the classic (neutral) to the alternative (acidic) 464 pathway, due to the blockade of CYP7A1, but not of cholesterol 7β-hydroxylase 465 (CYP7B1). Therefore, high levels of FGF-19 are probably insufficient to counteract 466 the increased bile acids synthesis induced by alcohol in alcoholic liver disease. 467 Moreover, it promotes the shift of the bile acids pool towards more hydrophobic, toxic 468

species. 469

There was also an increase in total plasma UDCA and TUDCA levels in Cir_sAH 470 patients relative to Cir_noAH patients. These bile acids have hepatoprotective 471 effects. However, TUDCA exerts this effect by replenishing hepatic mitochondrial 472 glutathione [START_REF] Colell | Tauroursodeoxycholic acid protects hepatocytes from ethanol-fed rats against tumor 697 necrosis factor-induced cell death by replenishing mitochondrial glutathione[END_REF]. Thus, the increase in glutathione metabolism of the intestinal 473 microbiota, which may decrease its bioavailability to the mitochondria, combined with 474 an intestinal microbiota-associated decrease in the levels of biotin, an essential 475 cofactor of mitochondrial metabolism, could explain why the increased TUDCA levels 476 seen in Cir_sAH patients does not have a hepatoprotective effect. This is supported 477 by the fact that UDCA showed hepatoprotective effects in in vitro studies and early 478 stages of alcoholic liver disease, but not in severe alcoholic hepatitis patients with 479 cholestasis [START_REF] Pelletier | A randomized 700 controlled trial of ursodeoxycholic acid in patients with alcohol-induced cirrhosis and 701 jaundice[END_REF]. 480 22 Specific bacteria may be involved in the production of UDCA in severe alcoholic 481 hepatitis patients. Plasma UDCA, that was increased in severe alcoholic hepatitis 482 patients, and that is only produced by bacteria in the gut from CDCA, positively 483 correlated with the abundance of Actinobacteria and Proteobacteria phyla. The 484 abundance of these phyla was increased in severe alcoholic hepatitis patients, as 485 was the abundance of the Bifidobacteria and Clostridium genera. Administration of 486 Bifidobacteria animalis, as a bile salt-hydrolysing bacteria, and Clostridium absonum, 487 as a CDCA to UDCA epimerizing bacteria, result in increased levels of faecal UDCA 488 in pigs [START_REF] Lepercq | Increasing 703 ursodeoxycholic acid in the enterohepatic circulation of pigs through the administration 704 of living bacteria[END_REF]. 489

An increased level of faecal bile acids was reported in cirrhotic patients with ongoing 490 alcohol consumption but, in these patients, the consequences of a potential liver 491 inflammatory process (ie alcoholic hepatitis) is unknown [START_REF] Kakiyama | Colonic 597 inflammation and secondary bile acids in alcoholic cirrhosis[END_REF][START_REF] Bajaj | Continued Alcohol 600 Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis[END_REF]. We now show that 492 total faecal bile acids levels probably decreased in Cir_sAH patients due to 493 decreased excretion of plasma bile acids in the bile, as discussed earlier. Faecal bile 494 acids shape the intestinal microbiota as deconjugation provides cellular carbon, 495 nitrogen, and sulfur for some bacterial species, especially Bacteroides and Bilophila 496 [START_REF] Van Eldere | Tauroconjugation of cholic 706 acid stimulates 7 alpha-dehydroxylation by fecal bacteria[END_REF]. Thus, the decrease in faecal bile acids levels in Cir_sAH patients may be 497 responsible for the decrease in the abundance of Bacteroidetes and Bilophila. 498 Moreover, primary faecal bile acids levels have been shown to increase intestinal 499 permeability [START_REF] Raimondi | 709 Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers 710 via EGFR activation[END_REF], which can increase PAMP release into the systemic circulation, 500 participating in the higher levels of endotoxemia observed in alcoholic liver disease 501 patients. Furthermore, bile acids bactericidal activity is related to their hydrophobicity, 502 which increases their affinity for the phospholipid bilayer of the bacterial cell 503 membrane, and unconjugated bile acids are week acids with strong bactericidal 504 activities. Among the bile acids, DCA is extremely toxic and inhibits the growth of 505 many intestinal bacteria, including Clostridium perfringens, Bacteroides fragilis, 506 Lactobacilli, and Bifidobacteria [START_REF] Floch | The effect of bile acids on intestinal 713 microflora[END_REF][START_REF] Kurdi | Mechanism of growth inhibition by free 715 bile acids in lactobacilli and bifidobacteria[END_REF]. In our study we observed a decrease in 507 unconjugated bile acids, total secondary bile acids and DCA in the feces, that are 508 highly hydrophobic. This may be responsible for the increase levels of Lactobacillus 509 and Bifidobacterium seen in the microbiota of these patients. It has also been 510

