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The metabolomic window into hepatobiliary disease

  • Diren Beyoğlu
    Affiliations
    Hepatology Research Group, Department of Clinical Research, University of Bern, Bern, Switzerland
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  • Jeffrey R. Idle
    Correspondence
    Corresponding author. Address: Hepatology Research Group, Department of Clinical Research, University of Bern, Floor F, Murtenstrasse 35, 3010 Bern, Switzerland. Tel.: +41 79 446 24 24; fax: +41 31 632 49 97.
    Affiliations
    Hepatology Research Group, Department of Clinical Research, University of Bern, Bern, Switzerland
    Search for articles by this author

      Summary

      The emergent discipline of metabolomics has attracted considerable research effort in hepatology. Here we review the metabolomic data for non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), alcoholic liver disease (ALD), hepatitis B and C, cholecystitis, cholestasis, liver transplantation, and acute hepatotoxicity in animal models. A metabolomic window has permitted a view into the changing biochemistry occurring in the transitional phases between a healthy liver and hepatocellular carcinoma or cholangiocarcinoma. Whether provoked by obesity and diabetes, alcohol use or oncogenic viruses, the liver develops a core metabolomic phenotype (CMP) that involves dysregulation of bile acid and phospholipid homeostasis. The CMP commences at the transition between the healthy liver (Phase 0) and NAFLD/NASH, ALD or viral hepatitis (Phase 1). This CMP is maintained in the presence or absence of cirrhosis (Phase 2) and whether or not either HCC or CCA (Phase 3) develops. Inflammatory signalling in the liver triggers the appearance of the CMP. Many other metabolomic markers distinguish between Phases 0, 1, 2 and 3. A metabolic remodelling in HCC has been described but metabolomic data from all four Phases demonstrate that the Warburg shift from mitochondrial respiration to cytosolic glycolysis foreshadows HCC and may occur as early as Phase 1. The metabolic remodelling also involves an upregulation of fatty acid β-oxidation, also beginning in Phase 1. The storage of triglycerides in fatty liver provides high energy-yielding substrates for Phases 2 and 3 of liver pathology. The metabolomic window into hepatobiliary disease sheds new light on the systems pathology of the liver.

      Keywords

      Metabolomics and the liver in brief

      Over the past decade or more, many authors have defined the terms metabolomics and metabonomics. It is unproductive and unnecessary to add further to these definitions here. All the reader needs to know from the point of view of hepatobiliary disease, is that metabolomics is a window that offers a view distinct from the lenses of genomics, transcriptomics, and proteomics. There can be no other organ where such a plethora of both lipids and water-soluble metabolites are metabolically interchanged. No other organ exceeds the rates of metabolism and energy production and consumption as found in the liver. Not only is the liver the source of myriad endogenous metabolites and precursors used by other organs, but also houses a vast array of detoxication enzymes that are crucial for rendering less toxic, more water-soluble and readily excretable the 1–3 million xenobiotics to which we are exposed in our lifetimes [
      • Idle J.R.
      • Gonzalez F.J.
      Metabolomics.
      ]. The hepatic metabolome is therefore a highly complex and dynamic flux of small metabolites (say, <1.5 kDa, to include the larger phospholipid species, such as cardiolipins). Metabolomics in its practice combines high-throughput analytical chemistry, typically, methodologies based upon mass spectrometry or nuclear magnetic resonance spectroscopy, with multivariate data analysis. These technologies permit comparison of “global” metabolite profiles in an “unbiased” fashion for two or more groups of samples. Of course, no metabolomic investigation has ever delivered a global metabolite profile for a sample set, as this would require employment of multiple analytical platforms and several sample preparation protocols that performed from millimolar down to sub-picomolar concentrations. Moreover, different analytical platforms combined with specific sample preparation procedures each provide a different metabolomic window in the metabolic life of the liver. Accordingly, metabolomic findings reported are always biased by the laboratory analytical procedures employed, often highly so.
      This notwithstanding, many metabolomic investigators in recent years have entered the field of hepatobiliary disease and a considerable volume of publications has appeared. This review is therefore timely and we will attempt to make sense of a large and heterogeneous set of published studies concerning the varied hepatobiliary elements of pathophysiology where metabolomics has had something to say. This metabolomic window on hepatobiliary disease has furnished an overabundance of potential disease biomarkers. More importantly, in our view, the metabolomic lens has begun to provide new insights into liver disease mechanisms, new understandings that may unmask potential therapeutic targets and, one day, new treatment modalities.

      The metabolomic window into non-alcoholic diseases of the liver

      Overview

      In this review and as depicted in Fig. 1, we will describe the extent to which metabolomics has informed on the progression from the healthy liver to hepatocellular carcinoma (HCC) through the various phases of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver cirrhosis. We will also examine what metabolomics has taught about the various influencing factors and putative risk factors for these diseases, such as obesity, diabetes, alcohol, hepatitis B and C virus (HBV, HCV) infection. In addition, we will also review what metabolomics has contributed to the understanding of the change in hepatic function after liver transplantation.
      Figure thumbnail gr1
      Fig. 1Major liver diseases and potential influencing factors. This schematic shows the development of NAFLD from a healthy liver and various influencing factors. Steatosis is shown in yellow. NAFLD mostly becomes isolated fatty liver, but some cases progress to NASH, showing both steatosis and inflammatory necrosis (shown in red and black). NASH may progress to cirrhosis and then to HCC or to HCC directly. HCC, cirrhosis, and decompensated cirrhosis may all be treated by liver transplantation. Chemical carcinogens, such as aflatoxin B1, together with alcohol and HBV and HCV infection, are all potential influencing factors. NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus.

      Non-alcoholic fatty liver disease (NAFLD)

      Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent condition that affects 15% to 45% persons in developed nations [
      • Farrell G.C.
      • van Rooyen D.
      • Gan L.
      • Chitturi S.
      NASH is an inflammatory disorder: pathogenic prognostic and therapeutic implications.
      ] and both children and adults from all ethnic groups [
      • Torres D.M.
      • Williams C.D.
      • Harrison S.A.
      Features, diagnosis, and treatment of non-alcoholic fatty liver disease.
      ]. A diagnosis of NAFLD implies an increased risk of such diseases as cardiovascular disease, diabetes, colonic adenomas, hypothyroidism, and polycystic ovary syndrome [
      • Torres D.M.
      • Williams C.D.
      • Harrison S.A.
      Features, diagnosis, and treatment of non-alcoholic fatty liver disease.
      ]. NAFLD is generally considered to be the hepatic manifestation of metabolic syndrome [
      • Vinaixa M.
      • Rodriguez M.A.
      • Rull A.
      • Beltran R.
      • Blade C.
      • Brezmes J.
      • et al.
      Metabolomic assessment of the effect of dietary cholesterol in the progressive development of fatty liver disease.
      ]. The reference standard for diagnosing hepatic steatosis remains liver biopsy [
      • Torres D.M.
      • Williams C.D.
      • Harrison S.A.
      Features, diagnosis, and treatment of non-alcoholic fatty liver disease.
      ]. Investigators have employed metabolomic protocols in an attempt to define biomarkers that might replace this invasive procedure for a disease of such high prevalence. Table 1 shows a summary of 11 studies with metabolomic components that inform regarding the formation of hepatic steatosis. Animal models and studies in living human subjects and human tissues have been employed. One common finding is that of increased lipid species in the liver and serum/plasma, including cholesterol esters [
      • van Ginneken V.
      • Verhey E.
      • Poelmann R.
      • Ramakers R.
      • van Dijk K.W.
      • Ham L.
      • et al.
      Metabolomics (liver and blood profiling) in a mouse model in response to fasting: a study of hepatic steatosis.
      ,
      • Hyde M.J.
      • Griffin J.L.
      • Herrera E.
      • Byrne C.D.
      • Clarke L.
      • Kemp P.R.
      Delivery by Caesarean section, rather than vaginal delivery, promotes hepatic steatosis in piglets.
      ], triacylglycerols [
      • Vinaixa M.
      • Rodriguez M.A.
      • Rull A.
      • Beltran R.
      • Blade C.
      • Brezmes J.
      • et al.
      Metabolomic assessment of the effect of dietary cholesterol in the progressive development of fatty liver disease.
      ,
      • van Ginneken V.
      • Verhey E.
      • Poelmann R.
      • Ramakers R.
      • van Dijk K.W.
      • Ham L.
      • et al.
      Metabolomics (liver and blood profiling) in a mouse model in response to fasting: a study of hepatic steatosis.
      ,
      • Hyde M.J.
      • Griffin J.L.
      • Herrera E.
      • Byrne C.D.
      • Clarke L.
      • Kemp P.R.
      Delivery by Caesarean section, rather than vaginal delivery, promotes hepatic steatosis in piglets.
      ,
      • Puri P.
      • Wiest M.M.
      • Cheung O.
      • Mirshahi F.
      • Sargeant C.
      • Min H.K.
      • et al.
      The plasma lipidomic signature of non-alcoholic steatohepatitis.
      ], diacylglycerols [
      • Vinaixa M.
      • Rodriguez M.A.
      • Rull A.
      • Beltran R.
      • Blade C.
      • Brezmes J.
      • et al.
      Metabolomic assessment of the effect of dietary cholesterol in the progressive development of fatty liver disease.
      ], sphingomyelins [
      • Vinaixa M.
      • Rodriguez M.A.
      • Rull A.
      • Beltran R.
      • Blade C.
      • Brezmes J.
      • et al.
      Metabolomic assessment of the effect of dietary cholesterol in the progressive development of fatty liver disease.
      ], various bile salts [
      • Garcia-Canaveras J.C.
      • Donato M.T.
      • Castell J.V.
      • Lahoz A.
      A comprehensive untargeted metabonomic analysis of human steatotic liver tissue by RP and HILIC chromatography coupled to mass spectrometry reveals important metabolic alterations.
      ,
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ,
      • Barr J.
      • Vazquez-Chantada M.
      • Alonso C.
      • Perez-Cormenzana M.
      • Mayo R.
      • Galan A.
      • et al.
      Liquid chromatography–mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of non-alcoholic fatty liver disease.
      ], together with lactate [
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ,
      • Li H.
      • Wang L.
      • Yan X.
      • Liu Q.
      • Yu C.
      • Wei H.
      • et al.
      A proton nuclear magnetic resonance metabonomics approach for biomarker discovery in non-alcoholic fatty liver disease.
      ,
      • Toye A.A.
      • Dumas M.E.
      • Blancher C.
      • Rothwell A.R.
      • Fearnside J.F.
      • Wilder S.P.
      • et al.
      Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice.
      ] and glutamate [
      • Li H.
      • Wang L.
      • Yan X.
      • Liu Q.
      • Yu C.
      • Wei H.
      • et al.
      A proton nuclear magnetic resonance metabonomics approach for biomarker discovery in non-alcoholic fatty liver disease.
      ,
      • Noguchi Y.
      • Young J.D.
      • Aleman J.O.
      • Hansen M.E.
      • Kelleher J.K.
      • Stephanopoulos G.
      Tracking cellular metabolomics in lipoapoptosis- and steatosis-developing liver cells.
      ]. In addition, cysteine-glutathione disulfide and both oxidized and reduced glutathione were all reported to be depressed in the liver and serum/plasma [
      • Garcia-Canaveras J.C.
      • Donato M.T.
      • Castell J.V.
      • Lahoz A.
      A comprehensive untargeted metabonomic analysis of human steatotic liver tissue by RP and HILIC chromatography coupled to mass spectrometry reveals important metabolic alterations.
      ,
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ]. Finally, where diets that instigate fatty liver had been used, depressed concentrations of glucose were reported both in rat liver [
      • Griffin J.L.
      • Scott J.
      • Nicholson J.K.
      The influence of pharmacogenetics on fatty liver disease in the Wistar and Kyoto rats: a combined transcriptomic and metabonomic study.
      ] and mouse serum [
      • Li H.
      • Wang L.
      • Yan X.
      • Liu Q.
      • Yu C.
      • Wei H.
      • et al.
      A proton nuclear magnetic resonance metabonomics approach for biomarker discovery in non-alcoholic fatty liver disease.
      ], but in one study, elevated plasma glucose was reported [
      • Toye A.A.
      • Dumas M.E.
      • Blancher C.
      • Rothwell A.R.
      • Fearnside J.F.
      • Wilder S.P.
      • et al.
      Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice.
      ]. Taken together with elevated mouse serum/plasma lactate [
      • Li H.
      • Wang L.
      • Yan X.
      • Liu Q.
      • Yu C.
      • Wei H.
      • et al.
      A proton nuclear magnetic resonance metabonomics approach for biomarker discovery in non-alcoholic fatty liver disease.
      ,
      • Toye A.A.
      • Dumas M.E.
      • Blancher C.
      • Rothwell A.R.
      • Fearnside J.F.
      • Wilder S.P.
      • et al.
      Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice.
      ], pyruvate and alanine [
      • Toye A.A.
      • Dumas M.E.
      • Blancher C.
      • Rothwell A.R.
      • Fearnside J.F.
      • Wilder S.P.
      • et al.
      Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice.
      ], and human plasma lactate [
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ], these results would suggest that NAFLD engages in cytosolic glycolysis. NAFLD is frequently associated with insulin resistance and insulin has been reported in mice to activate pyruvate kinase M2 [
      • Hines I.N.
      • Hartwell H.J.
      • Feng Y.
      • Theve E.J.
      • Hall G.A.
      • Hashway S.
      • et al.
      Insulin resistance and metabolic hepatocarcinogenesis with parent-of-origin effects in AxB mice.
      ], the enzyme switch to glycolysis involved in the Warburg effect and thus the production of lactate and alanine from glucose via pyruvate. Furthermore, the reduction in glutathione derivatives in human liver [
      • Garcia-Canaveras J.C.
      • Donato M.T.
      • Castell J.V.
      • Lahoz A.
      A comprehensive untargeted metabonomic analysis of human steatotic liver tissue by RP and HILIC chromatography coupled to mass spectrometry reveals important metabolic alterations.
      ] and plasma [
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ] in NAFLD is a clear sign of active oxidative stress in the liver.
      The lipidomic component of the observations summarized in Table 1 is of interest. Firstly, it has been reported that phosphocholine, choline, betaine, and trimethylamine N-oxide (TMAO) were upregulated metabolites in both the liver and plasma of rodents fed diets that provoked fatty liver [
      • Li H.
      • Wang L.
      • Yan X.
      • Liu Q.
      • Yu C.
      • Wei H.
      • et al.
      A proton nuclear magnetic resonance metabonomics approach for biomarker discovery in non-alcoholic fatty liver disease.
      ,
      • Toye A.A.
      • Dumas M.E.
      • Blancher C.
      • Rothwell A.R.
      • Fearnside J.F.
      • Wilder S.P.
      • et al.
      Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice.
      ]. This is a clear indication of an increased turnover of phosphatidylcholine and phosphatidylethanolamine species in the liver, thus releasing free fatty acids through the action of phospholipases A1 and A2. These fatty acids, if not catabolized by β-oxidation, will be stored in the liver as triacylglycerols. This is what was observed in the metabolomic studies of animals with fatty liver [
      • Vinaixa M.
      • Rodriguez M.A.
      • Rull A.
      • Beltran R.
      • Blade C.
      • Brezmes J.
      • et al.
      Metabolomic assessment of the effect of dietary cholesterol in the progressive development of fatty liver disease.
      ,
      • van Ginneken V.
      • Verhey E.
      • Poelmann R.
      • Ramakers R.
      • van Dijk K.W.
      • Ham L.
      • et al.
      Metabolomics (liver and blood profiling) in a mouse model in response to fasting: a study of hepatic steatosis.
      ,
      • Hyde M.J.
      • Griffin J.L.
      • Herrera E.
      • Byrne C.D.
      • Clarke L.
      • Kemp P.R.
      Delivery by Caesarean section, rather than vaginal delivery, promotes hepatic steatosis in piglets.
      ]. Therefore, fatty liver is not just a deposition of fat in the liver but rather a rearrangement and repartitioning of lipid stores as it has been proposed [
      • van Ginneken V.
      • Verhey E.
      • Poelmann R.
      • Ramakers R.
      • van Dijk K.W.
      • Ham L.
      • et al.
      Metabolomics (liver and blood profiling) in a mouse model in response to fasting: a study of hepatic steatosis.
      ]. Using a mouse 24-h starvation protocol, it was observed that the triacylglycerols TG(44:2) and TG(48:3) massively increased in the liver by 2427% and 1198%, respectively. These are the most abundant triacylglycerols in adipose tissue and these findings suggest that adipose may be a source of triacylglycerols deposited in the liver in NAFLD [
