Giacomo Zaccherini

Ferran Aguilar

Paolo Caraceni

Joan Clària

Juan José Lozano

François Fenaille

Florence Castelli

Christophe Junot

Anna Curto

Chiara Formentin

Emmanuel Weiss

Mauro Bernardi

Rajiv Jalan

Paolo Angeli

Richard Moreau
email: richard.moreau@inserm.fr

Vicente Arroyo

Assessing the role of amino acids in systemic inflammation and organ failure in patients with ACLF

Keywords: Multiorgan failure, Small-molecules, Biomass, Immunity, Anabolic metabolism, Dormancy

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

RESULTS:

The main findings in ACLF were: (1) Metabolite modules were increased in parallel with increased levels of markers of systemic inflammation and oxidative stress. (2) Seventy percent of proteinogenic amino acids were present and most were increased. (3) A metabolic network, comprising the amino acids aspartate, glutamate, the serine-glycine one-carbon metabolism (folate cycle), and methionine cycle, was activated, suggesting increased purine and pyrimidine nucleotide synthesis. (4) Cystathionine, L-cystine, glutamate, pyroglutamate, involved in the transsulfuration pathway (a methionine cycle branch) were increased, consistent with an increased synthesis of antioxidant glutathione. (5) Intermediates of the catabolism of 5 out of the 6 ketogenic amino acids were increased. ( 6) Spermidine (a polyamine inducer of autophagy with anti-inflammatory effects) had decreased levels.

CONCLUSIONS:

In ACLF, blood amino acids fueled protein and nucleotide synthesis required by intense systemic inflammatory responses. Ketogenic amino acids were extensively catabolized to produce energy substrates in peripheral organs, an effect that was insufficient because organs failed. Finally, the decrease in spermidine levels may cause a defect in autophagy contributing to proinflammatory phenotype in ACLF.

Lay Summary

This study is the first to provide a comprehensive view of the landscape of blood amino acids in patients with acutely decompensated cirrhosis, in particular among those with ACLF. The results show that these patients have features of intense skeletal muscle catabolism resulting in the mobilization of amino acids for anabolic and catabolic purposes. Importantly, in patients with ACLF, amino acids along with glucose, are used, for intense anabolic, energy-consuming metabolism which supports de novo nucleotide and protein synthesis, presumably in the activated innate immune system. We also found that the blood levels of the polyamine spermidine, an inducer of autophagy, were decreased and probably skewed toward GABA synthetic pathway.

Several studies, including the CANONIC study, [START_REF] Moreau | Acute-onchronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis[END_REF][START_REF] Clària | Systemic inflammation in decompensated cirrhosis. Characterization and role in acute-on-chronic liver failure[END_REF] the PREDICT study, [START_REF] Trebicka | The PREDICT study uncovers three clinical courses of acutely decompensated cirrhosis that have distinct pathophysiology[END_REF] and others, [START_REF] Bajaj | Survival in infection-related acute-on-chronic liver failure is defined by extrahepatic organ failures[END_REF][START_REF] Arroyo | Acute-on-chronic liver failure[END_REF] have shown that patients who are admitted to the hospital for an acutely decompensated cirrhosis associated with organ failure(s) and intense systemic inflammation have acute-on-chronic liver failure (ACLF). In ACLF, the presence of pathogen-associated molecular patterns (PAMPs) of bacterial origin, [START_REF] Arroyo | Acute-on-chronic liver failure[END_REF][START_REF] Arroyo | Acute-onchronic liver failure in cirrhosis[END_REF][START_REF] Moreau | Effects of Long-term Norfloxacin Therapy in Patients with Advanced Cirrhosis[END_REF] damageassociated molecular patterns (DAMPs) released by dying cells, [START_REF] Arroyo | Acute-on-chronic liver failure[END_REF] activates patternrecognition receptors of the innate immune system; this activation resulting in systemic inflammation. [START_REF] Arroyo | Acute-on-chronic liver failure[END_REF][START_REF] Arroyo | Acute-onchronic liver failure in cirrhosis[END_REF] Although systemic inflammation is certainly the primary cause of organ failures in ACLF, the mechanisms by which inflammation can perturb organ functions are still unclear. These mechanisms may include tissue hypoperfusion, [START_REF] Arroyo | Acute-on-chronic liver failure[END_REF] alterations in cardiac function, [START_REF] Jansen | Significant reduction in heart rate variability is a feature of acute decompensation of cirrhosis and predicts 90-day mortality[END_REF][START_REF] Praktiknjo | Cardiodynamic state is associated with systemic inflammation and fatal acute-onchronic liver failure[END_REF] and immune-cell-mediated tissue damage. [START_REF] Arroyo | Acute-on-chronic liver failure[END_REF] Moreover, recent results of metabolomics suggested that, in patients with ACLF, systemic inflammatory responses were associated with a defect in mitochondrial fatty acid-derived energy production in peripheral organs which may contribute to organ system failures. [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF] During sepsis under "non-cirrhotic" conditions, innate immune cells are activated and have an anabolic, energy-consuming metabolism resulting in synthesis of nucleotides, proteins and lipids that support leukocyte proliferation (increased biomass) and biosynthesis of a myriad of biomolecules involved in host defense, including, for example, soluble proteins such as cytokines, chemokines, and acutephase proteins (Fig. S1). [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] Nutrients (e.g., glucose, amino acids) necessary to fuel immune activation [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] are mobilized from internal stores. [START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] In addition, in activated innate immune cells, some amino acids are produced by cell-intrinsic mechanisms. Amino acids are used as building blocks for protein synthesis; two nonessential amino acids (glutamine and aspartate) are also substrates for de novo synthesis of nucleotides, [START_REF] Pareek | Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells[END_REF][START_REF] Villa | Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides[END_REF] which are indispensable to RNA synthesis and therefore protein synthesis. Glucose extracted from blood is channeled to aerobic glycolysis to produce ATP, pyruvate, and lactate. [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] Glucose is also involved in nucleotide synthesis by giving rise to riboses (through the pentose phosphate pathway, PPP) and the nonessential amino acid serine (which is metabolized through the folate cycle). [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF][START_REF] Pareek | Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells[END_REF][START_REF] Villa | Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides[END_REF] The folate cycle, and the methionine cycle are two components of what is referred to as one-carbon metabolism network. [START_REF] Sanderson | Methionine metabolism in health and cancer: a nexus of diet and precision medicine[END_REF] Through the methionine cycle, the essential amino acid methionine can give rise to nucleotides among several other molecules. [START_REF] Sanderson | Methionine metabolism in health and cancer: a nexus of diet and precision medicine[END_REF][START_REF] Madeo | Spermidine in health and disease[END_REF] Collectively, these findings indicate that activation of the innate immune system requires the integrated metabolism of glucose, nonessential amino acids, and one-carbon metabolism, to produce nucleotides and proteins. The different metabolic pathways are detailed in Box 1 and Figs. S2 through S5. [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF][START_REF] Pareek | Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells[END_REF][START_REF] Villa | Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides[END_REF][START_REF] Sanderson | Methionine metabolism in health and cancer: a nexus of diet and precision medicine[END_REF][START_REF] Madeo | Spermidine in health and disease[END_REF][START_REF] Vander Heiden | Understanding the Intersections between Metabolism and Cancer Biology[END_REF][START_REF] Kanarek | Dietary modifications for enhanced cancer therapy[END_REF] Results of metabolomics performed in blood from patients with ACLF revealed features consistent with enhanced aerobic glycolysis and PPP in the innate immune system. [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF] Because of the integration of glucose and amino acid metabolisms in activated innate system (Box 1; Figs. S2 through S5), we hypothesized that alterations in glucose metabolism may be associated with changes in amino acid metabolism and contribute to immune activation in ACLF. On the other hand, evidence has also been shown in blood from patients with ACLF of an enhanced catabolism of the essential amino acid tryptophan through the kynurenine pathway, giving rise to metabolites that may perturb peripheral-organ functions, [START_REF] Clària | Orchestration of Tryptophan-Kynurenine pathway, acute decompensation and acute-on-chronic liver failure in cirrhosis[END_REF] therefore suggesting a role of certain amino acids in the development of organ system failures.