suggested that a decreased level of faecal bile acids stimulated the growth of gram-511 negative and conversely decreases the growth gram-positive bacteria (52). Indeed, 512 we observed in severe alcoholic hepatitis patients an increase in several taxa that are 513 gram-negative (eg Gammaproteobacteria) that could be secondary to the decrease 514 faecal bile acids level. Moreover, gram-negative bacteria produce LPS that was 515 related to increases alcoholic liver necrosis and inflammation [START_REF] Mathurin | 720 Exacerbation of alcoholic liver injury by enteral endotoxin in rats[END_REF]. We also observed 516 in our study a decrease in several taxa from the gram-positive Firmicutes phylum 517 (Christensenellaceae, Oscillospira) that 7α-dehydroxylate primary bile acids to toxic 518 secondary bile acids. Thus, the decrease and shift of the bile acids pool in the feces 519 could be responsible for the increase in LPS-producing bacteria and for the decrease 520 of gram-positive members of Firmicutes able to transform primary bile acids into 521 secondary bile acids. This hypothesis may explain the decrease in secondary bile 522 acids in severe alcoholic hepatitis patients observed in our study.

523

A limit of our study was a potential lack of power related to the small number of 524 patients, which did not allow us to identify changes in taxa with low counts. However, 525 the recruitment of severe alcoholic hepatitis patients for intestinal microbiota studies 526 is challenging, as most are rapidly treated (often by antibiotics to prevent or treat 527 complications). This bias did not occur in our patients as they were included before 528 any specific treatment for severe alcoholic hepatitis . Moreover, we did not exclude 529 patients with proton-pump inhibitors intake which was shown to alter the IM 530 24 composition (54). However, there was no difference in the use of proton-pump 531 inhibitors between groups, suggesting that they are not responsible for the results 532 observed in our study. 533

In conclusion, severe alcoholic hepatitis is associated with specific alterations of the 534 bile acids homeostasis and of the intestinal microbiota. These changes are 535

characterized by an increased level of hydrophobic bile acids and of Actinobacteria 536 and a decrease of Bacteroidetes. The increase and shift in the bile acids pool 537 towards hydrophobic and toxic species could be responsible for the specific intestinal 538 microbiota changes, including an increase in the LPS-producing gram-negative 539 bacteria such as Gammaproteobacteria and a decrease in certain gram-positive 540 bacteria capable to transform primary into secondary bile acids. Furthermore, the 541 changes in the intestinal microbiota were associated with a shift in its functions, 542 especially decreased biotin metabolism and increased glutathione metabolism, which 543 could play a role in the initiation and progression of severe alcoholic hepatitis, 544 through impairment of the protective effects of UDCA on mitochondrial metabolism. 545

Our study provides a new hypothesis for future studies to address bile acids and the 546 intestinal microbiota as new therapeutic targets to improve the management of 547 alcoholic liver disease patients. 548

Grant Support: 549

  However, FGF-19 positively correlated with the MELD score (r = 0.49, p = 0.04), but 353 not the Maddrey or AH histological scores. 354 These results suggest that FXR is activated in Cir_sAH patients independently of the 355 faecal bile acids concentration in the gut. 356 17 DISCUSSION 357