      • van Ginneken V.
      • Verhey E.
      • Poelmann R.
      • Ramakers R.
      • van Dijk K.W.
      • Ham L.
      • et al.
      Metabolomics (liver and blood profiling) in a mouse model in response to fasting: a study of hepatic steatosis.
      ]. Secondly, elevated hepatic concentrations of various lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and phosphatidylcholine (PC) species have been reported for human steatotic vs. non-steatotic livers [
      • Garcia-Canaveras J.C.
      • Donato M.T.
      • Castell J.V.
      • Lahoz A.
      A comprehensive untargeted metabonomic analysis of human steatotic liver tissue by RP and HILIC chromatography coupled to mass spectrometry reveals important metabolic alterations.
      ]. These molecules are obvious candidates for the elevated choline and choline metabolites discussed above. Finally, three studies in humans reported elevated bile salts in the liver [
      • Garcia-Canaveras J.C.
      • Donato M.T.
      • Castell J.V.
      • Lahoz A.
      A comprehensive untargeted metabonomic analysis of human steatotic liver tissue by RP and HILIC chromatography coupled to mass spectrometry reveals important metabolic alterations.
      ] that spilled over to elevated bile acids in serum/plasma [
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ,
      • Barr J.
      • Vazquez-Chantada M.
      • Alonso C.
      • Perez-Cormenzana M.
      • Mayo R.
      • Galan A.
      • et al.
      Liquid chromatography–mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of non-alcoholic fatty liver disease.
      ]. Bile acids act as signaling molecules in the liver that regulate lipid and glucose homeostasis [
      • Torres D.M.
      • Williams C.D.
      • Harrison S.A.
      Features, diagnosis, and treatment of non-alcoholic fatty liver disease.
      ,
      • Wei J.
      • Qiu de K.
      • Ma X.
      Bile acids and insulin resistance: implications for treating non-alcoholic fatty liver disease.
      ]. Certain bile acids, in particular, chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA), are endogenous ligands that activate the farnesoid X receptor (FXR) [
      • Wang H.
      • Chen J.
      • Hollister K.
      • Sowers L.C.
      • Forman B.M.
      Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.
      ]. The nuclear receptor FXR modulates conversion of cholesterol to bile acids by the regulation of the expression of CYP7A1 [
      • Torres D.M.
      • Williams C.D.
      • Harrison S.A.
      Features, diagnosis, and treatment of non-alcoholic fatty liver disease.
      ]. Moreover, FXR reduces lipogenesis by downregulating expression of SREBP-1, activates the nuclear receptor PPARα causing an increase in β-oxidation of free fatty acids (FFA), both of which processes reduce hepatic FFA levels [
      • Torres D.M.
      • Williams C.D.
      • Harrison S.A.
      Features, diagnosis, and treatment of non-alcoholic fatty liver disease.
      ,
      • Wei J.
      • Qiu de K.
      • Ma X.
      Bile acids and insulin resistance: implications for treating non-alcoholic fatty liver disease.
      ]. There is a single report of elevated hepatic levels of the bile salts glycochenodeoxycholate 3-sulfate (GCDCA-3S) and taurochenodeoxycholate (TCDCA) in human fatty liver [
      • Garcia-Canaveras J.C.
      • Donato M.T.
      • Castell J.V.
      • Lahoz A.
      A comprehensive untargeted metabonomic analysis of human steatotic liver tissue by RP and HILIC chromatography coupled to mass spectrometry reveals important metabolic alterations.
      ]. TCDCA is a relatively weak activator of FXR [
      • Wang H.
      • Chen J.
      • Hollister K.
      • Sowers L.C.
      • Forman B.M.
      Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.
      ] and GCDCA-3S appears not to have been studied in this regard. It is curious that NAFLD existed in the presence of increased serum/plasma concentrations of glycocholate, taurocholate, glycochenodeoxycholate [
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ], and deoxycholate [
      • Barr J.
      • Vazquez-Chantada M.
      • Alonso C.
      • Perez-Cormenzana M.
      • Mayo R.
      • Galan A.
      • et al.
      Liquid chromatography–mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of non-alcoholic fatty liver disease.
      ], which may not reflect hepatic concentrations of the FXR activators CDCA and DCA. This theme will be returned to in the next section.

      Non-alcoholic steatohepatitis (NASH)

      NASH is a more advanced stage of NAFLD with a major inflammatory component [
      • Farrell G.C.
      • van Rooyen D.
      • Gan L.
      • Chitturi S.
      NASH is an inflammatory disorder: pathogenic prognostic and therapeutic implications.
      ]. NAFLD may progress to NASH, but >80% of cases remain as isolated fatty liver (IFL) with no or minimal progression to cirrhosis and no increased risk of death relative to the general population [
      • Torres D.M.
      • Williams C.D.
      • Harrison S.A.
      Features, diagnosis, and treatment of non-alcoholic fatty liver disease.
      ]. It has been estimated that ∼11% of NASH cases develop cirrhosis over 15 years and ∼7% progress to hepatocellular carcinoma (HCC) over 6.5 years, either via cirrhosis or sometimes directly [
      • Torres D.M.
      • Williams C.D.
      • Harrison S.A.
      Features, diagnosis, and treatment of non-alcoholic fatty liver disease.
      ] (Fig. 1). The origins of the hepatic inflammation in NASH continues to involve a major research effort and one theory posits that hepatitis originates in visceral adipose, which is intrinsically pro-inflammatory [
      • Farrell G.C.
      • van Rooyen D.
      • Gan L.
      • Chitturi S.
      NASH is an inflammatory disorder: pathogenic prognostic and therapeutic implications.
      ]. A study in mice fed a high-fat diet supports this theory [
      • Stanton M.C.
      • Chen S.C.
      • Jackson J.V.
      • Rojas-Triana A.
      • Kinsley D.
      • Cui L.
      • et al.
      Inflammatory Signals shift from adipose to liver during high fat feeding and influence the development of steatohepatitis in mice.
      ].
      There have been relatively few metabolomic studies addressing the pathobiology of NASH and its progression from simple NAFLD and all these have examined serum/plasma only. Five studies are summarized in Table 2. As with NAFLD, triacylglycerols and several fatty acids were elevated in plasma [
      • Puri P.
      • Wiest M.M.
      • Cheung O.
      • Mirshahi F.
      • Sargeant C.
      • Min H.K.
      • et al.
      The plasma lipidomic signature of non-alcoholic steatohepatitis.
      ] and like NAFLD, several other fatty acids and LPCs were attenuated in plasma [
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ]. When a small series of NASH was compared with NAFLD, significant changes in serum concentrations of only three phospholipids were reported [
      • Barr J.
      • Vazquez-Chantada M.
      • Alonso C.
      • Perez-Cormenzana M.
      • Mayo R.
      • Galan A.
      • et al.
      Liquid chromatography–mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of non-alcoholic fatty liver disease.
      ]. A study using NMR, which, unlike mass-spectrometry-based platforms does not have the power of detecting a large range of molecules [
      • Beyoglu D.
      • Idle J.R.
      Metabolomics and its potential in drug development.
      ], contributed raised serum concentrations of glucose, glutamate and taurine [
      • Li H.
      • Wang L.
      • Yan X.
      • Liu Q.
      • Yu C.
      • Wei H.
      • et al.
      A proton nuclear magnetic resonance metabonomics approach for biomarker discovery in non-alcoholic fatty liver disease.
      ]. The greatest metabolomic insights into NASH pathogenesis come from a recent study that combined high-end analytics and targeted gene expression by qPCR [
      • Tanaka N.
      • Matsubara T.
      • Krausz K.W.
      • Patterson A.D.
      • Gonzalez F.J.
      Disruption of phospholipid and bile acid homeostasis in mice with non-alcoholic steatohepatitis.
      ]. In this study, NASH was generated in mice fed a methionine- and choline-deficient (MCD) diet. UPLC-ESI-TOFMS metabolomics revealed a statistically significant depression of LPC (16:0), LPC (18:0) and LPC (18:1) in serum with a significant rise in tauro-β-muricholate, taurocholate, and 12-HETE for MCD fed mice compared with mice on a normal diet. As a positive control, genetically obese ob/ob mice with severe steatosis were administered galactosamine (GalN), which provoked severe inflammation and hepatocyte injury with marked upregulation of hepatic mRNAs coding for TNFα and TGFβ1. Serum of GalN-injected ob/ob steatotic mice compared with saline-injected ob/ob steatotic mice displayed the same changes in LPCs and bile acids as the MCD fed mice. Thus, the decline in serum LPC and rise in serum bile acids are a signature of the inflammatory component of NASH, rather than the steatotic component. To investigate further the mechanisms involved in these perturbations of LPC and bile acid homeostasis in the NASH model, hepatic mRNA levels were determined by qPCR for genes involved in the metabolism and transport of LPC, bile acids and 12-HETE. Lysophosphatidylcholine acyltransferases (LPCAT) that convert LPC to PC [
      • Zhao Y.
      • Chen Y.Q.
      • Bonacci T.M.
      • Bredt D.S.
      • Li S.
      • Bensch W.R.
      • et al.
      Identification and characterization of a major liver lysophosphatidylcholine acyltransferase.
      ] were all upregulated with two- to four-fold elevations in hepatic Lpcat1, Lpcat2, and Lpcat3 mRNAs in the NASH model. Additionally, the transporters SLC10A1 and SLCO1A1 that uptake bile salts into hepatocytes and the transporters ABCC1 and ABCC4 that export bile acids from the liver were highly downregulated and upregulated, respectively [
      • Tanaka N.
      • Matsubara T.
      • Krausz K.W.
      • Patterson A.D.
      • Gonzalez F.J.
      Disruption of phospholipid and bile acid homeostasis in mice with non-alcoholic steatohepatitis.
      ]. Taken together, these observations explain how the inflammatory phenotype of NASH in a mouse model results in the changes in serum metabolites described in Table 2 and this is shown in Fig. 2. Importantly, similar perturbations have been observed in NASH patients [
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ], suggesting that similar mechanisms may operate in humans. Finally, it should be stated that biomarkers for NASH are limited and therapeutic options are poorly developed, which serves to emphasize the need for further metabolomic research in this area.
      Figure thumbnail gr2
      Fig. 2Mechanisms leading to lowered LPCs and elevated bile acids in serum in NASH. Reproduced with permission from Tanaka et al.
      [
      • Tanaka N.
      • Matsubara T.
      • Krausz K.W.
      • Patterson A.D.
      • Gonzalez F.J.
      Disruption of phospholipid and bile acid homeostasis in mice with non-alcoholic steatohepatitis.
      ]
      . LPC, lysophosphatidylcholine.

