Elsevier

Journal of Hepatology

Volume 74, Issue 5, May 2021, Pages 1117-1131
Journal of Hepatology

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

https://doi.org/10.1016/j.jhep.2020.11.035Get rights and content

Highlights

  • In ACLF, skeletal muscle catabolism results in the release of amino acids that are used for anabolic and catabolic purposes.

  • In ACLF, nonessential amino acids and glucose are used for de novo nucleotide and protein synthesis.

  • In ACLF, the essential amino acid methionine fuels the transsulfuration pathway.

  • In ACLF, blood levels of the polyamine spermidine, an inducer of autophagy, are decreased.

Background & Aims

Systemic inflammation and organ failure(s) are the hallmarks of acute-on-chronic liver failure (ACLF), yet their pathogenesis remains uncertain. Herein, we aimed to assess the role of amino acids in these processes in patients with ACLF.

Methods

The blood metabolomic database of the CANONIC study (comprising 137 metabolites, with 43% related to amino acids) – obtained in 181 patients with ACLF and 650 with acute decompensation without ACLF (AD) – was reanalyzed with a focus on amino acids, in particular 9 modules of co-regulated metabolites. We also compared blood metabolite levels between ACLF and AD.

Results

The main findings in ACLF were: i) Metabolite modules were increased in parallel with increased levels of markers of systemic inflammation and oxidative stress. ii) Seventy percent of proteinogenic amino acids were present and most were increased. iii) 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. iv) Cystathionine, L-cystine, glutamate and pyroglutamate, which are involved in the transsulfuration pathway (a methionine cycle branch) were increased, consistent with increased synthesis of the antioxidant glutathione. v) Intermediates of the catabolism of 5 out of the 6 ketogenic amino acids were increased. vi) The levels of spermidine (a polyamine inducer of autophagy with anti-inflammatory effects) were decreased.

Conclusions

In ACLF, blood amino acids fueled protein and nucleotide synthesis required for the intense systemic inflammatory response. 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 the proinflammatory phenotype in ACLF.

Lay summary

Systemic inflammation and organ failures are hallmarks of acute-on-chronic liver failure (ACLF). Herein, we aimed to characterize the role of amino acids in these processes. The blood metabolome of patients with acutely decompensated cirrhosis, and particularly those with ACLF, reveals evidence of intense skeletal muscle catabolism. Importantly, amino acids (along with glucose), are used for intense anabolic, energy-consuming metabolism in patients with ACLF, presumably to support de novo nucleotide and protein synthesis in the activated innate immune system.

Introduction

Several studies, including the CANONIC study,1,2 the PREDICT study,3 and others,4,5 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, pathogen-associated molecular patterns (PAMPs) of bacterial origin[5], [6], [7] and damage-associated molecular patterns (DAMPs) released by dying cells5 activate pattern-recognition receptors of the innate immune system, resulting in systemic inflammation.5,6 Although systemic inflammation is certainly the primary cause of organ failures in ACLF, the mechanisms by which inflammation can perturb organ function are still unclear. These mechanisms may include tissue hypoperfusion,5 alterations in cardiac function,8,9 and immune-cell-mediated tissue damage.5 Moreover, recent metabolomic results 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.10

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 acute-phase proteins (Fig. S1).[11], [12], [13] Nutrients (e.g. glucose, amino acids) necessary to fuel immune activation[11], [12], [13] are mobilized from internal stores.13 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; 2 nonessential amino acids (glutamine and aspartate) are also substrates for de novo synthesis of nucleotides,14,15 which are indispensable for RNA synthesis and therefore protein synthesis. Glucose extracted from blood is channeled to aerobic glycolysis to produce ATP, pyruvate, and lactate.11,13 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).[11], [12], [13], [14], [15] The folate cycle, and the methionine cycle are components of what is referred to as the one-carbon metabolism network.16 Through the methionine cycle, the essential amino acid methionine can give rise to nucleotides among several other molecules.16,17 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.[11], [12], [13], [14], [15], [16], [17], [18], [19]

Results of metabolomics performed in blood from patients with ACLF revealed features consistent with enhanced aerobic glycolysis and PPP in the innate immune system.10 Because of the integration of glucose and amino acid metabolism in the 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. There is also evidence of enhanced catabolism of the essential amino acid tryptophan, through the kynurenine pathway, in the blood of patients with ACLF; this could give rise to metabolites that perturb peripheral-organ functions,20 indicating a potential role of certain amino acids in the development of organ system failures. Of note, patients with ACLF have sarcopenia,21,22 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 of assessing the potential role of major amino acid metabolic pathways in systemic inflammatory responses and organ failures in patients with acutely decompensated cirrhosis, with or without ACLF. Because amino acid and glucose metabolism are interrelated11,12 (Fig. S2A), we used the entire metabolic data set to perform correlative and integrative analyses. We also leveraged the fact that the blood metabolome may capture important metabolic changes that occur in tissues with highly active metabolism such as activated immune cells23 and cancer cells.24

Section snippets

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.10,20

Metabolomic data set

The data set comprised 137 well-annotated blood metabolites that had been identified using untargeted metabolomics by liquid chromatography coupled to high-resolution mass spectrometry, as previously described.10 The 137 metabolites are

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.

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

Discussion

This study reanalyzed the metabolomic data set (comprising 137 metabolites) obtained from samples of the CANONIC study,10 a study which enrolled a large cohort of patients with acutely decompensated cirrhosis.1 Using the entire metabolomic data set obtained in the whole cohort, we identified 9 metabolite modules, each containing highly correlated molecules and each receiving an arbitrary color label. Unlike other metabolites, amino acids were present in every module, highlighting the important

Financial support

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.

Authors’ contributions

Study concept and design (RM, VA); acquisition of clinical data and samples (GZ, PC, MB, PA, RJ); bioinformatic and statistical analyses (FA, JJL, AC, FF, FC, CJ); acquisition of metabolomic data (FF, FC, CJ), integration of clinical and biological results and interpretation of data (GZ, FA, JJL, AC, JC, RM); drafting of the manuscript (RM, VA); critical revision of the manuscript for important intellectual content (GZ, FA, PC, JC, FF, CJ, CF, EW, MB, PA, RJ); study supervision (RM, VA).

Data availability statement

The metabolomic data set used in this study is available in J Hepatol 2020;72:688-701.

Conflicts of interest

Dr. Jalan has research collaborations with Yaqrit and Takeda. Dr. Jalan is the inventor of OPA, which has been patented by UCL and licensed to Mallinckrodt Pharma. He is also the founder of Yaqrit limited, a spin out company from University College London and Thoeris Ltd. Dr. Bernardi is part of the speakers’ bureau for Grifols SA, Octapharma AG, Shire/Takeda, CLS Behring GmbH, and PPTA, and is a consultant for Grifols SA, CLS Behring GmbH, Martin Pharmaceuticals and Shire/Takeda.

The remaining

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