32 Figure legends 730 Figure 1 :Figure 2 :Figure 3 :

 32730123 Figure legends 730Figure 1: Intestinal microbiota profiles and its metabolic functions. (A) 731

Figure 4 :Figure 5 :Figure 6 :

 456 Figure 4: Faecal bile acids profiles in alcoholic liver disease. (A) PCA ordination 769

Figure 1 :Figure 2 :Figure 3 :Figure 4 :

 1234 Figure 1: Intestinal microbiota profiles and its metabolic functions. (A) Weighted UniFrac distances (quantitative method reflecting the structure of the intestinal microbiota) and (B) Unweighted UniFrac distances (qualitative method reflecting the composition of the intestinal microbiota) showing a difference in the structure of the intestinal microbiota only between Cir_sAH patients (blue) and Cir_noAH patients (red, p < 0.05 for Weighted UniFrac distances). Each point represents a subject and the distance between the points is proportional to the similarity of the intestinal microbiota. Cladogram showing the taxa with the largest differences in abundance between (C) Cir_sAH patients (green) and Cir_noAH patients (red) and (D) noCir_AH patients (red) and noCir_noAH patients. The size of the circle in the cladogram plot is proportional to bacterial abundance. From inside to outside, the circles represent phylum, class, order, family, and genus. Only taxa with a LDA score > 2 and p < 0.05, determined by the Wilcoxon signed rank test, are shown. (E) LDA Effect Size (LEfSe) for the predicted metagenome metabolic pathways (KEGG modules) increased in Cir_sAH (green) and Cir_noAH patients (red) (LDA score > 2.0, p < 0.05 determined by the Wilcoxon signed rank test).254x190mm (300 x 300 DPI)

Figure 5 :Figure 6 :

 56 Figure 5: Specificity of faecal bile acids profiles depending on alcoholic-induced liver inflammation. (A) PLS-DA score plot of faecal bile acids concentrations of Cir_sAH vs. Cir_noAH patients with 95% confidence ellipse showing a significant difference between the two groups (R2 = 0.8, Q2 = 0.6, p = 0.001). (B) Variable importance in projection (VIP) of PLS-DA showing the faecal bile acids that discriminate Cir_sAH from Cir_noAH patients (VIP score >1). The colored boxes on the right indicate the relative concentrations of the corresponding faecal bile acids in each group. 254x190mm (300 x 300 DPI)

  bile acids, total lithocholate (LCA) (LCA, glycolithocholate (GLCA), 278

	279	lithocholate-3-sulfate	(LCA3s),	taurolithocholate-3-sulfate	(TLCA3s),	and
	280	glycolithocholate-3-sulfate	(GLCA3s)),	total	deoxycholate	(DCA)	(DCA;
	281	glycodeoxycholate (GDCA), and taurodeoxycholate (TDCA)), chenodeoxycholic acid
	282	3-sulfate (CDCA3s), and glycoursodeoxycholate-3-sulfate (GUDCA3s) than

  In bold bile acids that were different between the groups (p<0.05 using a Mann Whitney test with a FDR correction for multiple comparaissons). In bold bile acids were different between the groups (p<0.05 using a Mann Whitney test with a FDR correction for multiple comparaissons).