      Fibrosis and cirrhosis

      Liver fibrosis is a scarring process involving the deposition of excess connective tissue in response to injury. Cirrhosis may be considered as the end stage of this reaction, comprising formation of fibrous septa and hepatocyte nodules. Oxidative stress provokes the inflammatory reactions and apoptosis involved in the generation of cirrhosis [
      • Constantinou M.A.
      • Theocharis S.E.
      • Mikros E.
      Application of metabonomics on an experimental model of fibrosis and cirrhosis induced by thioacetamide in rats.
      ]. It is now clear that NAFLD/NASH may develop into cirrhosis, although the histological features of precursor NASH in the cirrhotic liver may be challenging to diagnose [
      • Yeh M.M.
      • Brunt E.M.
      Pathology of non-alcoholic fatty liver disease.
      ]. Cirrhosis may arise due to a large number of causes, principal among which are not only NAFLD/NASH but also alcoholic fatty liver disease and viral hepatitis B or C (Fig. 1). There are no cut-off values for laboratory analyses that give a diagnosis of cirrhosis [
      • Wiegand J.
      • Berg T.
      The etiology, diagnosis and prevention of liver cirrhosis: part 1 of a series on liver cirrhosis.
      ] and so the generation of novel metabolomic biomarkers to detect early cirrhosis has become a justifiable aim. Table 3 summarizes such studies.
      Three studies have been conducted in rats administered hepatotoxins to provoke fibrosis and cirrhosis. Histopathology confirmed that rats exposed to thioacetamide in their drinking water developed hepatic fibrosis after one month and cirrhosis after three months. Liver extracts examined by NMR had higher levels of lactate [
      • Constantinou M.A.
      • Theocharis S.E.
      • Mikros E.
      Application of metabonomics on an experimental model of fibrosis and cirrhosis induced by thioacetamide in rats.
      ], suggesting a degree of anaerobic metabolism within the fibrotic liver. Two studies treated rats with carbon tetrachloride (CCl4), which induced fibrosis [
      • Gou X.
      • Tao Q.
      • Feng Q.
      • Peng J.
      • Sun S.
      • Cao H.
      • et al.
      Urinary metabonomics characterization of liver fibrosis induced by CCl(4) in rats and intervention effects of Xia Yu Xue Decoction.
      ,
      • Zhang A.
      • Sun H.
      • Dou S.
      • Sun W.
      • Wu X.
      • Wang P.
      • et al.
      Metabolomics study on the hepatoprotective effect of scoparone using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry.
      ], and the authors also evaluated treatment with the Chinese medicine Xia Yu Xue Decoction [
      • Gou X.
      • Tao Q.
      • Feng Q.
      • Peng J.
      • Sun S.
      • Cao H.
      • et al.
      Urinary metabonomics characterization of liver fibrosis induced by CCl(4) in rats and intervention effects of Xia Yu Xue Decoction.
      ] or scoparone, a drug isolated from a medicinal plant [
      • Zhang A.
      • Sun H.
      • Dou S.
      • Sun W.
      • Wu X.
      • Wang P.
      • et al.
      Metabolomics study on the hepatoprotective effect of scoparone using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry.
      ]. Many metabolomic signals were reported after CCl4 administration, including decreases in the urinary excretion of certain amino acids and gut flora metabolites (which were mostly reversed by Xia Yu Xue Decoction) [
      • Gou X.
      • Tao Q.
      • Feng Q.
      • Peng J.
      • Sun S.
      • Cao H.
      • et al.
      Urinary metabonomics characterization of liver fibrosis induced by CCl(4) in rats and intervention effects of Xia Yu Xue Decoction.
      ] and an increased urinary excretion of glycocholate [
      • Zhang A.
      • Sun H.
      • Dou S.
      • Sun W.
      • Wu X.
      • Wang P.
      • et al.
      Metabolomics study on the hepatoprotective effect of scoparone using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry.
      ]. Neither serum nor liver tissue was examined in these studies. Thus, hepatic fibrosis provoked in a normal, rather than fatty, rat liver, is associated with somewhat minor changes in the urinary metabolome.
      Eight metabolomic investigations of hepatic cirrhosis have all been performed on human materials, six on serum [
      • Gao H.
      • Lu Q.
      • Liu X.
      • Cong H.
      • Zhao L.
      • Wang H.
      • et al.
      Application of 1H NMR-based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis.
      ,
      • Waldhier M.C.
      • Almstetter M.F.
      • Nurnberger N.
      • Gruber M.A.
      • Dettmer K.
      • Oefner P.J.
      Improved enantiomer resolution and quantification of free d-amino acids in serum and urine by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.
      ,
      • Lian J.S.
      • Liu W.
      • Hao S.R.
      • Guo Y.Z.
      • Huang H.J.
      • Chen D.Y.
      • et al.
      A serum metabonomic study on the difference between alcohol- and HBV-induced liver cirrhosis by ultraperformance liquid chromatography coupled to mass spectrometry plus quadrupole time-of-flight mass spectrometry.
      ,
      • Du Z.
      • Zhang L.
      • Liu S.
      Application of liquid chromatography–mass spectrometry in the study of metabolic profiling of cirrhosis in different grades.
      ,
      • Qi S.W.
      • Tu Z.G.
      • Peng W.J.
      • Wang L.X.
      • Ou-Yang X.
      • Cai A.J.
      • et al.
      (1)H NMR-based serum metabolic profiling in compensated and decompensated cirrhosis.
      ,
      • Lin X.
      • Zhang Y.
      • Ye G.
      • Li X.
      • Yin P.
      • Ruan Q.
      • et al.
      Classification and differential metabolite discovery of liver diseases based on plasma metabolic profiling and support vector machines.
      ], one on liver biopsies [
      • Martinez-Granados B.
      • Morales J.M.
      • Rodrigo J.M.
      • Del Olmo J.
      • Serra M.A.
      • Ferrandez A.
      • et al.
      Metabolic profile of chronic liver disease by NMR spectroscopy of human biopsies.
      ], and one on faeces [
      • Huang H.J.
      • Zhang A.Y.
      • Cao H.C.
      • Lu H.F.
      • Wang B.H.
      • Xie Q.
      • et al.
      Metabolomic analyses of faeces reveals malabsorption in cirrhotic patients.
      ]. No clear picture emerges from these studies. An increased serum concentration of non-essential amino acids [
      • Gao H.
      • Lu Q.
      • Liu X.
      • Cong H.
      • Zhao L.
      • Wang H.
      • et al.
      Application of 1H NMR-based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis.
      ] and certain d-amino acids [
      • Waldhier M.C.
      • Almstetter M.F.
      • Nurnberger N.
      • Gruber M.A.
      • Dettmer K.
      • Oefner P.J.
      Improved enantiomer resolution and quantification of free d-amino acids in serum and urine by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.
      ] and a decreased serum concentration of essential amino acids [
      • Gao H.
      • Lu Q.
      • Liu X.
      • Cong H.
      • Zhao L.
      • Wang H.
      • et al.
      Application of 1H NMR-based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis.
      ,
      • Waldhier M.C.
      • Almstetter M.F.
      • Nurnberger N.
      • Gruber M.A.
      • Dettmer K.
      • Oefner P.J.
      Improved enantiomer resolution and quantification of free d-amino acids in serum and urine by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.
      ,
      • Qi S.W.
      • Tu Z.G.
      • Peng W.J.
      • Wang L.X.
      • Ou-Yang X.
      • Cai A.J.
      • et al.
      (1)H NMR-based serum metabolic profiling in compensated and decompensated cirrhosis.
      ] suggest that the cirrhotic liver has an impaired ability to metabolize both protein and d-amino acids. Other notable observations include the decrease in several LPCs in serum of cirrhotics versus healthy volunteers, whether cirrhosis was due to alcohol or hepatitis B [
      • Lian J.S.
      • Liu W.
      • Hao S.R.
      • Guo Y.Z.
      • Huang H.J.
      • Chen D.Y.
      • et al.
      A serum metabonomic study on the difference between alcohol- and HBV-induced liver cirrhosis by ultraperformance liquid chromatography coupled to mass spectrometry plus quadrupole time-of-flight mass spectrometry.
      ]. This pattern is similar to that observed for NASH (Table 2), although the cirrhotic patients studied had a background of alcohol abuse or hepatitis B. Moreover, glycochenodeoxycholic acid and glycocholic acid concentrations were also elevated in serum [
      • Lian J.S.
      • Liu W.
      • Hao S.R.
      • Guo Y.Z.
      • Huang H.J.
      • Chen D.Y.
      • et al.
      A serum metabonomic study on the difference between alcohol- and HBV-induced liver cirrhosis by ultraperformance liquid chromatography coupled to mass spectrometry plus quadrupole time-of-flight mass spectrometry.
      ]. Clearly, the mechanism proposed by Gonzalez and colleagues [
      • Tanaka N.
      • Matsubara T.
      • Krausz K.W.
      • Patterson A.D.
      • Gonzalez F.J.
      Disruption of phospholipid and bile acid homeostasis in mice with non-alcoholic steatohepatitis.
      ] shown in Fig. 2 may apply not only to NASH but to other inflammatory liver diseases.
      Selective impairment of hepatic β-oxidation was apparent from a reduced serum carnitine and increased serum palmitoleoylcarnitine (16:1) and oleoylcarnitine (18:1) concentrations [
      • Lin X.
      • Zhang Y.
      • Ye G.
      • Li X.
      • Yin P.
      • Ruan Q.
      • et al.
      Classification and differential metabolite discovery of liver diseases based on plasma metabolic profiling and support vector machines.
      ]. Impaired ammonium detoxication in cirrhosis is implied from a reported shift from hepatic levels of glutamine and glucose to glutamate [
      • Martinez-Granados B.
      • Morales J.M.
      • Rodrigo J.M.
      • Del Olmo J.
      • Serra M.A.
      • Ferrandez A.
      • et al.
      Metabolic profile of chronic liver disease by NMR spectroscopy of human biopsies.
      ]. Finally, a very interesting report catalogued changes in the faecal metabolome between 24 healthy volunteers and 17 cirrhotics [
      • Huang H.J.
      • Zhang A.Y.
      • Cao H.C.
      • Lu H.F.
      • Wang B.H.
      • Xie Q.
      • et al.
      Metabolomic analyses of faeces reveals malabsorption in cirrhotic patients.
      ]. In faeces from cirrhotic patients, there was an increased concentration of the major LPCs (16:0, 18:0, 18:1, 18:2) and a decreased faecal excretion of chenodeoxycholic acid and 7-ketolithocholic acid, the latter reported as a gut flora metabolite of the former by Bacteroides intestinalis [
      • Fukiya S.
      • Arata M.
      • Kawashima H.
      • Yoshida D.
      • Kaneko M.
      • Minamida K.
      • et al.
      Conversion of cholic acid and chenodeoxycholic acid into their 7-oxo derivatives by Bacteroides intestinalis AM-1 isolated from human feces.
      ]. The data on faecal excretion of LPCs and bile acids further supports and enhances the mechanism outlined in Fig. 2.