Of note, patients with ACLF have sarcopenia, [START_REF] Praktiknjo | Sarcopenia Is Associated With Development of Acute-on-Chronic Liver Failure in Decompensated Liver Cirrhosis Receiving Transjugular Intrahepatic Portosystemic Shunt[END_REF][START_REF] Praktiknjo | Fatfree muscle mass in magnetic resonance imaging predicts acute-on-chronic liver failure and survival in decompensated cirrhosis[END_REF] indicating intense skeletal muscle catabolism that may increase amino acid release and availability for anabolic and catabolic purposes.

In the current study we reanalyzed the blood metabolome data set obtained in the CANONIC study with the aim to assess the potential role of major amino acid metabolic pathways in systemic inflammatory responses and organ failures among patients with acutely decompensated cirrhosis, with or without ACLF. Because metabolisms of amino acids and glucose are interrelated [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF] (Fig. S2A), we used the entire metabolic data set to perform correlative and integrative analyses. We also leveraged the fact that blood metabolome may capture important metabolic changes that occur in tissues with highly active metabolism such as activated immune cells [START_REF] Langley | An integrated clinico-metabolomic model improves prediction of death in sepsis[END_REF] and cancer cells. [START_REF] Gao | Dietary methionine influences therapy in mouse cancer models and alters human metabolism[END_REF]

Patients and methods

Patients

The present study used metabolomic data obtained in biobanked serum samples collected at enrollment in the CANONIC study of 831 patients with acutely decompensated cirrhosis. Part of the results of this metabolomic data set have been previously published. [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF][START_REF] Clària | Orchestration of Tryptophan-Kynurenine pathway, acute decompensation and acute-on-chronic liver failure in cirrhosis[END_REF]

Methods

Metabolomic data set

The data set comprised 137 well-annotated blood metabolites that had been identified using untargeted metabolomics by liquid chromatography coupled to highresolution mass spectrometry, as previously described. [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF] The 137 metabolites are described in Table 1 and Table S1.

Analysis of the data set

The strategy of analysis is described in the Supplementary Appendix, in particular the use of weighted gene co-expression network analysis (WGCNA) [START_REF] Langfelder | WGCNA: an R package for weighted correlation network analysis[END_REF][START_REF] Pei | WGCNA Application to Proteomic and Metabolomic Data Analysis[END_REF] and the computation of 9 eigenmetabolites (each being representative of a single module). [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF][START_REF] Chialvo | Eigenvector metabolite analysis reveals dietary effects on the association among metabolite correlation patterns, gene expression, and phenotypes[END_REF]

Results

Characteristics of the study population at enrollment

There were 181 patients who had ACLF and 650 who had acute decompensation without ACLF (a group hereafter called "AD"). Table S2 summarizes baseline characteristics of patients.

Identifying modules of related blood metabolites in acutely decompensated cirrhosis at enrollment

Reducing the dimension of the metabolomic data set

First, we explored the possibility that significant correlations existed between metabolites of the entire data set obtained in the whole cohort of patients with acutely decompensated cirrhosis. In fact, a large number of metabolite pairs were linked by a positive and significant correlation, with a R coefficient of 0.5 or more (Fig. S6A).

Moreover, several metabolites were involved in more than one pair of significant correlations, suggesting the existence of groups of positively co-regulated metabolites (Fig. S6A). In contrast, very few metabolite pairs were negatively and significantly correlated, and even less were interconnected (Fig. S6B). Next, using WGCNA in the whole metabolomic data set, we identified 9 modules of co-regulated metabolites (Fig. S6C); a metabolite contained in a given module being not present in another module. Each module received a color name, which was arbitrarily given by the algorithm; the smallest module (magenta) contained 5 metabolites and the largest (turquoise) 28 metabolites (Table 1; Fig. 1).