		Median Min Max Median Min Max Median Min Max Median Min Max Median Min Max Median Min Max Median Min Max Median Min Max
	CA CA	0 0.07	0 0 11.66 0.51	0.00 0.10	0 0	0.90 1.02	0 0.03	0 0 49.09 0.33	0.19 0.09 0.01 1.56 0 1.38
	GCA CA_3s	0.07 0	0 0 0.32 7.10	0.26 0.02 0.001 0.002 5.36 0.05	0.36 0.02	0 0	0.54 1.14	3.11 0.001	0 0 0.08 9.30
	TCA GCA	0.03 0.01 0.001 3.17 0 6.44	0.01 0.01	0 0.01	0.74 0.13	0.16 0.01	0 0	2.78 7.62	4.48 0	0 0 0.01 25.46
	Total CA TCA	0.16 0.01	0 0 1.90 13.54	0.32 0	0.003 6.25 0 0.04	0.59 0.01	0 0 10.07 3.05	8.46 0.01	0 0 0.08 34.82
	CDCA Total CA	0.07 0.17 0.003 12.13 0 17.95	0.03 0.14	0 0.02	8.37 1.07	0.02 0.23	0 0 66.82 0.63	1.49 0.10 0.01 1.58 0 9.15
	CDCA_3S CDCA	0 0.16 0.01 6.82 0 0.16	0.00 0.08	0 0.01	0.28 2.73	0.02 0.05 0.005 34.24 0 0.07	0.02 0.11 0.04 6.77 0 0.22
	GCDCA CDCA_3S	1.05 0.02	0 0 4.16 56.08	1.49 0.07 0.003 0.01 32.55 0.36	2.49 0.32	0.002 4.10 0 1.30	34.89 0.01	7.36 57.37 0 0.18
	TCDCA GCDCA	0.06 0.05	0 0 4.35 12.81	0.05 0.05	0 0.01	1.83 0.13	0.48 0.02 0.002 0	9.05 5.44	21.97 0.01 0.001 0.04 5.02 45.01
	Total CDCA TCDCA	1.73 0.01	0.001 68.89 0 0.94	1.85 0.01	0.01 42.94 0 0.03	4.00 0.01 0.001 0.002 11.39 0.05	61.35 0.02 0.004 0.03 12.38 81.23
	Total Primary Total CDCA	1.90 0.45 0.05 9.61 0.002 82.43	2.30 0.33	0.01 43.24 0.11 2.93	4.74 0.64 0.01 40.34 0.002 14.44	70.67 0.31 0.05 6.84 16.57 99.42
	DCA Total Primary	0.11 0.78 0.07 20.72 0 67.94	0.24 0.53	0 0.13	17.99 4.00	0.16 0.89 0.01 107.16 0.001 0.55	0.01 0.38 0.08 7.75 0 0.88
	DCA_3S DCA	0 3.78 0.01 24.12 0 0.07	0 5.61	0 0.28 22.36 0.24	0 3.76 0.62 25.27 0 0.05	0 0.13 0.01 2.61 0 0.10
	GDCA DCA_3S	0.21 0.14 0.002 22.06 0 23.50	0.75 0.30	0 0.01	10.59 4.45	0.50 0.07	0.002 1.19 0 9.86	0.16 0	0 0 1.24 6.98
	TDCA GDCA	0.02 0.02	0 0 1.05 6.30	0.00 0.03	0 0	0.31 0.12	0.14 0.01	0 0	2.01 0.10	0.17 0	0 0 0.003 4.51
	Total DCA TDCA	0.47 0.01	0 0 0.41 70.94	1.15 0.01	0 0	28.58 0.04	1.02 0.01	0.002 2.51 0 0.08	0.36 0	0 0 0.002 12.19
	LCA Total DCA	0 4.54 0.05 46.49 0 0.03	0 7.43	0 0.36 22.74 0.19	0.02 6.49 0.63 25.55 0 0.08	0.01 0.13 0.01 2.61 0 0.11
	LCA_3S LCA	0 1.96 0.003 8.74 0 0.11	0 1.96	0 0.38 20.00 0.07	0.06 1.95 0.02 0	0.23 5.43	0.04 0.58 0.005 2.93 0 0.48
	GLCA LCA_3S	0 0.17	0 0 18.64 0.07	0 0.60 0.004 0	0.04 7.82	0.05 0.21	0 0 18.31 0.10	0.04 0	0 0 0.25 0.17
