High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy of Paired Clinical Liver Tissue Samples from Hepatocellular Cancer and Surrounding Region.

The global burden of liver cancer is increasing. Timely diagnosis is important for optimising the limited available treatment options. Understanding the metabolic consequences of hepatocellular carcinoma (HCC) may lead to more effective treatment options. We aimed to document metabolite differences between HCC and matched surrounding tissues of varying aetiology, obtained at the time of liver resection, and to interpret metabolite changes with clinical findings. High-resolution magic angle spinning nuclear magnetic resonance (HRMAS-NMR) spectroscopy analyses of N = 10 paired HCC and surrounding non-tumour liver tissue samples were undertaken. There were marked HRMAS-NMR differences in lipid levels in HCC tissue compared to matched surrounding tissue and more subtle changes in low-molecular-weight metabolites, particularly when adjusting for patient-specific variability. Differences in lipid-CH3, lipid-CH2, formate, and acetate levels were of particular interest. The obvious differences in lipid content highlight the intricate interplay between metabolic adaptations and cancer cell survival in the complex microenvironment of liver cancer. Differences in formate and acetate might relate to bacterial metabolites. Therefore, documentation of metabolites in HCC tissue according to histology findings in patients is of interest for personalised medicine approaches and for tailoring targeted treatment strategies.

Clinical, experimental and pathophysiological effects of Yaq-001: a non-absorbable, gut-restricted adsorbent in models and patients with cirrhosis.

OBJECTIVE: Targeting bacterial translocation in cirrhosis is limited to antibiotics with risk of antimicrobial resistance. This study explored the therapeutic potential of a non-absorbable, gut-restricted, engineered carbon bead adsorbent, Yaq-001 in models of cirrhosis and acute-on-chronic liver failure (ACLF) and, its safety and tolerability in a clinical trial in cirrhosis. DESIGN: Performance of Yaq-001 was evaluated in vitro. Two-rat models of cirrhosis and ACLF, (4 weeks, bile duct ligation with or without lipopolysaccharide), receiving Yaq-001 for 2 weeks; and two-mouse models of cirrhosis (6-week and 12-week carbon tetrachloride (CCl4)) receiving Yaq-001 for 6 weeks were studied. Organ and immune function, gut permeability, transcriptomics, microbiome composition and metabolomics were analysed. The effect of faecal water on gut permeability from animal models was evaluated on intestinal organoids. A multicentre, double-blind, randomised, placebo-controlled clinical trial in 28 patients with cirrhosis, administered 4 gr/day Yaq-001 for 3 months was performed. RESULTS: Yaq-001 exhibited rapid adsorption kinetics for endotoxin. In vivo, Yaq-001 reduced liver injury, progression of fibrosis, portal hypertension, renal dysfunction and mortality of ACLF animals significantly. Significant impact on severity of endotoxaemia, hyperammonaemia, liver cell death, systemic inflammation and organ transcriptomics with variable modulation of inflammation, cell death and senescence in the liver, kidneys, brain and colon was observed. Yaq-001 reduced gut permeability in the organoids and impacted positively on the microbiome composition and metabolism. Yaq-001 regulated as a device met its primary endpoint of safety and tolerability in the clinical trial. CONCLUSIONS: This study provides strong preclinical rationale and safety in patients with cirrhosis to allow clinical translation. TRIAL REGISTRATION NUMBER: NCT03202498.

Rat liver extracellular matrix and perfusion bioreactor culture promote human amnion epithelial cell differentiation towards hepatocyte-like cells.

