Background/Aims:Steatotic liver disease (SLD) including metabolic dysfunction-associated and alcoholic liver disease, is the most prevalent chronic liver condition globally. Liver biopsy remains the gold standard for fibrosis assessment, but traditional staging is difficult even for highly skilled experts. Collagen proportionate area (CPA), a quantitative digital pathology measure, may offer an alternative, yet its prognostic value in population-based settings is unclear. Methods:Liver biopsies from a cohort of 166 adults with biopsy-confirmed SLD, diagnosed between 1995 and 2008, were retrieved. The liver biopsies underwent fibrosis staging (stages F0-F4) by a pathologist and digital CPA quantification. Clinical outcomes and mortality were tracked via national registries over a median follow-up of 14.8 years. Associations between fibrosis stage, CPA, and all-cause mortality and liver-related events were assessed using Cox regression and cause-specific hazard models, adjusting for alcohol use and age. Predictive performance was evaluated with area under the receiver operating characteristic curve (AUC), index of prediction accuracy (IPA), and calibration plots, internally validated by bootstrapping. Results:CPA correlated significantly with fibrosis stage (Spearman's ρ = 0.63), particularly in advanced fibrosis. Higher fibrosis stages and greater CPA were associated with increased all-cause mortality and liver-related events, but the associations for CPA were nonsignificant after adjustment for age and alcohol use. Scaled estimates of HRs from CPA were lower compared to HRs from fibrosis stages. Predictive models for mortality demonstrated comparable and moderate discrimination for CPA and fibrosis stage, improving with age adjustment. IPA values and calibration plots indicated positive predictive accuracy for mortality but poor performance for liver-specific outcomes. Conclusions:CPA closely correlates with fibrosis stage and has comparable long-term prognostic value in SLD. Interpretation of study results is limited by the small sample size and low number of liver-specific events. Larger studies are needed to validate CPA's clinical utility and explore its integration into routine practice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a systemic fibroinflammatory condition with elevated cardiovascular risk. This study primarily evaluated cardiac fibrosis using MRI-derived extracellular volume fraction (ECV), with hepatic and pancreatic fibrosis assessed as secondary aims, examining whether ECV mapping of the heart, liver, and pancreas reflects extracellular matrix remodeling in relation to liver fibrosis severity. Thirty-nine adults with biopsy-confirmed MASLD without clinical cardiovascular disease and 10 healthy controls underwent 3.0-Tesla MRI with contrast-enhanced T1 mapping to quantify ECV in the myocardium (n = 49), liver (n = 49), and pancreas (n = 29). In MASLD patients, fibrosis markers included the Fibrosis-4 index (FIB-4), liver stiffness measurement (LSM), and Enhanced Liver Fibrosis (ELF) test. Myocardial ECV was higher in patients with advanced fibrosis, defined by FIB-4 ≥ 2.67 (p = 0.046), LSM ≥ 12.5 kPa (p = 0.030), and ELF ≥ 9.8 (p = 0.036), and correlated with FIB-4 (r = 0.39, p = 0.012). Liver ECV was elevated in advanced fibrosis and correlated with portal pressure (r = 0.63), LSM (r = 0.60), and histological stage (r = 0.48). Pancreatic ECV was unrelated to liver fibrosis but correlated with HbA1c (r = 0.40). These findings suggest that multi-organ ECV mapping may reflect matrix remodeling and support MASLD risk stratification.