      Hepatocellular carcinoma (HCC)

      More than half a million people are diagnosed each year with hepatocellular carcinoma (HCC). The disease has a poor prognosis, generally because of its late presentation and its incidence is growing in developed countries. There has been considerable research effort to try to define biomarkers that would aid earlier detection and thus improve patient outcomes. Many researchers, particularly in China, have employed metabolomic protocols towards this end. Table 4 contains details of 24 metabolomic investigations of human HCC [
      • Gao H.
      • Lu Q.
      • Liu X.
      • Cong H.
      • Zhao L.
      • Wang H.
      • et al.
      Application of 1H NMR-based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis.
      ,
      • Lin X.
      • Zhang Y.
      • Ye G.
      • Li X.
      • Yin P.
      • Ruan Q.
      • et al.
      Classification and differential metabolite discovery of liver diseases based on plasma metabolic profiling and support vector machines.
      ,
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Pang T.
      • Lv S.
      • et al.
      Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases.
      ,
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Wang C.
      • Zhao X.
      • et al.
      Strategy for metabonomics research based on high-performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry.
      ,
      • Yang Y.
      • Li C.
      • Nie X.
      • Feng X.
      • Chen W.
      • Yue Y.
      • et al.
      Metabonomic studies of human hepatocellular carcinoma using high-resolution magic-angle spinning 1H NMR spectroscopy in conjunction with multivariate data analysis.
      ,
      • Yin P.
      • Wan D.
      • Zhao C.
      • Chen J.
      • Zhao X.
      • Wang W.
      • et al.
      A metabonomic study of hepatitis B-induced liver cirrhosis and hepatocellular carcinoma by using RP-LC and HILIC coupled with mass spectrometry.
      ,
      • Wu H.
      • Xue R.
      • Dong L.
      • Liu T.
      • Deng C.
      • Zeng H.
      • et al.
      Metabolomic profiling of human urine in hepatocellular carcinoma patients using gas chromatography/mass spectrometry.
      ,
      • Chen J.
      • Wang W.
      • Lv S.
      • Yin P.
      • Zhao X.
      • Lu X.
      • et al.
      Metabonomics study of liver cancer based on ultra performance liquid chromatography coupled to mass spectrometry with HILIC and RPLC separations.
      ,
      • Wang J.
      • Zhang S.
      • Li Z.
      • Yang J.
      • Huang C.
      • Liang R.
      • et al.
      (1)H-NMR-based metabolomics of tumor tissue for the metabolic characterization of rat hepatocellular carcinoma formation and metastasis.
      ,
      • Chen T.
      • Xie G.
      • Wang X.
      • Fan J.
      • Qiu Y.
      • Zheng X.
      • et al.
      Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma.
      ,
      • Chen F.
      • Xue J.
      • Zhou L.
      • Wu S.
      • Chen Z.
      Identification of serum biomarkers of hepatocarcinoma through liquid chromatography/mass spectrometry-based metabonomic method.
      ,
      • Patterson A.D.
      • Maurhofer O.
      • Beyoglu D.
      • Lanz C.
      • Krausz K.W.
      • Pabst T.
      • et al.
      Aberrant lipid metabolism in hepatocellular carcinoma revealed by plasma metabolomics and lipid profiling.
      ,
      • Tan Y.
      • Yin P.
      • Tang L.
      • Xing W.
      • Huang Q.
      • Cao D.
      • et al.
      Metabolomics study of stepwise hepatocarcinogenesis from the model rats to patients: potential biomarkers effective for small hepatocellular carcinoma diagnosis.
      ,
      • Wang B.
      • Chen D.
      • Chen Y.
      • Hu Z.
      • Cao M.
      • Xie Q.
      • et al.
      Metabonomic profiles discriminate hepatocellular carcinoma from liver cirrhosis by ultraperformance liquid chromatography–mass spectrometry.
      ,
      • Zhou L.
      • Wang Q.
      • Yin P.
      • Xing W.
      • Wu Z.
      • Chen S.
      • et al.
      Serum metabolomics reveals the deregulation of fatty acids metabolism in hepatocellular carcinoma and chronic liver diseases.
      ,
      • Ye G.
      • Zhu B.
      • Yao Z.
      • Yin P.
      • Lu X.
      • Kong H.
      • et al.
      Analysis of urinary metabolic signatures of early hepatocellular carcinoma recurrence after surgical removal using gas chromatography–mass spectrometry.
      ,
      • Lin X.
      • Yang F.
      • Zhou L.
      • Yin P.
      • Kong H.
      • Xing W.
      • et al.
      A support vector machine-recursive feature elimination feature selection method based on artificial contrast variables and mutual information.
      ,
      • Ressom H.W.
      • Xiao J.F.
      • Tuli L.
      • Varghese R.S.
      • Zhou B.
      • Tsai T.H.
      • et al.
      Utilization of metabolomics to identify serum biomarkers for hepatocellular carcinoma in patients with liver cirrhosis.
      ,
      • Xiao J.F.
      • Varghese R.S.
      • Zhou B.
      • Nezami Ranjbar M.R.
      • Zhao Y.
      • Tsai T.H.
      • et al.
      LC–MS based serum metabolomics for identification of hepatocellular carcinoma biomarkers in Egyptian cohort.
      ,
      • Zhang A.
      • Sun H.
      • Yan G.
      • Han Y.
      • Ye Y.
      • Wang X.
      Urinary metabolic profiling identifies a key role for glycocholic acid in human liver cancer by ultra-performance liquid-chromatography coupled with high-definition mass spectrometry.
      ,
      • Nahon P.
      • Amathieu R.
      • Triba M.N.
      • Bouchemal N.
      • Nault J.C.
      • Ziol M.
      • et al.
      Identification of serum proton NMR metabolomic fingerprints associated with hepatocellular carcinoma in patients with alcoholic cirrhosis.
      ,
      • Budhu A.
      • Roessler S.
      • Zhao X.
      • Yu Z.
      • Forgues M.
      • Ji J.
      • et al.
      Integrated metabolite and gene expression profiles identify lipid biomarkers associated with progression of hepatocellular carcinoma and patient outcomes.
      ,
      • Beyoglu D.
      • Imbeaud S.
      • Maurhofer O.
      • Bioulac-Sage P.
      • Zucman-Rossi J.
      • Dufour J.F.
      • et al.
      Tissue metabolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptomic classification.
      ,
      • Shariff M.I.
      • Gomaa A.I.
      • Cox I.J.
      • Patel M.
      • Williams H.R.
      • Crossey M.M.
      • et al.
      Urinary metabolic biomarkers of hepatocellular carcinoma in an Egyptian population: a validation study.
      ], three of chemically-induced rat HCC [
      • Wang J.
      • Zhang S.
      • Li Z.
      • Yang J.
      • Huang C.
      • Liang R.
      • et al.
      (1)H-NMR-based metabolomics of tumor tissue for the metabolic characterization of rat hepatocellular carcinoma formation and metastasis.
      ,
      • Tan Y.
      • Yin P.
      • Tang L.
      • Xing W.
      • Huang Q.
      • Cao D.
      • et al.
      Metabolomics study of stepwise hepatocarcinogenesis from the model rats to patients: potential biomarkers effective for small hepatocellular carcinoma diagnosis.
      ,
      • Li Z.F.
      • Wang J.
      • Huang C.
      • Zhang S.
      • Yang J.
      • Jiang A.
      • et al.
      Gas chromatography/time-of-flight mass spectrometry-based metabonomics of hepatocarcinoma in rats with lung metastasis: elucidation of the metabolic characteristics of hepatocarcinoma at formation and metastasis.
      ] and two of hepatocellular adenomas in the flatfish Limanda limanda [
      • Southam A.D.
      • Easton J.M.
      • Stentiford G.D.
      • Ludwig C.
      • Arvanitis T.N.
      • Viant M.R.
      Metabolic changes in flatfish hepatic tumours revealed by NMR-based metabolomics and metabolic correlation networks.
      ,
      • Stentiford G.D.
      • Viant M.R.
      • Ward D.G.
      • Johnson P.J.
      • Martin A.
      • Wenbin W.
      • et al.
      Liver tumors in wild flatfish: a histopathological, proteomic, and metabolomic study.
      ]. Many investigators of human HCC employed healthy volunteers as a control group, especially for the collection of serum/plasma or urine [
      • Gao H.
      • Lu Q.
      • Liu X.
      • Cong H.
      • Zhao L.
      • Wang H.
      • et al.
      Application of 1H NMR-based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis.
      ,
      • Lin X.
      • Zhang Y.
      • Ye G.
      • Li X.
      • Yin P.
      • Ruan Q.
      • et al.
      Classification and differential metabolite discovery of liver diseases based on plasma metabolic profiling and support vector machines.
      ,
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Pang T.
      • Lv S.
      • et al.
      Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases.
      ,
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Wang C.
      • Zhao X.
      • et al.
      Strategy for metabonomics research based on high-performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry.
      ,
      • Yin P.
      • Wan D.
      • Zhao C.
      • Chen J.
      • Zhao X.
      • Wang W.
      • et al.
      A metabonomic study of hepatitis B-induced liver cirrhosis and hepatocellular carcinoma by using RP-LC and HILIC coupled with mass spectrometry.
      ,
      • Wu H.
      • Xue R.
      • Dong L.
      • Liu T.
      • Deng C.
      • Zeng H.
      • et al.
      Metabolomic profiling of human urine in hepatocellular carcinoma patients using gas chromatography/mass spectrometry.
      ,
      • Chen J.
      • Wang W.
      • Lv S.
      • Yin P.
      • Zhao X.
      • Lu X.
      • et al.
      Metabonomics study of liver cancer based on ultra performance liquid chromatography coupled to mass spectrometry with HILIC and RPLC separations.
      ,
      • Chen T.
      • Xie G.
      • Wang X.
      • Fan J.
      • Qiu Y.
      • Zheng X.
      • et al.
      Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma.
      ,
      • Chen F.
      • Xue J.
      • Zhou L.
      • Wu S.
      • Chen Z.
      Identification of serum biomarkers of hepatocarcinoma through liquid chromatography/mass spectrometry-based metabonomic method.
      ,
      • Patterson A.D.
      • Maurhofer O.
      • Beyoglu D.
      • Lanz C.
      • Krausz K.W.
      • Pabst T.
      • et al.
      Aberrant lipid metabolism in hepatocellular carcinoma revealed by plasma metabolomics and lipid profiling.
      ,
      • Wang B.
      • Chen D.
      • Chen Y.
      • Hu Z.
      • Cao M.
      • Xie Q.
      • et al.
      Metabonomic profiles discriminate hepatocellular carcinoma from liver cirrhosis by ultraperformance liquid chromatography–mass spectrometry.
      ,
      • Zhou L.
      • Wang Q.
      • Yin P.
      • Xing W.
      • Wu Z.
      • Chen S.
      • et al.
      Serum metabolomics reveals the deregulation of fatty acids metabolism in hepatocellular carcinoma and chronic liver diseases.
      ,
      • Ye G.
      • Zhu B.
      • Yao Z.
      • Yin P.
      • Lu X.
      • Kong H.
      • et al.
      Analysis of urinary metabolic signatures of early hepatocellular carcinoma recurrence after surgical removal using gas chromatography–mass spectrometry.
      ,
      • Lin X.
      • Yang F.
      • Zhou L.
      • Yin P.
      • Kong H.
      • Xing W.
      • et al.
      A support vector machine-recursive feature elimination feature selection method based on artificial contrast variables and mutual information.
      ,
      • Zhang A.
      • Sun H.
      • Yan G.
      • Han Y.
      • Ye Y.
      • Wang X.
      Urinary metabolic profiling identifies a key role for glycocholic acid in human liver cancer by ultra-performance liquid-chromatography coupled with high-definition mass spectrometry.
      ,
      • Shariff M.I.
      • Gomaa A.I.
      • Cox I.J.
      • Patel M.
      • Williams H.R.
      • Crossey M.M.
      • et al.
      Urinary metabolic biomarkers of hepatocellular carcinoma in an Egyptian population: a validation study.
      ], others used cirrhotics as a comparator group [
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Pang T.
      • Lv S.
      • et al.
      Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases.
      ,
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Wang C.
      • Zhao X.
      • et al.
      Strategy for metabonomics research based on high-performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry.
      ,
      • Yin P.
      • Wan D.
      • Zhao C.
      • Chen J.
      • Zhao X.
      • Wang W.
      • et al.
      A metabonomic study of hepatitis B-induced liver cirrhosis and hepatocellular carcinoma by using RP-LC and HILIC coupled with mass spectrometry.
      ,
      • Patterson A.D.
      • Maurhofer O.
      • Beyoglu D.
      • Lanz C.
      • Krausz K.W.
      • Pabst T.
      • et al.
      Aberrant lipid metabolism in hepatocellular carcinoma revealed by plasma metabolomics and lipid profiling.
      ,
      • Tan Y.
      • Yin P.
      • Tang L.
      • Xing W.
      • Huang Q.
      • Cao D.
      • et al.
      Metabolomics study of stepwise hepatocarcinogenesis from the model rats to patients: potential biomarkers effective for small hepatocellular carcinoma diagnosis.
      ,
      • Wang B.
      • Chen D.
      • Chen Y.
      • Hu Z.
      • Cao M.
      • Xie Q.
      • et al.
      Metabonomic profiles discriminate hepatocellular carcinoma from liver cirrhosis by ultraperformance liquid chromatography–mass spectrometry.
      ,
      • Zhou L.
      • Wang Q.
      • Yin P.
      • Xing W.
      • Wu Z.
      • Chen S.
      • et al.
      Serum metabolomics reveals the deregulation of fatty acids metabolism in hepatocellular carcinoma and chronic liver diseases.
      ,
      • Lin X.
      • Yang F.
      • Zhou L.
      • Yin P.
      • Kong H.
      • Xing W.
      • et al.
      A support vector machine-recursive feature elimination feature selection method based on artificial contrast variables and mutual information.
      ,
      • Ressom H.W.
      • Xiao J.F.
      • Tuli L.
      • Varghese R.S.
      • Zhou B.
      • Tsai T.H.
      • et al.
      Utilization of metabolomics to identify serum biomarkers for hepatocellular carcinoma in patients with liver cirrhosis.
      ,
      • Xiao J.F.
      • Varghese R.S.
      • Zhou B.
      • Nezami Ranjbar M.R.
      • Zhao Y.
      • Tsai T.H.
      • et al.
      LC–MS based serum metabolomics for identification of hepatocellular carcinoma biomarkers in Egyptian cohort.
      ,
      • Nahon P.
      • Amathieu R.
      • Triba M.N.
      • Bouchemal N.
      • Nault J.C.
      • Ziol M.
      • et al.
      Identification of serum proton NMR metabolomic fingerprints associated with hepatocellular carcinoma in patients with alcoholic cirrhosis.
      ], while others included acute hepatitis [
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Pang T.
      • Lv S.
      • et al.
      Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases.
      ,
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Wang C.
      • Zhao X.
      • et al.
      Strategy for metabonomics research based on high-performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry.
      ], chronic hepatitis [
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Pang T.
      • Lv S.
      • et al.
      Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases.
      ,
      • Yang J.
      • Xu G.
      • Zheng Y.
      • Kong H.
      • Wang C.
      • Zhao X.
      • et al.
      Strategy for metabonomics research based on high-performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry.
      ,
      • Tan Y.
      • Yin P.
      • Tang L.
      • Xing W.
      • Huang Q.
      • Cao D.
      • et al.
      Metabolomics study of stepwise hepatocarcinogenesis from the model rats to patients: potential biomarkers effective for small hepatocellular carcinoma diagnosis.
      ,
      • Zhou L.
      • Wang Q.
      • Yin P.
      • Xing W.
      • Wu Z.
      • Chen S.
      • et al.
      Serum metabolomics reveals the deregulation of fatty acids metabolism in hepatocellular carcinoma and chronic liver diseases.
      ,
      • Lin X.
      • Yang F.
      • Zhou L.
      • Yin P.
      • Kong H.
      • Xing W.
      • et al.
      A support vector machine-recursive feature elimination feature selection method based on artificial contrast variables and mutual information.
      ], benign liver tumours [
      • Chen T.
      • Xie G.
      • Wang X.
      • Fan J.
      • Qiu Y.
      • Zheng X.
      • et al.
      Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma.
      ], and acute myeloid leukemia [
      • Patterson A.D.
      • Maurhofer O.
      • Beyoglu D.
      • Lanz C.
      • Krausz K.W.
      • Pabst T.
      • et al.
      Aberrant lipid metabolism in hepatocellular carcinoma revealed by plasma metabolomics and lipid profiling.
      ] as comparator groups. These metabolomic comparisons have permitted insights into the biochemical transitions to HCC from various precursor states, at least as viewed through serum/plasma or urine. A relatively few studies have addressed the hepatic metabolome directly by interrogating tumour tissue and paired uninvolved liver for human HCC [
      • Yang Y.
      • Li C.
      • Nie X.
      • Feng X.
      • Chen W.
      • Yue Y.
      • et al.
      Metabonomic studies of human hepatocellular carcinoma using high-resolution magic-angle spinning 1H NMR spectroscopy in conjunction with multivariate data analysis.
      ,
      • Budhu A.
      • Roessler S.
      • Zhao X.
      • Yu Z.
      • Forgues M.
      • Ji J.
      • et al.
      Integrated metabolite and gene expression profiles identify lipid biomarkers associated with progression of hepatocellular carcinoma and patient outcomes.
      ,
      • Beyoglu D.
      • Imbeaud S.
      • Maurhofer O.
      • Bioulac-Sage P.
      • Zucman-Rossi J.
      • Dufour J.F.
      • et al.
      Tissue metabolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptomic classification.
      ], chemically-induced rat HCC [
      • Wang J.
      • Zhang S.
      • Li Z.
      • Yang J.
      • Huang C.
      • Liang R.
      • et al.
      (1)H-NMR-based metabolomics of tumor tissue for the metabolic characterization of rat hepatocellular carcinoma formation and metastasis.
      ] and fish hepatocellular adenoma [
      • Southam A.D.
      • Easton J.M.
      • Stentiford G.D.
      • Ludwig C.
      • Arvanitis T.N.
      • Viant M.R.
      Metabolic changes in flatfish hepatic tumours revealed by NMR-based metabolomics and metabolic correlation networks.
      ,
      • Stentiford G.D.
      • Viant M.R.
      • Ward D.G.
      • Johnson P.J.
      • Martin A.
      • Wenbin W.
      • et al.
      Liver tumors in wild flatfish: a histopathological, proteomic, and metabolomic study.
      ]. Two recent reports also combined transcriptomic and metabolomic analyses of human HCC [
      • Budhu A.
      • Roessler S.
      • Zhao X.
      • Yu Z.
      • Forgues M.
      • Ji J.
      • et al.
      Integrated metabolite and gene expression profiles identify lipid biomarkers associated with progression of hepatocellular carcinoma and patient outcomes.
      ,
      • Beyoglu D.
      • Imbeaud S.
      • Maurhofer O.
      • Bioulac-Sage P.
      • Zucman-Rossi J.
      • Dufour J.F.
      • et al.
      Tissue metabolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptomic classification.
      ]. As will be demonstrated below, comparison of the outputs of metabolomic investigations of NAFLD/NASH, cirrhosis, and HCC will permit a new understanding of the chain of biochemical events that lead from a healthy liver to HCC.