Analysis of module membership according to categories of metabolites

We identified the presence of important categories of metabolites, including molecules related to amino acids, fatty acids, polyamines; metabolites related to glycolysis and its branch PPP; metabolites related to purines and pyrimidines, acylcarnitines; and Krebs cycle intermediates (Fig. 1; Table 1). Metabolites related to amino acids represented 43% of the metabolic data set and were the only category to be present in each of the 9 modules, although their proportion varied across modules. Amino acids and related molecules were exclusive members of the yellow module and represented the majority of metabolite members of the pink and magenta modules (86% and 80%, respectively).

Metabolite modules are upregulated in acute-on-chronic liver failure (ACLF)

Comparing eigenmetabolite values (one per module) between patients with ACLF and those without, we found that each eigenmetabolite was significantly higher in patients with ACLF (Fig. 2). This finding is consistent with the observation that a prominent upregulation characterizes the blood metabolome of patients with ACLF. [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF] Of note, although in each module, most members had significantly higher blood levels in ACLF relative to AD, some were not different between the 2 groups or even significantly lower in ACLF (Table 1). These findings highlight the interest of using WGCNA for metabolomic data sets as it allowed to include in a same module metabolites that may be interrelated although their blood levels changed in opposite directions.

In ACLF, upregulation of metabolite modules correlates with intense systemic inflammation and oxidative stress

In patients of the CANONIC cohort, we had values for white-cell count, and plasma levels of 18 protein markers of inflammation [START_REF] Moreau | Acute-onchronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis[END_REF][START_REF] Clària | Systemic inflammation in decompensated cirrhosis. Characterization and role in acute-on-chronic liver failure[END_REF] (Table S1). We assessed the correlation of these variables with the 9 eigenmetabolites (Fig. 3A). Five modules or more were positively and significantly correlated with markers of systemic inflammation, including white-cell count; CRP; soluble CD163; soluble CD206; master cytokines such as interleukin (IL)-6, tumor necrosis factor α, IL-10; master chemokines IL-8 (also known as C-X-C motif chemokine 8) and C-C motif chemokine 2 (also known as MCP-1). Only 4 markers of inflammation were not positively correlated with metabolite modules. Together these findings indicate that, in ACLF, metabolite module upregulation correlated with intense systemic inflammation.

The levels of human nonmercaptalbumin-2 (HNA2, which assesses irreversibly oxidized albumin 2 ) that were also available, were increased in ACLF, consistent with systemic oxidative stress (Table S1). The 9 modules were positively correlated with HNA2 levels (Fig. 3A; p<0.05 for every correlation) indicating that, in ACLF, metabolite module upregulation correlated with systemic oxidative stress.

In ACLF, upregulation of metabolite modules correlates with organ system failures and severity

Correlations of the 9 eigenmetabolites with important clinical features are shown in Fig. 3B. Modules were variably correlated with age. A large number of modules were positively, and significantly, correlated with serum bilirubin, INR, and plasma renin concentration. Consistent with findings related to plasma renin, several modules were negatively and significantly correlated with mean arterial pressure. The results therefore indicated that upregulation of a large number of modules seen in ACLF, was closely associated with worsening in liver, coagulation (which primarily reflects to liver failure), kidney, and circulatory functions. Of note, 3 modules were correlated positively and significantly with increasing hepatic encephalopathy (HE) grades, indicating that changes in blood metabolome were less related to brain failure than the other major organ system failures. Finally, we found that the 9 modules were positively and significantly correlated with the "global" CLIF-C OF score, the increasing number of failing organ systems, and the increasing ACLF grade, confirming that module upregulations paralleled the increase in patients' severity.

Addressing the role of deteriorated kidney function in blood metabolite accumulation

Kidney dysfunction or failure being highly prevalent in patients with acutely decompensated cirrhosis (Table S2), a deterioration in kidney function might result in decreased renal metabolite excretion and accordingly contribute to blood metabolite accumulation. Indeed, eigenmetabolites were significantly correlated with serum creatinine (Fig. 3B) and increased in presence of kidney dysfunction (Fig. S7).

However, among patients with ACLF, only 2 eigenmetabolites (turquoise and pink modules) were upregulated in patients with single kidney failure, relative to those without kidney failure (Fig. S7). Moreover, the 9 eigenmetabolites were positively and significantly correlated with bilirubin and INR, suggesting a relationship with liver failure (Fig. 3B). Together, these findings suggested that the levels of blood metabolites may not depend only on the deterioration of kidney function. Therefore, we computed a multivariate analysis evaluating the independent correlations of each metabolite of each module with serum creatinine, serum bilirubin, and the grades of HE. We found that 50% to 100% of metabolites of each of the 9 modules (therefore including the yellow module) were independently and positively correlated with creatinine (Fig. S8A). We also found that 40% to 100% of metabolites of each of the 9 modules were independently and positively correlated with bilirubin (Fig. S8A). In addition, a variable proportion of the metabolites of 5 modules were independently and positively correlated with increasing HE grades (Fig. S8A). A large majority of metabolites (69%) were independently correlated with deterioration of two organs (50% of metabolites) or three (19% of metabolites) (Fig. S8B; Table S3). Nine percent of the 137 metabolites were not correlated with either of the three organs.

Collectively, these findings suggested that blood metabolite accumulation in ACLF was not a simple consequence of a reduction in renal excretion of metabolites but the result of several independent effects. The multivariate analysis also revealed that among the 137 metabolites, 78% were independently correlated with creatinine, 73% with bilirubin and 28% with HE grade, confirming that changes in blood metabolome were more closely associated with deterioration of kidney or liver functions than with deterioration of brain function.

Addressing the association with precipitating events

Among patients with ACLF, we compared the 9 eigenmetabolites measured at enrollment, between patients who had bacterial infection as a precipitating event and those without. Four eigenmetabolites (turquoise, magenta, green, and black) were significantly higher in patients with bacterial infection while the remaining eigenmetabolites did not differ between the two groups (Fig. S9A). We identified 13 metabolites of potential bacterial origin in 3 modules associated with infection (8 metabolites in the turquoise module, 4 in the green module, and one in the black module) (Table 1). Among patients with ACLF, we also did the comparison for two other precipitating events, excessive alcohol consumption and gastrointestinal hemorrhage, and found no differences in eigenmetabolites with each of these events (Fig. S9B andS9C).