Abstract Background & Aims Early identification of decompensation in patients with cirrhosis is important to enable timely detection, management of complications and for effective treatment. This study investigates the biology of decompensation and aim to identify protein biomarkers for identification of high-risk patients. Methods The primary analysis included plasma samples from 46 patients with metabolic dysfunction associated steatotic liver disease (MASLD) related cirrhosis. Plasma samples were depleted for the top 14 most abundant proteins and the proteome was measured by liquid chromatography tandem mass spectrometry. The dataset was divided into a training (14 compensated, 10 decompensated) and a test cohort of compensated patients (11 progressing to decompensation, 11 remaining compensated). Changes in protein levels were determined by ANCOVA and a prognostic model was developed using logistic regression. External validation was performed in an independent cohort of 120 patients with alcohol-related cirrhosis. Time-to-event analyses were conducted in this cohort using Cox regression. Results 52 proteins involved in impaired hepatic function, fibrogenesis, immune activation, and metabolic changes were significantly different between compensated and decompensated patients. A prognostic model with four proteins (NBL1, LTBP4, APOC4, GHR), demonstrated predictive ability for future decompensation (AUC=0.93, 73% sensitivity, 100% specificity). In the external validation cohort, the model demonstrated generalizability (AUC=0.78, 72% sensitivity, 82% specificity). Validation cohort time-to-event analyses showed that higher baseline scores were associated with shorter time to liver-related events (HR 1.32; log-rank p = 0.027), underscoring the panel’s prognostic value. Conclusion Our study indicates that patients with decompensated cirrhosis are characterized by proteomic signatures of fibrogenesis and metabolic dysfunction. Capturing these signatures could help identify patients at risk of complications and potentially those eligible for aetiology directed treatment. Impact and Implications Addressing a critical unmet need for early detection of cirrhosis decompensation, our proteomic study identifies a four-protein panel with predictive ability for decompensation. These findings hold significant implications for hepatologists, clinical researchers, and healthcare systems, offering a novel tool to enhance prognostication and refine treatment strategies, potentially facilitating targeted patient monitoring. However, considering the small discovery sample size and the distinct aetiology of the external validation cohort, further validation is essential before broad clinical integration. Graphical Abstract

Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of chronic liver disease worldwide. Progression to metabolic dysfunction-associated steatohepatitis (MASH) can result in fibrosis, cirrhosis, and increased mortality. MASLD and MASH are often under-diagnosed due to their asymptomatic nature, limited awareness, and stigma associated with both liver disease and increased body weight, often resulting in delayed diagnosis. Unclear referral pathways from general practice to specialists and dietitians are often delayed, limiting timely and coordinated care. Given the growing concern regarding MASH and its urgent challenges, the first national, structured, multidisciplinary expert group in Denmark was convened to identify recommendations by capturing a broad, practice-oriented perspective. Methods: Eleven experts with overlapping roles participated, including nine with scientific and clinical expertise in obesity, diabetes, and gastroenterology/hepatology, two from general practice, a dietitian, and three representing patient associations, ensuring both clinical and patient perspectives. Results: The expert panel identified seven focus areas for improving MASH diagnosis and management in Denmark. Key challenges included late diagnosis, low awareness, and suboptimal referral pathways. Moreover, the panel highlighted the need for standardised referral processes, interdisciplinary collaboration, and increased awareness in primary care to enable timely detection and coordinated care. The framework provides a practice-oriented foundation for national strategies, supporting structured patient pathways, coherent management, and future implementation initiatives. Conclusions: Taken together, this publication can serve as a reference for general practitioners, medical specialists, dietitians, professional societies, and healthcare authorities, to support the development of structured, sustainable pathways for MASH care in Denmark.

In clinical practice, assessing alcohol intake can be challenging because patients may under-report or misjudge their consumption, so biomarkers of chronic intake provide valuable additional information. Phosphatidylethanol (PEth) is a group of abnormal phospholipids that are formed in cell membranes only in the presence of ethanol (alcohol). We developed, analytically validated, and clinically implemented a rapid and robust LC-MS/MS-based method for quantifying PEth (16:0/18:1) in whole blood. The method demonstrates a limit of detection (LOD) of 0.01 μM and a limit of quantification (LOQ) of 0.03 μM, with confirmed linearity up to at least 0.8 μM. Validation using reference materials and samples from patients with liver disease and self-reported alcohol intake confirmed its accuracy. Clinical implementation over a two-month period, involving 20 analytical batches, demonstrated inter-assay precision below 15%. Our results support the clinical applicability of PEth use for accurate estimation of alcohol intake in humans.