Congenital and chronic liver diseases have a substantial health burden worldwide. The most effective treatment available for these patients is whole organ transplantation; however, due to the severely limited supply of donor livers and the side effects associated with the immunosuppressive regimen required to accept allograft, the mortality rate in patients with end-stage liver disease is annually rising. Stem cell-based therapy aims to provide alternative treatments by either cell transplantation or bioengineered construct transplantation. Human amnion epithelial cells (AEC) are a widely available, ethically neutral source of cells with the plasticity and potential of multipotent stem cells and immunomodulatory properties of perinatal cells. AEC have been proven to be able to achieve functional improvement towards hepatocyte-like cells, capable of rescuing animals with metabolic disorders; however, they showed limited metabolic activities in vitro. Decellularised extracellular matrix (ECM) scaffolds have gained recognition as adjunct biological support. Decellularised scaffolds maintain native ECM components and the 3D architecture instrumental of the organ, necessary to support cells' maturation and function. We combined ECM-scaffold technology with primary human AEC, which we demonstrated being equipped with essential ECM-adhesion proteins, and evaluated the effects on AEC differentiation into functional hepatocyte-like cells (HLC). This novel approach included the use of a custom 4D bioreactor to provide constant oxygenation and media perfusion to cells in 3D cultures over time. We successfully generated HLC positive for hepatic markers such as ALB, CYP3A4 and CK18. AEC-derived HLC displayed early signs of hepatocyte phenotype, secreted albumin and urea, and expressed Phase-1 and -2 enzymes. The combination of liver-specific ECM and bioreactor provides a system able to aid differentiation into HLC, indicating that the innovative perfusion ECM-scaffold technology may support the functional improvement of multipotent and pluripotent stem cells, with important repercussions in the bioengineering of constructs for transplantation.

The Role of Mass Spectrometry in Hepatocellular Carcinoma Biomarker Discovery.

Hepatocellular carcinoma (HCC) is the main liver malignancy and has a high mortality rate. The discovery of novel biomarkers for early diagnosis, prognosis, and stratification purposes has the potential to alleviate its disease burden. Mass spectrometry (MS) is one of the principal technologies used in metabolomics, with different experimental methods and machine types for different phases of the biomarker discovery process. Here, we review why MS applications are useful for liver cancer, explain the MS technique, and briefly summarise recent findings from metabolomic MS studies on HCC. We also discuss the current challenges and the direction for future research.

Partial MCT1 invalidation protects against diet-induced non-alcoholic fatty liver disease and the associated brain dysfunction.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has been associated with mild cerebral dysfunction and cognitive decline, although the exact pathophysiological mechanism remains ambiguous. Using a diet-induced model of NAFLD and monocarboxylate transporter-1 (Mct1+/-) haploinsufficient mice, which resist high-fat diet-induced hepatic steatosis, we investigated the hypothesis that NAFLD leads to an encephalopathy by altering cognition, behaviour, and cerebral physiology. We also proposed that global MCT1 downregulation offers cerebral protection. METHODS: Behavioural tests were performed in mice following 16 weeks of control diet (normal chow) or high-fat diet with high fructose/glucose in water. Tissue oxygenation, cerebrovascular reactivity, and cerebral blood volume were monitored under anaesthesia by multispectral optoacoustic tomography and optical fluorescence. Cortical mitochondrial oxygen consumption and respiratory capacities were measured using ex vivo high-resolution respirometry. Microglial and astrocytic changes were evaluated by immunofluorescence and 3D reconstructions. Body composition was assessed using EchoMRI, and liver steatosis was confirmed by histology. RESULTS: NAFLD concomitant with obesity is associated with anxiety- and depression-related behaviour. Low-grade brain tissue hypoxia was observed, likely attributed to the low-grade brain inflammation and decreased cerebral blood volume. It is also accompanied by microglial and astrocytic morphological and metabolic alterations (higher oxygen consumption), suggesting the early stages of an obesogenic diet-induced encephalopathy. Mct1 haploinsufficient mice, despite fat accumulation in adipose tissue, were protected from NAFLD and associated cerebral alterations. CONCLUSIONS: This study provides evidence of compromised brain health in obesity and NAFLD, emphasising the importance of the liver-brain axis. The protective effect of Mct1 haploinsufficiency points to this protein as a novel therapeutic target for preventing and/or treating NAFLD and the associated brain dysfunction. IMPACT AND IMPLICATIONS: This study is focused on unravelling the pathophysiological mechanism by which cerebral dysfunction and cognitive decline occurs during NAFLD and exploring the potential of monocarboxylate transporter-1 (MCT1) as a novel preventive or therapeutic target. Our findings point to NAFLD as a serious health risk and its adverse impact on the brain as a potential global health system and economic burden. These results highlight the utility of Mct1 transgenic mice as a model for NAFLD and associated brain dysfunction and call for systematic screening by physicians for early signs of psychological symptoms, and an awareness by individuals at risk of these potential neurological effects. This study is expected to bring attention to the need for early diagnosis and treatment of NAFLD, while having a direct impact on policies worldwide regarding the health risk associated with NAFLD, and its prevention and treatment.