Antecedence of metabolite module upregulation relative to ACLF development and death

Among the patients with AD (without ACLF) at enrollment, we wondered whether blood metabolome at that time differed between those who will develop ACLF by 28 days (n = 69) and those who will not (n = 536). We compared the 2 groups using the 9 eigenmetabolites measured at enrollment and found that, except for the redmodule eigenmetabolite, every eigenmetabolite was significantly higher among patients who developed ACLF by 28 days than among those who did not (Fig. S10).

We found that the same 8 eigenmetabolites were significantly higher at enrollment, among patients who died by 28 days (n = 79) than among those who survived by 28 days (n = 752) (Fig. S11). The remaining eigenmetabolite (red module) did not differ between the 2 groups. Collectively, these findings indicated that metabolite module upregulation at enrollment preceded the short-term occurrence of major outcomes such as the development of ACLF and death.

Antecedence of changes in individual metabolites relative to death

We asked whether the blood levels of paradigmatic metabolites assessed at enrollment would be associated with the patients' outcome. The levels of methionine, those related to aerobic glycolysis (pyruvate and lactate), and those related to PPP (pentose phosphates, glucuronate, galacturonate) were significantly higher among patients who died by 28 days than among those who survived (Fig. S12). In contrast, the levels of the polyamine spermidine were significantly lower among patients who died by 28 days (Fig. S12). These findings suggest an important prognostic role for the metabolism of methionine, glycolysis, PPP, and spermidine, in patients with acutely decompensated cirrhosis. In addition, our results indicate that the prognostic value of individual paradigmatic metabolites was generally related to the increases in their blood levels, and for spermidine to the decreases in their blood levels.

Potential multiple roles of amino acids in ACLF

Proteinogenic amino acids are mobilized in ACLF

We first analyzed the yellow module whose all members (n=16) were amino acids. Of these, 12 had blood levels higher in ACLF relative to AD (Table 1). Together, these findings suggested an orchestrated regulation of blood levels of yellow-module members. Among the yellow-module members, 11 (including serine, methionine, arginine, proline, phenylalanine, tyrosine, isoleucine, lysine, asparagine, histidine, and tryptophan) were proteinogenic amino acids, i.e., molecules that are incorporated into proteins under mRNA direction. In the context of inflammation, proteinogenic amino acids can also be used for catabolic purposes, as shown in other parts of the Results section and previous studies [START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] (Fig. S1).

Modeling the role of amino acids in nucleotide synthesis in ACLF

ACLF associates with enhanced aerobic glycolysis, PPP, and nucleotide synthesis. In ACLF, as expected, [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF] there was blood accumulation of pyruvate and lactate. Although pyruvate and lactate had different module membership (brown module and blue module, respectively [Table 1]), a finding that may be explained by the fact that pyruvate can give rise not only to lactate but also acetyl-CoA, and oxaloacetate, among others [START_REF] Kanarek | Dietary modifications for enhanced cancer therapy[END_REF] (Fig. S2A), lactate and pyruvate levels were positively and significantly correlated (Fig. S13A), indicating increased aerobic glycolysis (Fig. 4A). In addition, ACLF was associated with increased blood levels of metabolites related to the branch of glycolysis, PPP, including ribose-5-phosphate (green module; Fig. 4A), a precursor of PRPP, indicating that glucose-6-phosphate was also channeled to PPP (Fig. 4A). Of note, the levels of pyruvate (Fig. S13B) and lactate (Fig. S13C) were significantly and positively correlated with the levels of pentose phosphates and metabolites related to PPP (glucuronate, galacturonate, both greenmodule members), consistent with the simultaneous activation of aerobic glycolysis and PPP in ACLF. In addition, we found that N-formyl-L-methionine (green module), a reporter of N 10 -formyl tetrahydrofolate synthesis, [START_REF] Pareek | Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells[END_REF] was increased in ACLF, suggesting an increased conversion of serine to glycine and formate (one-carbon) production in the folate cycle (Fig. 5). In addition, the blue module was enriched with several molecules involved in nucleotide synthesis, including glutamine, aspartate, xanthine and hypoxanthine (purine bases involved in purine salvage; Fig. 4B); inosine (the hypoxanthine nucleoside); orotate (whose production is coupled to ETC and gives rises to pyrimidine ring; Fig. 4C); thymine (a pyrimidine base) and its derivative (dihydrothymine). Several Krebs cycle intermediates, e.g. α-ketoglutarate, oxaloacetate (which gives rise to aspartate [Fig. 4A]) were increased in ACLF and all in the brown module. Of note, investigating positively correlated eigenmetabolites, we identified an ordered network of metabolite modules showing, in particular, a structure with the following order yellow-blue-brown-green (Fig. S14), suggesting a close co-orchestration of these 4 modules. Of note, we found evidence in ACLF of increased methionine cycle and its branch the methionine salvage pathway (see below) which may contribute to the increased levels in ACLF of adenine (green module; Table 1; Fig. 5), a substrate for salvage purine synthesis (Fig. 4B).

Collectively, our findings suggested that, in ACLF, there was an orchestrated activation of aerobic glycolysis and PPP, one-carbon metabolic network (involving the folate cycle and methionine cycle), and Krebs cycle with the objective to fuel de novo and salvage purine synthesis, and de novo pyrimidine synthesis. These findings were reminiscent to those in activated innate immune cells that have a stimulation of aerobic glycolysis and nucleotide synthesis [START_REF] Pareek | Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells[END_REF] (Fig. S2A). Because innate immunity is activated in ACLF, blood metabolome in patients with ACLF may capture metabolites related to enhanced nucleotide synthesis required to produce biomass (leukocytosis) and a broad variety of inflammatory mediators in the innate immune system.