      Disease progression from fatty liver to hepatocellular carcinoma

      The metabolomic observations encompassed in Table 1, Table 2, Table 3, Table 4 have been combined into a visual format (Fig. 3) which permits a biochemical view of the changes occurring from fatty liver through cirrhosis to HCC. Only observations reported in at least two independent human studies have been entered into this Figure. The paramount conclusion is that elevated bile acids and lowered LPCs are common across all three groups of pathology. The bile acids affected include GCA, TCA, GDCA, and GCDCA. A whole range of LPCs, comprising saturated, monounsaturated, and polyunsaturated long-chain and very long-chain fatty acids are affected. The probable mechanisms by which these metabolic perturbations have occurred were discussed above and are shown, in part, in Fig. 2. Increased biliary excretion of phospholipids is an additional factor already discussed above. Of importance is that these alterations in hepatic metabolism would appear to occur very early in the chain of events leading from the normal liver to HCC (Fig. 1) and therefore must be maintained throughout the progression to HCC.
      Table 1Summary of metabolomic studies examining the development of NAFLD.
      HV, healthy volunteers; PA, palmitic acid; OA, oleic acid; NMR, nuclear magnetic resonance spectroscopy; FPLC, fast performance liquid chromatography; HPTLC, high performance thin-layer chromatography; LCMS, liquid chromatography–mass spectrometry; GCFID, gas chromatography with flame ionization detection; UPLC, ultraperformance liquid chromatography; ESI, electrospray ionization; TQMS, triple quadrupole mass spectrometry; QTOFMS, quadrupole time-of-flight mass spectrometry; TMAO, trimethylamine N-oxide; TG, triacylglycerol (triglyceride); FA, fatty acid; 15-HETE, (±)-15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (non-enzymic oxidation product of arachidonic acid); DCA, deoxycholic acid; GCA, glycocholic acid; TCA, taurocholic acid; GCDCA, glycochenodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; G-6-P, glucose-6-phosphate; G-1-P, glucose-1-phosphate; GNMT, glycine N-methyltransferase; PUFA, polyunsaturated fatty acids; MUFA, monounsaturated fatty acids; LPC, lysophosphocholine; SCD1, stearoyl-CoA desaturase-1.
      Table 2Summary of metabolomic studies examining the development of NASH.
      For abbreviations, see Table 1 footnotes.
      Table 3Summary of metabolomic studies examining the development of hepatic fibrosis and cirrhosis.
      LC, liver cirrhosis; CHB, chronic hepatitis B; TMA, trimethylamine; CCl4, carbon tetrachloride; UFA, unsaturated fatty acid units (-CH = CH-CH2-). GCxGC-TOFMS, Two-dimensional gas chromatography time-of-flight mass spectrometry. OPLS-DA, orthogonal partial least squares projection to latent structures-discriminant analysis, PE, phosphatidylethanolamine. For other abbreviations, see footnotes to Table 1.
      Table 4Summary of metabolomic studies examining the development of hepatocellular carcinoma.
      FTICR, Fourier transform ion cyclotron resonance mass spectrometry; MR, relative molecular mass (molecular weight); GCTOFMS, gas chromatography time-of-flight mass spectrometry; BLT, benign liver tumour; AA, arachidonic acid; EPA, 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid; DHA, 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid; TQMS, triple quadrupole mass spectrometry; AFP, α-fetoprotein; LPE, lysophosphoethanolamine; TCA, taurocholic acid; CHB, chronic hepatitis B; RPost, recurrent HCC post-surgery; NRPost, nonrecurrent HCC post-surgery; RPre, recurrent HCC pre-surgery; For other abbreviations, see footnotes to Table 1.
      Figure thumbnail gr3
      Fig. 3Venn diagram showing the up- and downregulated metabolites in NAFLD/NASH, cirrhosis, and HCC. Elevated bile acids and lowered lysophosphatidylcholines are common across the pathological evolution in humans and comprise a core metabolomic phenotype. For abbreviations, see .
      As shown in Fig. 3, NAFLD/NASH (Table 1, Table 2) is characterized by upregulation of lactate, glucose, glutamate and tyrosine, together with the downregulation of cortisone. This would suggest that, in the fatty liver states, hepatic glucose is mobilized from glycogen almost certainly due to insulin resistance [
      • Pessayre D.
      Role of mitochondria in non-alcoholic fatty liver disease.
      ]. The rise in lactate may be a sign of a degree of metabolic remodelling to aerobic glycolysis in response to elevated glucose, although there was little evidence of the other glycolytic metabolites, pyruvate and alanine [
      • Beyoglu D.
      • Imbeaud S.
      • Maurhofer O.
      • Bioulac-Sage P.
      • Zucman-Rossi J.
      • Dufour J.F.
      • et al.
      Tissue metabolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptomic classification.
      ], being elevated in NAFLD/NASH. The rise in glutamate is a sign of reduced cytosolic glutamine synthesis and thus an impairment of ammonium detoxication [
      • Adeva M.M.
      • Souto G.
      • Blanco N.
      • Donapetry C.
      Ammonium metabolism in humans.
      ]. This is usually associated with cirrhosis and liver failure, but may also be manifest in NASH [
      • Felipo V.
      • Urios A.
      • Montesinos E.
      • Molina I.
      • Garcia-Torres M.L.
      • Civera M.
      • et al.
      Contribution of hyperammonemia and inflammatory factors to cognitive impairment in minimal hepatic encephalopathy.
      ]. The upregulation of tyrosine in NAFLD/NASH is at odds with a single report in which plasma tyrosine was lower in NAFLD than controls [
      • Mukherjee S.
      • Vaidyanathan K.
      • Vasudevan D.M.
      • Das S.K.
      Role of plasma amino acids and gaba in alcoholic and non-alcoholic fatty liver disease-a pilot study.
      ]. Elevated tyrosine is more likely to be correct as the metabolomic report [
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ] employed a more specific analysis. Finally, the metabolomic investigations of NAFLD and NASH [
      • Kalhan S.C.
      • Guo L.
      • Edmison J.
      • Dasarathy S.
      • McCullough A.J.
      • Hanson R.W.
      • et al.
      Plasma metabolomic profile in non-alcoholic fatty liver disease.
      ] have reported that plasma cortisone is downregulated. This is consistent with the upregulation of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) in visceral obesity, metabolic syndrome, and type 2 diabetes [
      • Seckl J.R.
      • Walker B.R.
      Minireview: 11beta-hydroxysteroid dehydrogenase type 1 – a tissue-specific amplifier of glucocorticoid action.
      ,
      • Cooper M.S.
      • Stewart P.M.
      11Beta-hydroxysteroid dehydrogenase type 1 and its role in the hypothalamus–pituitary–adrenal axis, metabolic syndrome, and inflammation.
      ] and its role in NAFLD [
      • Moon S.S.
      • Lee Y.S.
      • Kim J.G.
      • Lee I.K.
      Association of 11beta-hydroxysteroid dehydrogenase type 1 gene polymorphisms with serum alanine aminotransferase activity.
      ]. HSD11B1 activates cortisone to cortisol in liver and adipose tissue. Thus, the aforementioned metabolomic signals are consistent with known biochemical characteristics of the fatty liver. In particular, elevated serum bile acids and reduced LPCs are in accord with known changes in gene expression in NASH (Fig. 2).
      As shown in Table 3, a relatively small number of metabolomic studies have addressed the conversion of either normal or fatty human liver states to cirrhosis. Only two metabolomic markers specific to cirrhosis could therefore be defined, downregulation of the branched-chain amino acids (BCAAs) valine and isoleucine. Lowered plasma BCAAs in cirrhosis was first observed almost six decades ago [
      • Muting D.
      • Wortmann V.
      Amino acid metabolism in liver diseases.
      ] and is due to hepatic metabolism of BCAAs to provide carbon skeletons for the TCA cycle [
      • Dam G.
      • Ott P.
      • Aagaard N.K.
      • Vilstrup H.
      Branched-chain amino acids and muscle ammonia detoxification in cirrhosis.
      ]. Noteworthy is the carry forward from NAFLD/NASH into cirrhosis of elevated bile acids and reduced LPCs (Fig. 3).
      The greatest number of human metabolomic studies was conducted in HCC and, not surprisingly, there occur a large number of metabolomic changes in HCC relative to cirrhosis or to control subjects (Table 4). As shown in Fig. 3, there are many signs of a metabolic remodelling in the livers of HCC patients, detected by metabolomics. For example, the decrease in glucose, citrate, and glycerol 3-phosphate coupled with an increase in pyruvate are all signs of the Warburg effect [
      • Warburg O.
      On respiratory impairment in cancer cells.
      ], a switch from mitochondrial respiration to cytosolic aerobic glycolysis [
      • Cairns R.A.
      • Harris I.S.
      • Mak T.W.
      Regulation of cancer cell metabolism.
      ,
      • Vander Heiden M.G.
      • Cantley L.C.
      • Thompson C.B.
      Understanding the Warburg effect: the metabolic requirements of cell proliferation.
      ]. By a metabolomic comparison of paired HCC biopsies and uninvolved liver tissues, we have calculated that the switch to the aerobic glycolysis in HCC is no more than four-fold [
      • Beyoglu D.
      • Imbeaud S.
      • Maurhofer O.
      • Bioulac-Sage P.
      • Zucman-Rossi J.
      • Dufour J.F.
      • et al.
      Tissue metabolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptomic classification.
      ]. Although tumours are generally considered to synthesize fatty acids de novo from citrate via acetyl-CoA [
      • Vander Heiden M.G.
      • Cantley L.C.
      • Thompson C.B.
      Understanding the Warburg effect: the metabolic requirements of cell proliferation.
      ], the accumulated metabolomic data in HCC (Fig. 3) tend to point to increased fatty acid β-oxidation, with elevated acetate and 2-oxoglutarate (immediate precursor of carnitine) and reduced free fatty acids, carnitine and carnitine esters. Furthermore, some transcriptomic types of HCC, in particular G1 and G3, displayed markedly reduced 1-palmitoylglycerol, 1-stearoylglycerol and palmitate compared with surrounding uninvolved liver tissue [
      • Beyoglu D.
      • Imbeaud S.
      • Maurhofer O.
      • Bioulac-Sage P.
      • Zucman-Rossi J.
      • Dufour J.F.
      • et al.
      Tissue metabolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptomic classification.
      ]. Thus, metabolic reprogramming in HCC appears to comprise a modest Warburg shift to glycolysis and a major upregulation of fatty acid catabolism in some tumour types.

      The metabolomic window into other hepatobiliary diseases

      Alcoholic liver disease

      The consumption of alcoholic beverages leads to exposure of the liver to ethanol. While many consider the pharmacological effects of ethanol consumption enjoyable, ethanol is nevertheless a solvent that can exhibit potent toxicological effects, in particular, on the liver. Alcohol exposure to laboratory animals can provoke a range of pathologies that parallel non-alcoholic liver disease. For example, 20 to 40 kg micropigs voluntarily consume an ethanol-supplemented diet (40% daily energy needs), developing peak blood ethanol levels >200 mg/dl and, within 6 months, hepatic steatosis, inflammation, and fibrosis. Alcoholic micropigs displayed increased hepatic TG levels relative to controls with elevated fatty acid ratios of 16:1n7/16:0 and 18:1n9/18:0, due to increased stearoyl-CoA desaturase activity. The authors concluded that increased de novo lipogenesis and reduced LPC synthesis and export were responsible for the accumulation of TG during alcoholic steatohepatitis (ASH) [
      • Zivkovic A.M.
      • Bruce German J.
      • Esfandiari F.
      • Halsted C.H.
      Quantitative lipid metabolomic changes in alcoholic micropigs with fatty liver disease.
      ]. Athymic nude mice gavaged with ethanol solutions from 5% gradually to 40%, developed mild hepatic hemorrhage, with elevated serum PC, decreased saturated and monounsaturated LPC, and elevated polyunsaturated LPC levels [
      • Li S.
      • Liu H.
      • Jin Y.
      • Lin S.
      • Cai Z.
      • Jiang Y.
      Metabolomics study of alcohol-induced liver injury and hepatocellular carcinoma xenografts in mice.
      ]. Similarly, rats fed 5% ethanol developed fatty infiltration after 2 months with mild inflammation and oxidative stress after 3 months. NMR metabolomics suggested that hepatic fatty acids and TG increased and plasma fatty acids and PC decreased [
      • Fernando H.
      • Bhopale K.K.
      • Kondraganti S.
      • Kaphalia B.S.
      • Shakeel Ansari G.A.
      Lipidomic changes in rat liver after long-term exposure to ethanol.
      ]. These contradictions may reflect a species difference but more likely underscore the relative weakness of NMR as a lipidomic tool.
      Another approach to study alcohol-induced liver disease (ALD) has been to employ the Ppara-null mouse, since the nuclear receptor PPARα is a master regulator of hepatic lipid metabolism whose biochemical effects can be detected through metabolomics, both in humans [
      • Patterson A.D.
      • Slanar O.
      • Krausz K.W.
      • Li F.
      • Hofer C.C.
      • Perlik F.
      • et al.
      Human urinary metabolomic profile of PPARalpha induced fatty acid beta-oxidation.
      ] and in mice [
      • Zhen Y.
      • Krausz K.W.
      • Chen C.
      • Idle J.R.
      • Gonzalez F.J.
      Metabolomic and genetic analysis of biomarkers for peroxisome proliferator-activated receptor alpha expression and activation.
      ]. Ppara-null and control mice were fed a 4% ethanol-containing liquid diet and an isocaloric control diet, respectively. After one month, steatosis with elevated hepatic TGs was observed for the Ppara-null mice only. Metabolomic analysis revealed elevated indole-3-lactic acid associated with the development of ALD in ethanol-treated Ppara-null mice [
      • Manna S.K.
      • Patterson A.D.
      • Yang Q.
      • Krausz K.W.
      • Li H.
      • Idle J.R.
      • et al.
      Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse.
      ]. In an enlarged study, these authors reported that indole-3-lactic acid and phenyllactic acid were potential biomarkers for early ALD [
      • Manna S.K.
      • Patterson A.D.
      • Yang Q.
      • Krausz K.W.
      • Idle J.R.
      • Fornace A.J.
      • et al.
      UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model.
      ]. CYP2E1 is the principal ethanol-inducible hepatic enzyme responsible for ethanol metabolism and hepatotoxicity [
      • Lieber C.S.
      Metabolism of alcohol.
      ]. A metabolomic study in Cyp2e1-null and control mice reported that the ethanol metabolite acetate can acetylate taurine in the liver, leading to ethanol-dose-dependent production of N-acetyltaurine [
      • Shi X.
      • Yao D.
      • Chen C.
      Identification of N-acetyltaurine as a novel metabolite of ethanol through metabolomics-guided biochemical analysis.
      ], a potential biomarker of ethanol hepatotoxicity. This reaction was found only in wild type animals with hepatic CYP2E1.