Chronically elevated branched chain amino acid levels are pro-arrhythmic.

AIMS: Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death (SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach. METHODS AND RESULTS: We employed a phenotype-driven N-ethyl-N-nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6-9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4-5 weeks, Bcat2p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs-leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation. CONCLUSIONS: Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure.

Mapping of population disparities in the cholangiocarcinoma urinary metabolome.

Phenotypic diversity in urinary metabolomes of different geographical populations has been recognized recently. In this study, urinary metabolic signatures from Western (United Kingdom) and South-East Asian (Thai) cholangiocarcinoma patients were characterized to understand spectral variability due to host carcinogenic processes and/or exogenous differences (nutritional, environmental and pharmaceutical). Urinary liquid chromatography mass spectroscopy (LC-MS) spectral profiles from Thai (healthy = 20 and cholangiocarcinoma = 14) and UK cohorts (healthy = 22 and cholangiocarcinoma = 10) were obtained and modelled using chemometric data analysis. Healthy metabolome disparities between the two distinct populations were primarily related to differences in dietary practices and body composition. Metabolites excreted due to drug treatment were dominant in urine specimens from cholangiocarcinoma patients, particularly in Western individuals. Urine from participants with sporadic (UK) cholangiocarcinoma contained greater levels of a nucleotide metabolite (uridine/pseudouridine). Higher relative concentrations of 7-methylguanine were observed in urine specimens from Thai cholangiocarcinoma patients. The urinary excretion of hippurate and methyladenine (gut microbial-host co-metabolites) showed a similar pattern of lower levels in patients with malignant biliary tumours from both countries. Intrinsic (body weight and body composition) and extrinsic (xenobiotic metabolism) factors were the main causes of disparities between the two populations. Regardless of the underlying aetiology, biological perturbations associated with cholangiocarcinoma urine metabolome signatures appeared to be influenced by gut microbial community metabolism. Dysregulation in nucleotide metabolism was associated with sporadic cholangiocarcinoma, possibly indicating differences in mitochondrial energy production pathways between cholangiocarcinoma tumour subtypes. Mapping population-specific metabolic disparities may aid in interpretation of disease processes and identification of candidate biomarkers.

A Double-Blind, Randomized Placebo-Controlled Trial of Probiotic Lactobacillus casei Shirota in Stable Cirrhotic Patients.

Background: In cirrhosis, a pathological gut microbiome has been linked with immune dysfunction. A pilot study of probiotic Lactobacillus casei Shirota (LcS) in alcoholic cirrhosis demonstrated significant improvement in neutrophil function. This study aimed to evaluate the efficacy of LcS on neutrophil function and significant infection rates in patients with cirrhosis. Methods: 92 cirrhotic patients (Child-Pugh score ≤10) were randomized to receive LcS or placebo, three times daily for six months. Primary end-points were incidence of significant infection and neutrophil function. Secondary end-points were cytokine profile, endotoxin, bacterial DNA positivity, intestinal permeability and quality of life. Results: Rates of infection, decompensation or neutrophil function did not differ between placebo and probiotic groups. LcS significantly reduced plasma monocyte chemotactic protein-1 and, on subgroup analysis, plasma interleukin-1ß (alcoholic cirrhosis), interleukin-17a and macrophage inflammatory protein-1ß (non-alcoholic cirrhosis), compared with placebo. No significant differences in intestinal permeability, bacterial translocation or metabolomic profile were observed. Conclusion: LcS supplementation in patients with early cirrhosis is safe. Although no significant infections were observed in either group, LcS improved cytokine profile towards an anti-inflammatory phenotype, an effect which appears to be independent of bacterial translocation.