Modeling methionine metabolism in ACLF

Next, we investigated the methionine cycle, the second component of the one-carbon metabolic network [START_REF] Sanderson | Methionine metabolism in health and cancer: a nexus of diet and precision medicine[END_REF] (Fig. 5).

ACLF associates with activation of the transsulfuration and methionine salvage pathways. Our patients with ACLF had several features consistent with the engagement of the methionine cycle. First, they had blood accumulation of methionine (yellow module). Second, they had evidence of activated transsulfuration pathway indicated by blood accumulation of cystathionine (magenta module) and Lcystine, pyroglutamate, and glutamate (all involved in glutathione synthesis [Fig. 5] and blue-module members). Taurine, the other product of the transsulfuration pathway, was a blue-module member but its levels did not significantly differ between ACLF and AD (Table 1), suggesting that glutathione production was prioritized in ACLF. Together, our results suggest that part of homocysteine was skewed towards cystathionine and the transsulfuration pathway. Third, patients with ACLF had increased blood levels of 5'-Deoxy-5'-(methylthio)adenosine (MTA) indicating engagement of the methionine salvage pathway. Collectively, our results suggest that, in ACLF, priority was given to both the production of the antioxidant glutathione via the transsulfuration pathway and methionine renewal via the methionine salvage pathway. Our finding that choline was contained in the blue module might suggest that choline was channeled to produce methionine and therefore contribute to methionine re-synthesis (Fig. 5). The engagement of metabolic pathways whose purpose is methionine renewal suggests a pivotal role of methionine in ACLF. In addition, the fact that adenine levels were increased in ACLF and that both MTA and adenine were green-module members, suggest that the methionine salvage pathway was used to fuel the purine salvage pathway (Fig. 5). Of note, our finding of a network comprising the yellow-blue-brown-green modules (Fig. S14), was also consistent with methionine fueling the flux backbone of one-carbon metabolism.

Decreased levels of the polyamine spermidine and accumulation of

GABA precursors in blood from patients with ACLF. In ACLF, the levels of the putrescine-precursor ornithine, and those of the polyamine synthesis byproduct MTA, were increased, suggesting an increased polyamine synthesis (Fig. 5). However, in ACLF, spermidine levels were not increased but significantly decreased (Table 1)

and spermine was undetectable. The decrease in spermidine may be explained, at least in part by the conversion of spermidine into N8-acetylspermidine (black module; Table 1; Fig. 5). Of note, N8-acetylspermidine and putrescine can give rise to 4acetamidobutanoate (turquoise module; Fig. 5) whose levels were increased in ACLF relative to AD (Table 1). 4-acetamidobutanoate is an immediate precursor of 4aminobutanoate (also known as GABA), a master mediator of GABAergic neurons.

Neuroinflammation-induced activation of the GABAergic system has been shown contributing to features of hepatic encephalopathy. [START_REF] Hernandez-Rabaza | Neuroinflammation increases GABAergic tone and membrane expression. Reversal by sulforaphane by promoting M2 polarization of microglia[END_REF] Collectively, these findings might suggest that N8-acetylspermidine via 4-acetamidobutanoate might fuel the GABAergic nervous system in acutely decompensated cirrhosis. However, this hypothesis would deserve confirmation because in the metabolite module network (Fig. S14), the modules containing each of these metabolites (black and turquoise, respectively) were not significantly correlated. Moreover, of the two metabolites, only N8-acetylspermidine but not 4-acetamidobutanoate, was independently and significantly correlated with HE grade (Table S3).

ACLF associates with blood accumulation of ketogenic amino acids

In ACLF, glucose is prioritized to immune cells at the expense of peripheral organs. [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF] In the context of PAMP-or DAMP-induced systemic inflammation, ketone bodies (acetoacetate, β-hydroxybutyrate) become an important source of energy in peripheral organs. [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF] Oxidation of fatty acids and amino acids, which are mobilized from adipose tissue and skeletal muscle, respectively, usually support ketogenesis. [START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] Our patients with ACLF had lipolysis as indicated by blood accumulation of free fatty acids, for example 2-Hydroxyhexadecanoate, a product of lipolysis; green module).

However, these patients also had blood accumulation of acylcarnitines (i.e., fatty acids attached to carnitine) (Table 1), indicating the inhibition, in peripheral organs, of the translocation of these molecules into mitochondria. [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF][START_REF] Langley | An integrated clinico-metabolomic model improves prediction of death in sepsis[END_REF] In peripheral organs, therefore, there was a suppression of fatty acid β-oxidation and ATP synthesis, which may contribute to organ failures. In addition to fatty acids, amino acids, including isoleucine, lysine, tryptophan, leucine, tyrosine, and phenylalanine (Fig. S15A), can serve as substrates for synthesis of ketone bodies. [START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] In our study, all ketogenic amino acids but leucine were in the yellow module; of these isoleucine, lysine, tyrosine, and phenylalanine had increased blood levels in ACLF while levels of tryptophan did not differ in ACLF versus AD (Table 1). Moreover, in ACLF, there was blood accumulation of catabolites of phenylalanine and tyrosine (Fig. S15B), tryptophan (Fig. S16), and lysine (Table 1). However, in ACLF, although there was extensive ketogenic amino acid catabolism, the canonical ketone body, βhydroxybutyrate, [START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] was not identified in our metabolomic data set, raising the hypothesis that ACLF was associated with a defect in or an inhibition of ketone body production from amino acids.

Discussion

This study reanalyzed the metabolomic data set (comprising 137 metabolites) obtained with samples of the CANONIC study, [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF] a study which enrolled a large cohort of patients with acutely decompensated cirrhosis. [START_REF] Moreau | Acute-onchronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis[END_REF] Using the entire metabolomic data set obtained in the whole cohort, we identified 9 metabolite modules; each containing highly correlated molecules and each receiving arbitrarily a name of color. Unlike other metabolites, amino acids were present in every module highlighting the important contribution of these molecules to blood metabolome. Each module, assessed via its eigenmetabolite, was upregulated in ACLF relative to AD, confirming that blood metabolome changes in ACLF are characterized by prominent metabolite accumulation, although some individual metabolites decreased (e.g., spermidine). [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF] Several eigenmetabolites were positively and significantly correlated with bona fide markers of systemic inflammation, indicating that metabolic changes paralleled the intensity of systemic inflammation. The 9 eigenmetabolites were positively correlated with markers of overall severity. Among patients with ACLF, some eigenmetabolites were higher in infected than in noninfected modules and the corresponding modules contained metabolites of potential bacterial origin. Finally, we found that 8 eigenmetabolites measured at enrollment in patients with AD, were significantly higher among those who developed ACLF during follow-up than among those who remained free of ACLF. These 8 eigenmetabolites were significantly higher in patients who died within 28 days than among patients who remained alive.