      Viral hepatitis B and C

      Evaluation of liver disease in patients with hepatitis B or C is essential to identify patients who require antiviral therapy and to determine prognosis. Staging of liver fibrosis and the occurrence of cirrhosis associated with HBV or HCV infection are traditionally done by biopsy, but now there has been a move towards the use of non-invasive biomarkers [
      • Castera L.
      Noninvasive methods to assess liver disease in patients with hepatitis B or C.
      ]. None of the serum biomarkers that were originally developed for hepatitis C involve small molecules. Metabolomic studies in hepatitis B and C patients are very timely. The first study of its kind to evaluate deteriorating liver function in chronic hepatitis B using metabolomics was conducted in China, where HBV infection occurs in 80–90% of HCC cases [
      • Yin P.
      • Wan D.
      • Zhao C.
      • Chen J.
      • Zhao X.
      • Wang W.
      • et al.
      A metabonomic study of hepatitis B-induced liver cirrhosis and hepatocellular carcinoma by using RP-LC and HILIC coupled with mass spectrometry.
      ]. Using LCMS, they established a decline in serum LPC(16:0), LPC(18:0), LPC(18:1), and LPC(18:2), together with an elevation of GCDCA (or its isomer GDCA) [
      • Yang J.
      • Zhao X.
      • Liu X.
      • Wang C.
      • Gao P.
      • Wang J.
      • et al.
      High performance liquid chromatography–mass spectrometry for metabonomics: potential biomarkers for acute deterioration of liver function in chronic hepatitis B.
      ]. Another Chinese study reported similar results when examining the progression of chronic hepatitis B to cirrhosis [
      • Zhang L.
      • Jia X.
      • Peng X.
      • Ou Q.
      • Zhang Z.
      • Qiu C.
      • et al.
      Development and validation of a liquid chromatography–mass spectrometry metabonomic platform in human plasma of liver failure caused by hepatitis B virus.
      ]. This, of course, is the same fingerprint as seen in NALFD/NASH, cirrhosis and HCC (Fig. 2, Fig. 3). It was also reported that serum GCA, GCDCA, and TCA were elevated in hepatitis B-induced cirrhosis [
      • Yin P.
      • Wan D.
      • Zhao C.
      • Chen J.
      • Zhao X.
      • Wang W.
      • et al.
      A metabonomic study of hepatitis B-induced liver cirrhosis and hepatocellular carcinoma by using RP-LC and HILIC coupled with mass spectrometry.
      ]. There do not appear to be metabolomic studies comparing HBV-positive and HBV-negative subjects. It should also be pointed out that HBV may cause HCC in the absence of cirrhosis. Currently, there are no biomarkers for predicting HCC development in HBV-positive patients without cirrhosis and this should be a priority for metabolomic research.
      HCV infection accounts for 70% of chronic hepatitis and 30% of liver transplants in developed countries [
      • Sherman M.
      • Shafran S.
      • Burak K.
      • Doucette K.
      • Wong W.
      • Girgrah N.
      • et al.
      Management of chronic hepatitis C: consensus guidelines.
      ,
      • Sherman M.
      • Shafran S.
      • Burak K.
      • Doucette K.
      • Wong W.
      • Girgrah N.
      • et al.
      Management of chronic hepatitis B: consensus guidelines.
      ,
      • Davis G.L.
      • Albright J.E.
      • Cook S.F.
      • Rosenberg D.M.
      Projecting future complications of chronic hepatitis C in the United States.
      ]. Regarding HCV, atomic emission spectroscopy on scalp hair has been performed in 73 HCV-positive and 82 HCV-negative subjects, the hair concentrations of Ca, Cu, Fe, Mg, Mn, and Zn determined and data analyzed by multivariate data analysis [
      • Lloyd G.R.
      • Ahmad S.
      • Wasim M.
      • Brereton R.G.
      Pattern recognition of inductively coupled plasma atomic emission spectroscopy of human scalp hair for discriminating between healthy and hepatitis C patients.
      ]. This metallomics [
      • Sperling M.
      • Karst U.
      Metallomics: an emerging interdisciplinary science.
      ] study showed that Mg, Ca, and Zn were most closely associated with HCV infection. No biological discussion of the findings was made. There has been a claim that NMR metabolomics on urine can distinguish HCV-infected from uninfected persons [
      • Godoy M.M.
      • Lopes E.P.
      • Silva R.O.
      • Hallwass F.
      • Koury L.C.
      • Moura I.M.
      • et al.
      Hepatitis C virus infection diagnosis using metabonomics.
      ], although little data were provided. A metabolomic comparison of HCV-infected and mock-infected hepatocytes revealed small but significant increases in alanine, tyrosine, and adenosine with HCV infection [
      • Roe B.
      • Kensicki E.
      • Mohney R.
      • Hall W.W.
      Metabolomic profile of hepatitis C virus-infected hepatocytes.
      ]. Interestingly, similar elevations have been recorded for NAFLD/NASH (tyrosine) and HCC (adenosine) (Fig. 3). Preliminary findings in HCV-infected tree shrews (Tupaia belangeri chinensis) suggested that HCV affects many pathways in the liver, with alterations in LPCs and bile acids (as for other liver diseases, 3), carnitine esters, fatty acids, and LPEs [
      • Sun H.
      • Zhang A.
      • Yan G.
      • Piao C.
      • Li W.
      • Sun C.
      • et al.
      Metabolomic analysis of key regulatory metabolites in HCV-infected tree shrews.
      ]. It is clear, therefore, that both HBV and HCV infections, together with NASH, trigger similar molecular events represented by the mechanisms shown in Fig. 2. Moreover, both alcohol- and HBV-induced cirrhosis displayed higher bile acids and lower LPCs than healthy controls in an almost identical manner [
      • Lian J.S.
      • Liu W.
      • Hao S.R.
      • Guo Y.Z.
      • Huang H.J.
      • Chen D.Y.
      • et al.
      A serum metabonomic study on the difference between alcohol- and HBV-induced liver cirrhosis by ultraperformance liquid chromatography coupled to mass spectrometry plus quadrupole time-of-flight mass spectrometry.
      ]. It would appear that depressed LPCs and elevated bile acids in serum represent a phenotype of hepatitis and cirrhosis independent of etiological origin, and that this phenotype is carried forward into any resultant HCC.

      Cholangiocarcinoma

      Cholangiocarcinoma (CCA) is an aggressive cancer originating from the biliary tract. It would appear that obesity, diabetes, hepatitis B and C, alcohol use, and cirrhosis are all major risk factors for CCA, suggesting a common pathogenesis with HCC [
      • Palmer W.C.
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      Are common factors involved in the pathogenesis of primary liver cancers? A meta-analysis of risk factors for intrahepatic cholangiocarcinoma.
      ]. It has also been proposed that genetically impaired biliary excretion of phospholipids underlies CCA [
      • Khan S.A.
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      Cholangiocarcinoma.
      ,
      • Komichi D.
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      Glycochenodeoxycholate plays a carcinogenic role in immortalized mouse cholangiocytes via oxidative DNA damage.
      ]. Metabolomic investigations support this view, with lower phosphatidylcholine and elevated glycine- and taurine-conjugated bile acids reported in the bile of CCA patients [
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      Metabolic profiling of bile in cholangiocarcinoma using in vitro magnetic resonance spectroscopy.
      ,
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      Differences in phosphatidylcholine and bile acids in bile from Egyptian and UK patients with and without cholangiocarcinoma.
      ].

      Cholestasis and cholecystitis

      Interruption of bile flow may have an extrahepatic and obstructive or an intrahepatic and biochemical basis. An NMR metabolomic study has been performed in rats in an attempt to use urinary biomarkers to distinguish the two mechanisms [
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      ]. Metabolomics revealed that cholestasis induced in Fxr-null mice by a cholic acid diet resulted in increased urinary excretion of bile salt tetrols, predominantly 3α,6,7α,12α-tetrahydroxy-5β-cholestan-26-oyltaurine, due to an adaptive upregulation of the steroid-hydroxylating cytochrome P450 CYP3A11 in these mice [
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      ]. An adaptive response was also characterized in a rat cholestasis model, with a shift from cytotoxic to cytoprotective bile acids in plasma and urine [
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      • Sogame Y.
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      Metabolomic investigation of cholestasis in a rat model using ultra-performance liquid chromatography/tandem mass spectrometry.
      ].
      Injection of Escherichia coli into the rabbit gallbladder produces a model for acalculous cholecystitis (AAC). Compared to saline-injected controls, AAC animals displayed increased serum LDL and VLDL, with decreased serum phospholipids, lactate, 3-hydroxybutyrate, citrate, lysine, asparagine, histidine, and glucose as demonstrated by NMR metabolomics [
      • Li Z.
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      • Zhang Y.
      • Lu M.
      • Qiao X.
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      Metabolomic study of serum from rabbits with acute acalculous cholecystitis.
      ]. These observations need to be refined with the use of LCMS-based metabolomics.

      Liver transplantation

      As shown in Fig. 1, several end-stage liver diseases require transplantation. A metabolomic study of a single patient with hepatitis B and HCC, who underwent two consecutive liver transplants, showed that the first failed graft was associated with elevated blood lactate, uric acid, citrate, glutamine and methionine, diagnostic of dysfunctional hepatic metabolic fluxes [
      • Serkova N.J.
      • Zhang Y.
      • Coatney J.L.
      • Hunter L.
      • Wachs M.E.
      • Niemann C.U.
      • et al.
      Early detection of graft failure using the blood metabolic profile of a liver recipient.
      ]. A series of 15 HCC patients displayed increased valine, alanine, acetone, succinate, glutamine, choline, lactate, and glucose one day after transplantation. After 7 days, lipids and choline increased while glucose and amino acids decreased [
      • Qi S.W.
      • Tu Z.G.
      • Peng W.J.
      • Wang L.X.
      • Ou-Yang X.
      • Cai A.J.
      • et al.
      (1)H NMR-based serum metabolic profiling in compensated and decompensated cirrhosis.
      ]. The metabolomic window appears to offer new insights into specific hepatic metabolic changes in the transplantation perioperative period.

      Miscellaneous other hepatobiliary diseases

      Metabolomic studies have been reported that are of relevance to Wilson’s disease [
      • Wilmarth P.A.
      • Short K.K.
      • Fiehn O.
      • Lutsenko S.
      • David L.L.
      • Burkhead J.L.
      A systems approach implicates nuclear receptor targeting in the Atp7b(−/−) mouse model of Wilson’s disease.
      ,
      • Santos E.M.
      • Ball J.S.
      • Williams T.D.
      • Wu H.
      • Ortega F.
      • van Aerle R.
      • et al.
      Identifying health impacts of exposure to copper using transcriptomics and metabolomics in a fish model.
      ], primary biliary cirrhosis [
      • Trottier J.
      • Bialek A.
      • Caron P.
      • Straka R.J.
      • Heathcote J.
      • Milkiewicz P.
      • et al.
      Metabolomic profiling of 17 bile acids in serum from patients with primary biliary cirrhosis and primary sclerosing cholangitis: a pilot study.
      ], primary sclerosing cholangitis [
      • Trottier J.
      • Bialek A.
      • Caron P.
      • Straka R.J.
      • Heathcote J.
      • Milkiewicz P.
      • et al.
      Metabolomic profiling of 17 bile acids in serum from patients with primary biliary cirrhosis and primary sclerosing cholangitis: a pilot study.
      ], the hepatic stage of malaria [
      • Sengupta A.
      • Basant A.
      • Ghosh S.
      • Sharma S.
      • Sonawat H.M.
      Liver metabolic alterations and changes in host intercompartmental metabolic correlation during progression of malaria.
      ,
      • Sengupta A.
      • Ghosh S.
      • Basant A.
      • Malusare S.
      • Johri P.
      • Pathak S.
      • et al.
      Global host metabolic response to Plasmodium vivax infection: a 1H NMR based urinary metabonomic study.
      ,
      • Ghosh S.
      • Sengupta A.
      • Sharma S.
      • Sonawat H.M.
      Metabolic fingerprints of serum, brain, and liver are distinct for mice with cerebral and noncerebral malaria: a (1)H NMR spectroscopy-based metabonomic study.
      ], as well as various aspects of hepatic encephalopathy [
      • Barba I.
      • Chatauret N.
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      • Cordoba J.
      A 1H nuclear magnetic resonance-based metabonomic approach for grading hepatic encephalopathy and monitoring the effects of therapeutic hypothermia in rats.
      ,
      • Williams H.R.
      • Cox I.J.
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      Metabonomics in hepatic encephalopathy: lucidity emerging from confusion.
      ,
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      ,
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      • et al.
      A longitudinal systems biology analysis of lactulose withdrawal in hepatic encephalopathy.
      ].