Characterisation of the Serum Metabolic Signature of Cholangiocarcinoma in a United Kingdom Cohort.

BACKGROUND: A distinct serum metabonomic pattern has been previously revealed to be associated with various forms of liver disease. Here, we aimed to apply mass spectrometry to obtain serum metabolomic profiles from individuals with cholangiocarcinoma and benign hepatobiliary diseases to gain an insight into pathogenesis and search for potential early-disease biomarkers. METHODS: Serum samples were profiled using a hydrophilic interaction liquid chromatography platform, coupled to a mass spectrometer. A total of 47 serum specimens from 8 cholangiocarcinoma cases, 20 healthy controls, 8 benign disease controls (bile duct strictures) and 11 patients with hepatocellular carcinoma (as malignant disease controls) were included. Data analysis was performed using univariate and multivariate statistics. RESULTS: The serum metabolome disparities between the metabolite profiles from healthy controls and patients with hepatobiliary disease were predominantly related to changes in lipid and lipid-derived compounds (phospholipids, bile acids and steroids) and amino acid metabolites (phenylalanine). A metabolic pattern indicative of inflammatory response due to cirrhosis and cholestasis was associated with the disease groups. The abundance of phospholipid metabolites was altered in individuals with liver disease, particularly cholangiocarcinoma, but no significant difference was seen between profiles from patients with benign biliary strictures and cholangiocarcinoma. CONCLUSION: The serum metabolome in cholangiocarcinoma exhibited changes in metabolites related to inflammation, altered energy production and phospholipid metabolism. This study serves to highlight future avenues for biomarker research in large-scale studies.

Metabolomics and microbial composition increase insight into the impact of dietary differences in cirrhosis.

BACKGROUND & AIMS: Dietary changes can modulate gut microbiota and interact with cirrhosis. Our prior study demonstrated that microbial diversity was higher in cirrhotics from Turkish vs the USA, which was associated with lower risk of 90-day hospitalizations. We aimed to define gut microbial functional and metabolomic changes to increase insight into benefits of the Mediterranean compared to Western diets. METHODS: In all, 139 Turkish (46 controls/50 compensated/43 decompensated) and 157 American subjects (48 controls/59 compensated/50 decompensated) were studied. Turkish subjects consumed a modified Mediterranean diet with daily fermented milk intake, whereas Americans consumed a Western diet. Predicted gut microbial functionalities and plasma metabolomics were compared between/within countries. Correlation network differences between microbiota and metabolites in cirrhotics from Turkey vs the USA were evaluated. RESULTS: Predicted microbial function showed lower amino acid, bioenergetics and lipid pathways, with functions related to vitamin B, glycan, xenobiotic metabolism, DNA/RNA synthesis, in cirrhotics from Turkey compared to the USA. Plasma metabolomics demonstrated higher relative lactate levels in Turkey vs the USA. The metabolite changes in decompensated cirrhosis, compared to controls, showed similar trends in Turkey and the USA, with reduced lipids and phosphocholines. Phosphocholines were significantly lower in patients hospitalized in 90 days (P = .03). Correlation networks in cirrhotics demonstrated linkage differences between beneficial taxa, Blautia and Oscillispira, and lactate and unsaturated lipids, in Turkey compared to American patients. CONCLUSIONS: A modified Mediterranean diet was associated with altered plasma metabolomics and beneficially alters microbiota functionality and correlations compared to Western diet in cirrhosis. These altered diet-microbial interactions could potentially affect the 90-day hospitalization risk.

Microbial functional change is linked with clinical outcomes after capsular fecal transplant in cirrhosis.