Moreover, we found that some individual paradigmatic metabolites significantly differed between patients who died and those who remained alive. These findings indicated the antecedence of eigenmetabolite upregulation, or changes in the expression of individual metabolites, relative to poor clinical outcome and suggesting that blood metabolites should be evaluated as biomarkers for outcome in future studies.

In univariate analysis, 8 eigenmetabolites were positively correlated with kidney failure suggesting that blood metabolite accumulation in ACLF may be a passive result of defective renal excretion. However, this hypothesis was not entirely supported by the results of multivariate analysis assessing the independent positive correlations of each metabolite of each module with serum creatinine, serum bilirubin, and HE grade. Indeed, we found that 69% of metabolites were independently correlated with deterioration of the function of two organs (50% of metabolites) or three (19% of metabolites), indicating the blood metabolite accumulation was a result of several independent mechanisms. Moreover, the mechanisms of blood metabolite accumulation in patients with ACLF and kidney failure may be complex. In these patients, indeed, blood metabolite accumulation may be a result of both renal hypofiltration and increased metabolite production by renal cells. [START_REF] Clària | Orchestration of Tryptophan-Kynurenine pathway, acute decompensation and acute-on-chronic liver failure in cirrhosis[END_REF] Of note, the multivariate analyses also showed that changes in blood metabolome were more closely associated with deterioration of kidney or liver functions that with deterioration of brain function.

Our results strongly suggested a model in which changes in blood metabolites, in particular amino acids, may be best explained by a "metabolic reprogramming" whose primary purpose, as discussed below, was to fuel the intense systemic inflammatory response.

An important finding was that a single module (the yellow one) contained exclusively amino acids, of which most had higher blood levels in ACLF relative to AD. In the context of acute systemic inflammation, the activation of the hypothalamicpituitary-adrenal axis results in glucocorticoid release that causes muscle proteolysis [START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF] and mobilization of amino acids. [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF][START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF] Accordingly, fact that the yellowmodule membership exclusively included amino acids may be explained, at least in part, by their orchestrated mobilization from skeletal muscles.

In ACLF, activated innate immune responses included, for example, stimulation of bone marrow myelopoiesis (a proliferative process) resulting in peripheral-blood leukocytosis; activation of innate immune cells to produce a myriad of inflammatory molecules; and stimulation of hepatocytes to produce a broad variety of acute-phase proteins involved in the innate immune response. [START_REF] Moreau | Acute-onchronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis[END_REF][START_REF] Clària | Systemic inflammation in decompensated cirrhosis. Characterization and role in acute-on-chronic liver failure[END_REF] Execution of these immune responses is known to rely on anabolic, energy-consuming, cellintrinsic metabolic pathways involved in de novo synthesis of molecules including nonessential amino acids, purine and pyrimidine nucleotides, and ultimately proteins. [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF] In innate immune cells, stimulation of PAMP receptors [START_REF] Koyasu | The role of PI3K in immune cells[END_REF] or of cytokine receptors [START_REF] Koyasu | The role of PI3K in immune cells[END_REF] transduce signals that activate glycolysis and de novo nucleotide synthesis [START_REF] Villa | Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides[END_REF][START_REF] Saxton | mTOR Signaling in Growth, Metabolism, and Disease[END_REF] (Fig. S17A-C). In our study, 70% of the yellow-module amino acid members were proteinogenic amino acids, i.e., amino acids that can be incorporated into proteins under mRNA direction. Therefore, their mobilization from skeletal muscle in ACLF, was probably explained by the necessity to fuel de novo protein synthesis in cells involved in innate immune response. Moreover, features of increased glycolysis (and PPP), extraction of Krebs cycle intermediates and nucleotide synthesis, found in blood from patients with ACLF may also be explained by the necessity to fuel immune activation. Ketone bodies can be produced from the hepatic catabolism of fatty acids and of 6 amino acids [START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] (Fig. S15A). In acute systemic inflammation, including ACLF, the hepatic production of ketone bodies relies on catabolism of ketogenic amino acids because of the inhibition of fatty acid oxidation, which was witnessed by blood accumulation of acylcarnitines. [START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] In our study, 5 ketogenic amino acids were in the yellow module, and we found features indicating their enhanced catabolism in ACLF (Figs. S15B,S16; Table 1). However, despite this extensive catabolism, βhydroxybutyrate was not detectable in our patients, suggesting a defect or an inhibition of ketogenesis from amino acids. If this was the case, patients with ACLF might benefit from β-hydroxybutyrate administration because of the protective effects of this ketone body.

Our study provided other interesting results. In ACLF, we identified features suggesting the activation of the transsulfuration pathway which is a branch of the methionine cycle (a component of one-carbon metabolism network). [START_REF] Sanderson | Methionine metabolism in health and cancer: a nexus of diet and precision medicine[END_REF] Because the transsulfuration pathway gives rise to the antioxidant glutathione (Fig. 5), activation of this pathway makes sense in ACLF where systemic oxidative stress occurred as indicated by increased HNA2 levels. [START_REF] Clària | Systemic inflammation in decompensated cirrhosis. Characterization and role in acute-on-chronic liver failure[END_REF] This study showed that the polyamine spermidine, which is a product of methionine and ornithine metabolism, had decreased blood levels in ACLF that may be explained by polyamine skewing towards GABA synthesis. Because spermidine is an inducer of autophagy, [START_REF] Madeo | Spermidine in health and disease[END_REF] a decrease in in spermidine may play a role in the decrease in autophagy recently found in ACLF. [START_REF] Wan | LC3-associated phagocytosis protects against inflammation and liver fibrosis via immunoreceptor inhibitory signaling[END_REF] Collectively, these findings may have potential therapeutic consequences (Supplementary discussion). Several other questions regarding changes in amino acid metabolism found in patients with ACLF, in particular those related to methionine metabolism, tryptophan catabolism, urea cycle, and the potential impact of metabolic costs of immunity on peripheral-organ functions, need to be addressed in future studies (Supplementary discussion).