      The metabolomic window into acute liver toxicity in animal models

      High-throughput metabolomic screening of hepatotoxins in laboratory animals first used NMR and pattern recognition algorithms [
      • Robertson D.G.
      • Reily M.D.
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      • Paterson D.A.
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      ,
      • Nicholson J.K.
      • Connelly J.
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      Metabonomics: a platform for studying drug toxicity and gene function.
      ,
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      Spectral editing and pattern recognition methods applied to high-resolution magic-angle spinning 1H nuclear magnetic resonance spectroscopy of liver tissues.
      ] but, in early studies, also employed Fourier-transform infrared spectroscopy [
      • Harrigan G.G.
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      Application of high-throughput Fourier-transform infrared spectroscopy in toxicology studies: contribution to a study on the development of an animal model for idiosyncratic toxicity.
      ]. Metabolomic profiles of numerous hepatotoxins in laboratory animals have been described, and include hydrazine [
      • Bollard M.E.
      • Keun H.C.
      • Beckonert O.
      • Ebbels T.M.
      • Antti H.
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      Comparative metabonomics of differential hydrazine toxicity in the rat and mouse.
      ], bromobenzene [
      • Heijne W.H.
      • Lamers R.J.
      • van Bladeren P.J.
      • Groten J.P.
      • van Nesselrooij J.H.
      • van Ommen B.
      Profiles of metabolites and gene expression in rats with chemically induced hepatic necrosis.
      ,
      • Waters N.J.
      • Waterfield C.J.
      • Farrant R.D.
      • Holmes E.
      • Nicholson J.K.
      Integrated metabonomic analysis of bromobenzene-induced hepatotoxicity: novel induction of 5-oxoprolinosis.
      ], methapyrilene [
      • Craig A.
      • Sidaway J.
      • Holmes E.
      • Orton T.
      • Jackson D.
      • Rowlinson R.
      • et al.
      Systems toxicology: integrated genomic, proteomic and metabonomic analysis of methapyrilene induced hepatotoxicity in the rat.
      ], methylenedianiline [
      • Ishihara K.
      • Katsutani N.
      • Aoki T.
      A metabonomics study of the hepatotoxicants galactosamine, methylene dianiline and clofibrate in rats.
      ], D-galactosamine [
      • Ishihara K.
      • Katsutani N.
      • Aoki T.
      A metabonomics study of the hepatotoxicants galactosamine, methylene dianiline and clofibrate in rats.
      ,
      • Feng B.
      • Wu S.M.
      • Lv S.
      • Liu F.
      • Chen H.S.
      • Gao Y.
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      A novel scoring system for prognostic prediction in d-galactosamine/lipopolysaccharide-induced fulminant hepatic failure BALB/c mice.
      ,
      • Coen M.
      • Goldfain-Blanc F.
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      • Walther B.
      • Robertson D.G.
      • Holmes E.
      • et al.
      Pharmacometabonomic investigation of dynamic metabolic phenotypes associated with variability in response to galactosamine hepatotoxicity.
      ], clofibrate [
      • Ishihara K.
      • Katsutani N.
      • Aoki T.
      A metabonomics study of the hepatotoxicants galactosamine, methylene dianiline and clofibrate in rats.
      ], allyl formate [
      • Yap I.K.
      • Clayton T.A.
      • Tang H.
      • Everett J.R.
      • Hanton G.
      • Provost J.P.
      • et al.
      An integrated metabonomic approach to describe temporal metabolic disregulation induced in the rat by the model hepatotoxin allyl formate.
      ], the anti-HBV compound Bay41-4109 [
      • Shi C.
      • Wu C.Q.
      • Cao A.M.
      • Sheng H.Z.
      • Yan X.Z.
      • Liao M.Y.
      NMR-spectroscopy-based metabonomic approach to the analysis of Bay41-4109, a novel anti-HBV compound, induced hepatotoxicity in rats.
      ], paracetamol [
      • Sun J.
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      • Holland R.D.
      • Schmitt T.C.
      • Cantor G.H.
      • Dragan Y.P.
      • et al.
      Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS.
      ,
      • Chen C.
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      • Shah Y.M.
      • Idle J.R.
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      Serum metabolomics reveals irreversible inhibition of fatty acid beta-oxidation through the suppression of PPARalpha activation as a contributing mechanism of acetaminophen-induced hepatotoxicity.
      ,
      • Cheng J.
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      • Idle J.R.
      • Gonzalez F.J.
      Rifampicin-activated human pregnane X receptor and CYP3A4 induction enhance acetaminophen-induced toxicity.
      ,
      • Winnike J.H.
      • Li Z.
      • Wright F.A.
      • Macdonald J.M.
      • O’Connell T.M.
      • Watkins P.B.
      Use of pharmaco-metabonomics for early prediction of acetaminophen-induced hepatotoxicity in humans.
      ,
      • Fukuhara K.
      • Ohno A.
      • Ando Y.
      • Yamoto T.
      • Okuda H.
      A 1H NMR-based metabolomics approach for mechanistic insight into acetaminophen-induced hepatotoxicity.
      ,
      • Kumar B.S.
      • Chung B.C.
      • Kwon O.S.
      • Jung B.H.
      Discovery of common urinary biomarkers for hepatotoxicity induced by carbon tetrachloride, acetaminophen and methotrexate by mass spectrometry-based metabolomics.
      ,
      • Prot J.M.
      • Bunescu A.
      • Elena-Herrmann B.
      • Aninat C.
      • Snouber L.C.
      • Griscom L.
      • et al.
      Predictive toxicology using systemic biology and liver microfluidic “on chip” approaches: application to acetaminophen injury.
      ,
      • Shintu L.
      • Baudoin R.
      • Navratil V.
      • Prot J.M.
      • Pontoizeau C.
      • Defernez M.
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      Metabolomics-on-a-chip and predictive systems toxicology in microfluidic bioartificial organs.
      ], isoniazid [
      • Liao Y.
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      • Yan X.Z.
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      Metabonomics profile of urine from rats administrated with different treatment period of isoniazid.
      ,
      • Li F.
      • Lu J.
      • Cheng J.
      • Wang L.
      • Matsubara T.
      • Csanaky I.L.
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      Human PXR modulates hepatotoxicity associated with rifampicin and isoniazid co-therapy.
      ], carbon tetrachloride [
      • Kumar B.S.
      • Chung B.C.
      • Kwon O.S.
      • Jung B.H.
      Discovery of common urinary biomarkers for hepatotoxicity induced by carbon tetrachloride, acetaminophen and methotrexate by mass spectrometry-based metabolomics.
      ,
      • Huang X.
      • Shao L.
      • Gong Y.
      • Mao Y.
      • Liu C.
      • Qu H.
      • et al.
      A metabonomic characterization of CCl4-induced acute liver failure using partial least square regression based on the GC/MS metabolic profiles of plasma in mice.
      ,
      • Yang L.
      • Xiong A.
      • He Y.
      • Wang Z.
      • Wang C.
      • Li W.
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      Bile acids metabonomic study on the CCl4- and alpha-naphthylisothiocyanate-induced animal models: quantitative analysis of 22 bile acids by ultraperformance liquid chromatography–mass spectrometry.
      ,
      • Lin Y.
      • Si D.
      • Zhang Z.
      • Liu C.
      An integrated metabonomic method for profiling of metabolic changes in carbon tetrachloride induced rat urine.
      ], α-naphthylisothiocyanate [
      • Yang L.
      • Xiong A.
      • He Y.
      • Wang Z.
      • Wang C.
      • Li W.
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      Bile acids metabonomic study on the CCl4- and alpha-naphthylisothiocyanate-induced animal models: quantitative analysis of 22 bile acids by ultraperformance liquid chromatography–mass spectrometry.
      ], perfluorododecanoic acid [
      • Ding L.
      • Hao F.
      • Shi Z.
      • Wang Y.
      • Zhang H.
      • Tang H.
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      Systems biological responses to chronic perfluorododecanoic acid exposure by integrated metabonomic and transcriptomic studies.
      ], valproate [
      • Lee M.S.
      • Jung B.H.
      • Chung B.C.
      • Cho S.H.
      • Kim K.Y.
      • Kwon O.S.
      • et al.
      Metabolomics study with gas chromatography–mass spectrometry for predicting valproic acid-induced hepatotoxicity and discovery of novel biomarkers in rat urine.
      ], Huang-yao-zi [
      • Liu Y.
      • Huang R.
      • Liu L.
      • Peng J.
      • Xiao B.
      • Yang J.
      • et al.
      Metabonomics study of urine from Sprague–Dawley rats exposed to Huang-yao-zi using (1)H NMR spectroscopy.
      ], dimethylnitrosamine [
      • Sun C.
      • Teng Y.
      • Li G.
      • Yoshioka S.
      • Yokota J.
      • Miyamura M.
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      Metabonomics study of the protective effects of Lonicera japonica extract on acute liver injury in dimethylnitrosamine treated rats.
      ], polychlorinated biphenyls [
      • Lu C.
      • Wang Y.
      • Sheng Z.
      • Liu G.
      • Fu Z.
      • Zhao J.
      • et al.
      NMR-based metabonomic analysis of the hepatotoxicity induced by combined exposure to PCBs and TCDD in rats.
      ,
      • Shi X.
      • Wahlang B.
      • Wei X.
      • Yin X.
      • Falkner K.C.
      • Prough R.A.
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      Metabolomic analysis of the effects of polychlorinated biphenyls in non-alcoholic fatty liver disease.
      ], 2,3,7,8-tetrachlorodibenzo-p-dioxin [
      • Lu C.
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      • Fu Z.
      • Zhao J.
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      NMR-based metabonomic analysis of the hepatotoxicity induced by combined exposure to PCBs and TCDD in rats.
      ], methamphetamine [
      • Shima N.
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      • Bando K.
      • Horie H.
      • Zaitsu K.
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      Influences of methamphetamine-induced acute intoxication on urinary and plasma metabolic profiles in the rat.
      ], (+)-usnic acid [
      • Lu X.
      • Zhao Q.
      • Tian Y.
      • Xiao S.
      • Jin T.
      • Fan X.
      A metabonomic characterization of (+)-usnic acid-induced liver injury by gas chromatography–mass spectrometry-based metabolic profiling of the plasma and liver in rat.
      ], pentamethychromanol [
      • Parman T.
      • Bunin D.I.
      • Ng H.H.
      • McDunn J.E.
      • Wulff J.E.
      • Wang A.
      • et al.
      Toxicogenomics and metabolomics of pentamethylchromanol (PMCol)-induced hepatotoxicity.
      ] and methotrexate [
      • Kumar B.S.
      • Chung B.C.
      • Kwon O.S.
      • Jung B.H.
      Discovery of common urinary biomarkers for hepatotoxicity induced by carbon tetrachloride, acetaminophen and methotrexate by mass spectrometry-based metabolomics.
      ]. Detailed analysis of these drug-induced liver injury (DILI) studies falls beyond the scope of this review. However, the reader is directed to The Liver Toxicity Biomarker Study on DILI and closely related topics that have been reviewed [
      • McBurney R.N.
      • Hines W.M.
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      • Moland C.L.
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      The liver toxicity biomarker study: phase I design and preliminary results.
      ,
      • Watkins P.B.
      Biomarkers for the diagnosis and management of drug-induced liver injury.
      ,
      • Beger R.D.
      • Sun J.
      • Schnackenberg L.K.
      Metabolomics approaches for discovering biomarkers of drug-induced hepatotoxicity and nephrotoxicity.
      ,
      • O’Connell T.M.
      • Watkins P.B.
      The application of metabonomics to predict drug-induced liver injury.
      ,
      • Ellinger-Ziegelbauer H.
      • Adler M.
      • Amberg A.
      • Brandenburg A.
      • Callanan J.J.
      • Connor S.
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      The enhanced value of combining conventional and “omics” analyses in early assessment of drug-induced hepatobiliary injury.
      ,
      • Gonzalez E.
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      • Conde-Vancells J.
      • Gutierrez-de Juan V.
      • Perez-Cormenzana M.
      • Mayo R.
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      Serum UPLC–MS/MS metabolic profiling in an experimental model for acute-liver injury reveals potential biomarkers for hepatotoxicity.
      ].

      A proposed metabolomics-based model for major liver disease

      Based upon a review of the available literature, we propose a three-stage progression from hepatic insult of the healthy liver to carcinoma (Fig. 4). A core metabolomic phenotype (CMP) arises early in this progression and comprises readily discernible changes in bile acids and phospholipids (Table 1, Table 2, Table 3, Table 4, Fig. 3). The CMP is maintained whether or not cirrhosis arises and/or HCC or CCA develops (Stages 2 and 3, respectively). This CMP is common to all etiologies in Stage 1, including NAFLD/NASH, ALD, and viral hepatitis. Other metabolomic perturbations distinguish the different stages (Fig. 3). We also propose that the metabolic remodelling described for HCC [
      • Beyoglu D.
      • Imbeaud S.
      • Maurhofer O.
      • Bioulac-Sage P.
      • Zucman-Rossi J.
      • Dufour J.F.
      • et al.
      Tissue metabolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptomic classification.
      ] begins at the transition from Phase 0 to Phase 1 as a consequence of the presence of inflammatory signalling in the liver, as outlined in Fig. 2. Thus, this body of accumulated metabolomic data may begin to cast further light on hepatobiliary diseases.
      Figure thumbnail gr4
      Fig. 4Diverse hepatic insults leading to a core metabolomic phenotype en route from the healthy liver to HCC and CCA. Elevated serum bile acids and urinary bile salts together with decreased serum lysophosphatidylcholines represent the core metabolomic phenotype (CMP). A metabolic remodelling begins in the transition from the healthy liver (Phase 0) to NAFLD/NASH, ALD or viral hepatitis (Phase 1). During Stage 1 there occurs a Warburg shift from mitochondrial respiration to cytosolic glycolysis, together with an increase in fatty acid β-oxidation. This metabolic remodelling persists through cirrhosis (Phase 2) and into carcinoma (Phase 3). Note that the sum of the carcinoma energy production D + E + F is greater than the summed energy production A + B + C in the healthy liver. CCA, cholangiocarcinoma; CMP, core metabolomic phenotype; ALD, alcoholic liver disease. For other abbreviations, see .

      Financial support

      The authors wish to acknowledge the financial support of the National Institutes of Health/National Institute of Allergy and Infectious Diseases (grant U19 AI067773-07/08), the Hassan Badawi Foundation Against Liver Cancer and Imperial Tobacco Limited, UK.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Acknowledgement

      We would like to thank our colleague Professor Jean-François Dufour for encouraging us to write this review.

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