BACKGROUNDHepatic encephalopathy (HE) is associated with poor outcomes. A prior randomized, pilot trial demonstrated safety after oral capsular fecal microbial transplant (FMT) in HE, with favorable changes in microbial composition and cognition. However, microbial functional changes are unclear. The aim of this study was to determine the effect of FMT on the gut-brain axis compared with placebo, using microbial function based on bile acids (BAs), inflammation (serum IL-6, LPS-binding protein [LBP]), and their association with EncephalApp.METHODSTwenty cirrhotic patients were randomized 1:1 into groups that received 1-time FMT capsules from a donor enriched in Lachnospiraceae and Ruminococcaceae or placebo capsules, with 5-month follow-up for safety outcomes. Stool microbiota and BA; serum IL-6, BA, and LBP; and EncephalApp were analyzed at baseline and 4 weeks after FMT/placebo. Correlation networks among microbiota, BAs, EncephalApp, IL-6, and LBP were performed before/after FMT.RESULTSFMT-assigned participants had 1 HE recurrence and 2 unrelated infections. Six placebo-assigned participants developed negative outcomes. FMT, but not placebo, was associated with reduced serum IL-6 and LBP and improved EncephalApp. FMT-assigned participants demonstrated higher deconjugation and secondary BA formation in feces and serum compared with baseline. No change was seen in placebo. Correlation networks showed greater complexity after FMT compared with baseline. Beneficial taxa, such as Ruminococcaceae, Verrucomicrobiaceae, and Lachnospiraceae, were correlated with cognitive improvement and decrease in inflammation after FMT. Fecal/serum secondary/primary ratios and PiCRUST secondary BA pathways did not increase in participants who developed poor outcomes.CONCLUSIONGut microbial function in cirrhosis is beneficially affected by capsular FMT, with improved inflammation and cognition. Lower secondary BAs in FMT recipients could select for participants who develop negative outcomes.TRIAL REGISTRATIONClinicaltrials.gov NCT03152188.FUNDINGNational Center for Advancing Translational Sciences NIH grant R21TR002024, VA Merit Review grant 2I0CX001076, the United Kingdom National Institute for Health Research Biomedical Facility at Imperial College London, the British Heart Foundation, Wellcome Trust, and King's College London.

Cholangiocarcinoma: a guide for the nonspecialist.

Cholangiocarcinoma (CCA) is a tumor with increasing prevalence around the world. The prevalence of CCA is highest in East Asia and most significantly in the countries through which the Mekong River flows, owing to the presence of liver flukes, which are consumed in raw fish dishes. Outside Asia, the causes of bile duct cancers for the most part are unknown. In this review, we assess the current state of knowledge in both fluke-associated and sporadic CCA, from etiological, diagnostic, and treatment perspectives.

Alterations in gut microbial function following liver transplant.

Liver transplantation (LT) improves daily function and ameliorates gut microbial composition. However, the effect of LT on microbial functionality, which can be related to overall patient benefit, is unclear and could affect the post-LT course. The aims were to determine the effect of LT on gut microbial functionality focusing on endotoxemia, bile acid (BA), ammonia metabolism, and lipidomics. We enrolled outpatient patients with cirrhosis on the LT list and followed them until 6 months after LT. Microbiota composition (Shannon diversity and individual taxa) and function analysis (serum endotoxin, urinary metabolomics and serum lipidomics, and stool BA profile) and cognitive tests were performed at both visits. We enrolled 40 patients (age, 56 ± 7 years; mean Model for End-Stage Liver Disease score, 22.6). They received LT 6 ± 3 months after enrollment and were re-evaluated 7 ± 3 months after LT with a stable course. A significant improvement in cognition with increase in microbial diversity, increase in autochthonous and decrease in potentially pathogenic taxa, and reduced endotoxemia were seen after LT compared with baseline. Stool BAs increased significantly after LT, and there was evidence of greater bacterial action (higher secondary, oxo and iso-BAs) after LT although the levels of conjugated BAs remained similar. There was a reduced serum ammonia and corresponding rise in urinary phenylacetylglutamine after LT. There was an increase in urinary trimethylamine-N-oxide, which was correlated with specific changes in serum lipids related to cell membrane products. The ultimate post-LT lipidomic profile appeared beneficial compared with the profile before LT. In conclusion, LT improves gut microbiota diversity and dysbiosis, which is accompanied by favorable changes in gut microbial functionality corresponding to BAs, ammonia, endotoxemia, lipidomic, and metabolomic profiles. Liver Transplantation 24 752-761 2018 AASLD.