Our study has limitations because of its descriptive design. Indeed, our hypotheses were not challenged by mechanistic investigations in animal models and we did not explore blood metabolome in patients with acute systemic inflammation (e.g., sepsis) without cirrhosis. Nevertheless, analysis of the literature indicated that certain blood metabolic alterations were shared by patients without cirrhosis who had sepsis and poor outcome and our patients with ACLF. Indeed, like our patients with ACLF relative to those with AD, patients without cirrhosis who died from sepsis had higher baseline blood levels of pyruvate, lactate, α-ketoglutarate, oxaloacetate, and acylcarnitines than those who survived to sepsis. [START_REF] Langley | An integrated clinico-metabolomic model improves prediction of death in sepsis[END_REF] However, unlike patients without cirrhosis who had sepsis, [START_REF] Langley | An integrated clinico-metabolomic model improves prediction of death in sepsis[END_REF] our patients did not have blood accumulation of ketone bodies suggesting that this lack of accumulation may be a hallmark of ACLF.

Moreover, to our knowledge, the present study is the first to show in a large series of patients with acute systemic inflammation, the existence of multiple alterations in the one-carbon metabolism network, a decrease in spermidine levels and a possible connection with GABA synthesis.

In conclusion, our results suggest that, in ACLF, amino acids may be mobilized from skeletal muscles or derived from Krebs cycle intermediates to fuel anabolic programs, including protein and nucleotide synthesis, engaged by intense innate immune responses. Ketogenic amino acids were extensively catabolized to produce energy substrates in peripheral organs, an effect that was insufficient because organs failed. Finally, the polyamine spermidine which is a potent inducer of autophagy with anti-inflammatory effects, had decreased blood levels in ACLF that may be explained by polyamine skewing towards GABA synthesis. Future studies should investigate whether modifications of nutritional support could be used to reduce systemic inflammation and result in improvement of organ functions.

Figure legends

Fig. 1. Metabolite module membership. Distribution of metabolite members of each module according to metabolite categories detailed in Table 1. Metabolites related to amino acids is the only category to be present across the 9 modules. a Some isomeric metabolites could not be resolved using the liquid chromatography coupled to high-resolution mass spectrometry approach. 1

Box 1. Anabolic metabolism in activated innate immune cells.

Studies performed on sepsis under "non-cirrhotic" conditions have shown metabolic alterations, in particular alterations of the metabolisms of glucose and amino acids. At the onset of sepsis, innate immune cells, including myeloid cells (neutrophils, monocytes/macrophages, and dentritic cells) are activated because they recognize the presence of PAMPs. [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] Activated innate immune cells have an anabolic, energy-consuming metabolism resulting in synthesis of nucleotides, proteins and lipids that support leukocyte proliferation (increased biomass) and biosynthesis of a myriad of biomolecules involved in host defense, including, for example, soluble proteins such as cytokines, chemokines, and acute-phase proteins (these being produced and secreted by activated hepatocytes) (Fig. S1). [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] Nutrients (e.g., glucose, amino acids) are necessary to fuel immune activation. [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] Because sickness behavior of anorexia is often associated with sepsis, mobilization of stored fuels is critical for survival. [START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] Thus, during sepsis, there is activation of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system resulting in intense catabolic metabolism including, in particular, liver glycogenolysis (stimulated by adrenaline), and skeletal muscle proteolysis (stimulated by glucocorticoids), providing fuels (glucose and amino acids, respectively) to immunity (Fig. S1). [START_REF] Wang | An evolutionary perspective on immunometabolism[END_REF][START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Ganeshan | Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance[END_REF] In addition, in activated innate immune cells, some amino acids are produced by cell-intrinsic mechanisms. Whichever the origin of amino acids (blood, cell-intrinsic production), these nutrients are used as building blocks for protein synthesis and for some of them as substrates for de novo synthesis of nucleotides (see below), which are indispensable for RNA production and therefore protein synthesis.