	GLCA_3S GLCA	0.18 0	0 0 0.003 74.05	0.007 0	0 0	12.83 0.02	0.27 0.00	0 0	0.68 0	0.19 0	0 0	26.48 0
	TLCA GLCA_3S	0	0	0 0 0.06 0	0 0.01	0 0	0 0.21	0 0.00	0 0	0.20 0.03	0	0	0 0	0.10 0
	TLCA_3S TLCA	0	0	0 0	3.49 0	0 0.00	0 0 0.001 0.13	0.09 0.00	0 0	0.66 0	0.11 0	0 0	0.76 0
	Total LCA Total LCA	0.29 2.53 0.05 22.91 0 74.05	0.01 2.93	0 0.44 28.04 13.02	0.66 2.69 0.38 20.15 0.001 1.25	0.47 0.58 0.01 2.93 0 26.48
	UDCA UDCA	0.03 0.10	0 0 7.18 7.97	0.00 0.06	0 0	5.94 1.47	0.01 0.01 0.002 0	0.06 2.38	0.05 0.01 0.003 1.94 0 0.73
	UDCA_3S UDCA_3S	0 0.04	0 0 4.09 0.13	0 0.04	0 0	1.01 0.63	0 0.09	0 0	0.13 0.90	0	0	0 0 0.23 0.14
	GUDCA GUDCA	0.17 0	0 0 0.15 13.26	0.32 0.01	0.000 20.54 0 0.02	0.13 0	0 0	0.27 0.11	0.76 0	0 0	2.49 0
	GUDCA_3S GUDCA_3S	0.08 0	0 0 0.08 22.34	0.24 0	0 0	49.30 0	0.09 0	0 0	0.15 0	0.13 0	0 0	1.16 0
	TUDCA TUDCA	0	0	0 0 0.04 0.01	0	0	0 0 0.001 0.00	0	0	0 0	0.02 0	0.26 0	0 0	0.66 0
	Total UDCA Total UDCA	0.37 0.30 0.01 11.31 0 42.49	0.56 0.39	0.001 56.73 0.02 1.54	0.28 0.41 0.002 0	0.44 3.11	1.30 0.02 0.003 1.94 0 4.09
	Total Secondary Total Secondary	1.68 8.40 0.21 63.85 0.002 96.84	2.32 10.24	0.00 66.00 0.83 52.40	2.12 10.68 1.80 27.92 0.004 3.93	2.47 0.72 0.02 5.68 0.58 26.48
	Total Unconjugated Total Unconjugated	0.64 9.33 0.40 83.97 0 85.89	0.37 10.54	0 0.93 53.27 26.49	0.27 11.17 1.80 111.58 0.001 1.74	1.89 2.30 0.19 10.97 0 12.02
	Total Glycoconjugated Total Glycoconjugated	2.32 0.10 0.002 7.67 0.002 96.49	3.56 0.10	0.01 93.90 0.03 0.51	3.90 0.05 0.002 13.30 0.005 6.58	39.98 0.02 0.001 0.05 9.11 83.85
	Total Tauroconjugated Total Tauroconjugated	0.16 0.02	0 0 2.86 19.25	0.36 0.02	0.003 1.83 0 0.11	0.90 0.02 0.001 10.20 0 14.71	32.17 0.02 0.01 0.10 8.19 60.33
	Total Sulfoconjugated Total Sulfoconjugated	0.50 0.46 0.01 31.88 0 74.05	0.84 1.23	0.001 54.38 0.05 8.36	0.79 0.71	0.001 1.06 0 25.13	0.65 0.04	0.06 26.48 0 1.99
	Total Conjugated Total Conjugated	2.60 0.12 0.002 10.54 0.002 100	4.26 0.16	0.01 93.98 0.03 0.53	5.89 0.08 0.02 23.50 0.005 18.19	81.77 0.03 0.02 0.12 17.30 100
	Total Bile Acids Total BA	3.60 9.87 1.21 84.57 0.003 100	4.62 10.77	0.01 100 0.96 53.80	6.84 11.32 1.82 135.08 0.01 18.36	84.57 2.36 0.31 11.03 17.37 100