The Plasma and Serum Metabotyping of Hepatocellular Carcinoma in a Nigerian and Egyptian Cohort using Proton Nuclear Magnetic Resonance Spectroscopy.

BACKGROUND/AIMS: Previous studies have observed disturbances in the 1H nuclear magnetic resonance (NMR) blood spectral profiles in malignancy. No study has metabotyped serum or plasma of hepatocellular carcinoma (HCC) patients from two diverse populations. We aimed to delineate the HCC patient metabotype from Nigeria (mostly hepatitis B virus infected) and Egypt (mostly hepatitis C virus infected) to explore lipid and energy metabolite alterations that may be independent of disease aetiology, diet and environment. METHODS: Patients with HCC (53) and cirrhosis (26) and healthy volunteers (19) were recruited from Nigeria and Egypt. Participants provided serum or plasma samples, which were analysed using 600 MHz 1H NMR spectroscopy with nuclear Overhauser enhancement spectroscopy pulse sequences. Median group spectra comparison and multivariate analysis were performed to identify regions of difference. RESULTS: Significant differences between HCC patients and healthy volunteers were detected in levels of low density lipoprotein (P = 0.002), very low density lipoprotein (P < 0.001) and lactate (P = 0.03). N-acetylglycoproteins levels in HCC patients were significantly different from both healthy controls and cirrhosis patients (P < 0.001 and 0.001). CONCLUSION: Metabotype differences were present, pointing to disturbed lipid metabolism and a switch from glycolysis to alternative energy metabolites with malignancy, which supports the Warburg hypothesis of tumour metabolism.

Fecal microbiota transplant from a rational stool donor improves hepatic encephalopathy: A randomized clinical trial.

Recurrent hepatic encephalopathy (HE) is a leading cause of readmission despite standard of care (SOC) associated with microbial dysbiosis. Fecal microbiota transplantation (FMT) may improve dysbiosis; however, it has not been studied in HE. We aimed to define whether FMT using a rationally derived stool donor is safe in recurrent HE compared to SOC alone. An open-label, randomized clinical trial with a 5-month follow-up in outpatient men with cirrhosis with recurrent HE on SOC was conducted with 1:1 randomization. FMT-randomized patients received 5 days of broad-spectrum antibiotic pretreatment, then a single FMT enema from the same donor with the optimal microbiota deficient in HE. Follow-up occurred on days 5, 6, 12, 35, and 150 postrandomization. The primary outcome was safety of FMT compared to SOC using FMT-related serious adverse events (SAEs). Secondary outcomes were adverse events, cognition, microbiota, and metabolomic changes. Participants in both arms were similar on all baseline criteria and were followed until study end. FMT with antibiotic pretreatment was well tolerated. Eight (80%) SOC participants had a total of 11 SAEs compared to 2 (20%) FMT participants with SAEs (both FMT unrelated; P = 0.02). Five SOC and no FMT participants developed further HE (P = 0.03). Cognition improved in the FMT, but not the SOC, group. Model for End-Stage Liver Disease (MELD) score transiently worsened postantibiotics, but reverted to baseline post-FMT. Postantibiotics, beneficial taxa, and microbial diversity reduction occurred with Proteobacteria expansion. However, FMT increased diversity and beneficial taxa. SOC microbiota and MELD score remained similar throughout. CONCLUSION: FMT from a rationally selected donor reduced hospitalizations, improved cognition, and dysbiosis in cirrhosis with recurrent HE. (Hepatology 2017;66:1727-1738).