Glucose extracted from blood is channeled to aerobic glycolysis to produce pyruvate, lactate and ATP (two ATP per glucose molecule) (Fig. S2A). Very few pyruvate is metabolized through the mitochondrial Krebs cycle, illustrating the preference of activated cells to use glucose for anabolism (biomass buildup, production of inflammatory cues) rather than for energy production; this preference being known as Warburg effect. [START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF] Thus, in activated innate immune cells, glucose and some nonessential amino acids play a crucial role in de novo nucleotide synthesis. Glucose is channeled to the pentose phosphate pathway (PPP) to produce riboses and then 5-phosphoribose-1-pyrophosphate (PRPP; Fig. S2A), which is a key molecule for synthesis of purine (Fig. S2B; Fig. S3) and pyrimidine (Fig. S2C; Fig. S4) nucleotides. [START_REF] Pareek | Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells[END_REF][START_REF] Villa | Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides[END_REF] In addition, the nonessential amino acid serine, extracted from blood or produced from an intermediate of aerobic glycolysis (Figs. S2 andS5), is channeled to the folate cycle to give rise to one-carbon atom and the amino acid glycine, both used for nucleotide synthesis (Fig. S5). [START_REF] Sanderson | Methionine metabolism in health and cancer: a nexus of diet and precision medicine[END_REF] In activated innate immune cells, the nonessential amino acid glutamine extracted from blood is used for nucleotide synthesis both directly (Figs. S2A to S2C, S3, and S4) and indirectly. The indirect pathway leads to the production of the nonessential amino acid aspartate (Fig. S2A), which is a major contributor to de novo nucleotide production (Figs. S2A). [START_REF] Pareek | Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells[END_REF][START_REF] Villa | Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides[END_REF] Thus, glutamine through glutamate gives rise to α-ketoglutarate which is channeled to Krebs cycle to produce oxaloacetate used for de novo aspartate synthesis. Finally, it is noteworthy that the folate cycle, mentioned earlier, and the methionine cycle are two components of what is referred to as one-carbon metabolism. [START_REF] Sanderson | Methionine metabolism in health and cancer: a nexus of diet and precision medicine[END_REF] Through the methionine cycle, the essential amino acid methionine can give rise to a myriad of molecules which all are crucial for immune cells. [START_REF] Sanderson | Methionine metabolism in health and cancer: a nexus of diet and precision medicine[END_REF][START_REF] Madeo | Spermidine in health and disease[END_REF] These molecules include not only nucleotides but also the methyl donor S-adenosylmethionine (SAM, involved in epigenetic regulation), polyamines (that stimulate autophagy), and cystathionine (which fuels the transsulfuration pathway that gives rise to the antioxidant glutathione) (Fig. S5). Collectively, these findings indicate that activation of the innate immune system requires the integrated metabolism of glucose, nonessential amino acids, and one-carbon metabolism, to produce nucleotides and proteins, among other effects. It is noteworthy that cancer cells that proliferate are an interesting model of biomass buildup. Indeed, proliferation of these cells relies on external nutrient supply and the integrated metabolism of glucose, nonessential amino acids, and one-carbon embolism network, to produce nucleotides, proteins, and antioxidant molecules required for biomass production. [START_REF] Vander Heiden | Understanding the Intersections between Metabolism and Cancer Biology[END_REF][START_REF] Kanarek | Dietary modifications for enhanced cancer therapy[END_REF]

Fig. 2 .

 2 Fig. 2. Metabolite modules are upregulated in ACLF. Comparison of the

Fig. 3 .

 3 Fig. 3. In ACLF, upregulation of metabolite modules correlates with intense

Fig. 4 .

 4 Fig. 4. Modeling of nucleotide synthesis in ACLF. (A) Schematic of central carbon

Fig. 5 .

 5 Fig. 5. Modeling the one-carbon metabolic network in ACLF. The one-carbon

Figure 5

 5 Figure 1

Table 1 . Representative blood metabolites measured in patients with acutely decompensated cirrhosis according to their module membership, HMDB class, KEGG annotations, fold-changes in ACLF relative to AD.*

 1 This study is a reanalysis of the CANONIC metabolomic data set previously published.[START_REF] Moreau | Acute-onchronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis[END_REF] The comprehensive lists of the 137 metabolites included in the data set is provided in

				Fold-
				change
	Metabolite module membership	HMDB Class	KEGG annotations (comment)	ACLF/AD	p value
	Turquoise module (n=28)
	Phenylacetyl-L-glutamine	N-acyl-alpha amino acids	Phenylalanine metabolism	2.47	<0.05
	N-Acetyl-ornithine	N-acyl-alpha amino acids	Arginine biosynthesis	1.23	<0.05
	4-Acetamidobutanoate	Gamma amino acids and	Arginine and proline metabolism	3.60	<0.05
		derivatives
	N6,N6,N6-Trimethyl-L-lysine	L-alpha-amino acid	Lysine degradation	1.76	<0.05
	N-Acetyl-L-alanine	N-acyl-l-alpha-amino acids	No KEGG ID	1.85	<0.05
	Hippurate	Hippuric acid (acyl glycine)	Phenylalanine metabolism.	1.66	<0.05
	Quinate	Quinic acids	Phenylalanine, tyrosine and tryptophan biosynthesis.	-1.44	0.05
	Phenol	Phenols	No KEGG pathway (HMDB: May be a product of tyrosine	2.89	<0.05
			metabolism)
	p-Anisate	P-methoxybenzoic acids and	No KEGG pathway (HMDB: Tyrosine metabolism)	4.27	<0.05
		derivatives.
	Quinolinate	Pyridinecarboxylic acids	Tryptophan metabolism	3.55	<0.05
	Kynurenate	Quinoline carboxylic acids	Tryptophan metabolism	2.65	<0.05
	Indolelactate	Indolyl carboxylic acids and	No KEGG pathway (HMDB: Tryptophan catabolite)	2.39	<0.05
		derivatives
	Indoxyl sulfate	Arylsulfates	No KEGG ID (HMDB: Tryptophan catabolite)	1.66	<0.05
	Indole-3-propionate	Indolyl carboxylic acids and	No KEGG ID (HMDB: Tryptophan catabolite)	-4.53	<0.05
		derivatives
	Methylimidazoleacetate	Imidazolyl carboxylic acids	Histidine metabolism	2.67	<0.05
		and derivatives

*

Table S1 .

 S1 Metabolite annotation was obtained using HMDB, KEGG databases and references #10 through #20, #23, #24, #32, #34 through #37. Metabolites in bold are those of potential bacterial origin.

[START_REF] Moreau | Blood metabolomics uncovers inflammation-associated mitochondrial dysfunction as a potential mechanism underlying ACLF[END_REF]

p values were obtained using the Student's test for unpaired data. HMDB denotes Human Metabolome Database (www.hmdb.ca), KEGG Kyoto Encyclopedia of Genes and Genomes www.genome.jp/kegg), AD acute decompensation of cirrhosis, and ACLF acute-on-chronic liver failure.

and PPTA, and is a consultant for Grifols SA, CLS Behring GmbH, Martin Pharmaceuticals and Shire/Takeda. The study was supported by the European Foundation for the Study of Chronic Liver Failure (EF-Clif). The EF-Clif is a non-profit private organization. Emmanuel Weiss is an EF-Clif Visiting Professor. The EF-Clif receives unrestricted donations from Cellex Foundation and Grifols, and is partner or contributor in several EU Horizon 2020 program projects. The funders had no influence on study design, data collection and analysis, decision to publish or preparation of the manuscript.