A longitudinal study of patients with cirrhosis treated with L-ornithine L-aspartate, examined with magnetization transfer, diffusion-weighted imaging and magnetic resonance spectroscopy.

The presence of overt hepatic encephalopathy (HE) is associated with structural, metabolic and functional changes in the brain discernible by use of a variety of magnetic resonance (MR) techniques. The changes in patients with minimal HE are less well documented. Twenty-two patients with well-compensated cirrhosis, seven of whom had minimal HE, were examined with cerebral 3 Tesla MR techniques, including T1- and T2-weighted, magnetization transfer and diffusion-weighted imaging and proton magnetic resonance spectroscopy sequences. Studies were repeated after a 4-week course of oral L-ornithine L-aspartate (LOLA). Results were compared with data obtained from 22 aged-matched healthy controls. There was no difference in mean total brain volume between patients and controls at baseline. Mean cerebral magnetization transfer ratios were significantly reduced in the globus pallidus and thalamus in the patients with cirrhosis irrespective of neuropsychiatric status; the mean ratio was significantly reduced in the frontal white matter in patients with minimal HE compared with healthy controls but not when compared with their unimpaired counterparts. There were no significant differences in either the median apparent diffusion coefficients or the mean fractional anisotropy, calculated from the diffusion-weighted imaging, or in the mean basal ganglia metabolite ratios between patients and controls. Psychometric performance improved in 50 % of patients with minimal HE following LOLA, but no significant changes were observed in brain volumes, cerebral magnetization transfer ratios, the diffusion weighted imaging variables or the cerebral metabolite ratios. MR variables, as applied in this study, do not identify patients with minimal HE, nor do they reflect changes in psychometric performance following LOLA.

Urinary metabolic profiling by 1H NMR spectroscopy in patients with cirrhosis may discriminate overt but not covert hepatic encephalopathy.

To date urinary metabolic profiling has been applied to define a specific metabolic fingerprint of hepatocellular carcinoma on a background of cirrhosis. Its utility for the stratification of other complications of cirrhosis, such as hepatic encephalopathy (HE), remains to be established. Urinary proton nuclear magnetic resonance (1H-NMR) spectra were acquired and NMR data from 52 patients with cirrhosis (35 male; 17 female, median (range) age [60 (18-81) years]) and 17 controls were compared. A sub-set of 45 patients (33 male; 12 female, [60 (18-90) years, median model for end stage liver disease (MELD) score 11 (7-27)]) were fully characterised by West-Haven criteria, Psychometric Hepatic Encephalopathy Score (PHES) and electroencephalogram (EEG), and defined as overt HE (OHE, n = 21), covert HE (cHE, n = 7) or no HE (n = 17). Urinary proton nuclear magnetic resonance (1H-NMR) spectra were analysed by partial-least-squares discriminant analysis (PLS-DA). The results showed good discrimination between patients with cirrhosis (n = 52) and healthy controls (n = 17) (R2X = 0.66, R2Y = 0.47, Q2Y = 0.31, sensitivity-60 %, specificity-100 %) as the cirrhosis group had higher 1-methylnicotinamide with lower hippurate, acetate, phenylacetylglycine and N-methyl nicotinic acid levels. While patients with OHE could be discriminated from those with no HE, with higher histidine, citrate and creatinine levels, the best models lack robust validity (R2X = 0.65, R2Y = 0.48, Q2Y = 0.12, sensitivity-100 %, specificity-64 %) with the sample size used. Urinary 1H-NMR metabolic profiling did not discriminate patients with cHE from those without HE, nor discriminate subjects on the basis of PHES/EEG result or MELD score. In conclusion, patients with cirrhosis showed different urinary 1H-NMR metabolic profiles compared to healthy controls and those with OHE may be distinguished from those with no HE although larger studies are required. However, urinary 1H-NMR metabolic profiling did not discriminate patients with differing grades of HE or according to severity of underlying liver disease.