RATIONALE:Hepatic encephalopathy is a brain dysfunction characterised by neurological and psychiatric changes due to liver insufficiency or portal-systemic shunting. The severity ranges from minor symptoms to coma. The previous version of this updated review meta-analysed data from 16 randomised clinical trials on branched-chain amino acids (BCAAs) versus control interventions, and found that BCAAs did not affect mortality but had a beneficial effect on hepatic encephalopathy. As data on critical outcomes were insufficient and new trials were published, we updated the review again. OBJECTIVES:To assess the beneficial and harmful effects of BCAAs versus any control intervention for people with cirrhosis and hepatic encephalopathy. SEARCH METHODS:We identified trials through manual and electronic searches in the Cochrane Hepato-Biliary Group Controlled Trials Register, the Cochrane Central Register of Controlled Trials, MEDLINE, Embase, Latin American and Caribbean Health Science Information database (LILACS), Science Citation Index Expanded, and Conference Proceedings Citation Index - Science. We also searched other resources and contacted experts for additional published and unpublished trials. The latest search date was 12 December 2024. ELIGIBILITY CRITERIA:We included randomised clinical trials, irrespective of bias control, language, outcomes reported, or publication status, that compared any form of branched-chain amino acid with no intervention, placebo, diets, non-absorbable disaccharides, antibiotics, or any other intervention with a potential effect on hepatic encephalopathy, in children and adults with overt or minimal hepatic encephalopathy associated with acute or chronic liver disease. OUTCOMES:The critical outcomes were all-cause mortality, hepatic encephalopathy (number of people without improved manifestations of hepatic encephalopathy), and serious adverse events (including nausea and diarrhoea). The important outcomes were quality of life and markers of nutritional status, including serum albumin and nitrogen balance. We evaluated outcomes at the longest available follow-up duration. The longest follow-up was also our primary time point for analysis. RISK OF BIAS:We assessed the risk of bias (RoB) of the critical and important outcomes using the Cochrane RoB 2 tool. SYNTHESIS METHODS:We used standard Cochrane methodology. We performed meta-analyses, based on intention-to-treat, with risk ratios (RRs) for dichotomous outcomes, standardised mean differences (SMDs) for continuous outcomes in trials using different scales, and mean differences (MDs) for continuous outcomes when trials used the same scales, all with 95% confidence intervals (CI). We conducted the main analysis using a random-effects model. We assessed the overall certainty of the evidence per outcome with the GRADE approach, with five components (risk of bias, indirectness, heterogeneity, imprecision using the minimally contextualised approach, and dissemination bias). INCLUDED STUDIES:We added two new randomised clinical trials to this review update. Thus, the 18 trials, published between 1984 and 2023, included 934 adults (mean age 47 to 64 years). We found no trials in children. Participants had overt hepatic encephalopathy (13 trials) and covert hepatic encephalopathy (five trials). Ten trials assessed oral BCAA supplements and eight trials assessed intravenous BCAAs. The control groups received no intervention or placebo (two trials), diets (11 trials), lactulose (three trials), or neomycin (two trials). Most participants had cirrhosis (minimum 97%). One trial included participants with acute hepatitis. The follow-up periods were from four days to two years (104 weeks). Ten trials were conducted in Europe, four in Asia, two in the USA, and one each in Brazil and Australia. Three trials received support from for-profit organisations in the form of interventions. SYNTHESIS OF RESULTS:BCAAs may have little to no effect on all-cause mortality compared with the control interventions, but the evidence is very uncertain (RR 0.89, 95% CI 0.71 to 1.12; 17 studies, 867 participants; very low-certainty evidence). We found no evidence of small-study effects. All trials reported the effect of BCAAs on hepatic encephalopathy. The evidence suggests that BCAAs reduce hepatic encephalopathy (RR 0.79, 95% CI 0.64 to 0.96; 18 studies, 934 participants; low-certainty evidence). The follow-up ranges for the two outcomes were four days to two years. Nausea and diarrhoea, considered as serious adverse events, occurred in 58 of 477 (12%) participants in the BCAAs group and 16 of 538 (3%) participants in the control intervention group (diets). The evidence is very uncertain about the effect of BCAAs on nausea and diarrhoea (RR 2.05, 95% CI 0.40 to 10.58; 6 studies, 1015 participants; very low-certainty evidence). Three trials provided data on quality of life, but we were unable to meta-analyse the data because of the methods used to register the scores. All participants in the three trials had cirrhosis, but some did not have hepatic encephalopathy at baseline. When the analyses were limited to people with hepatic encephalopathy, none of the trials found beneficial or harmful effects of BCAAs on the global 36-item Short Form Health Survey (SF-36) score or any of the subscales. BCAAs may have little to no effect on albumin concentration (range 12 to 56 weeks) (MD 0.60, 95% CI -0.90 to 2.09; I² = 0%; 3 studies, 176 participants; very low-certainty evidence), but the evidence is uncertain. The evidence is very uncertain about the effect of BCAAs on nitrogen balance (range four days to two years) (SMD 0.82, 95% CI -1.01 to 2.64; 3 studies, 108 participants; very low-certainty evidence). We downgraded the certainty of evidence for risk of bias, indirectness, and imprecision. Three trials are ongoing. AUTHORS' CONCLUSIONS:We added two new trials to the analyses. The evidence suggests BCAAs reduce hepatic encephalopathy, but the certainty of evidence is low. We do not know if BCAAs, compared with controls, have any effect on all-cause mortality, nausea and diarrhoea, albumin, and nitrogen balance because of very low-certainty evidence. We could not meta-analyse the data on quality of life. Trials in children are lacking. We lack randomised clinical trials comparing BCAAs with interventions such as non-absorbable disaccharides, rifaximin, or other antibiotics. FUNDING:No funding REGISTRATION: Protocol (1997): Gluud C, Koretz RL. Branched-chain amino acids for hepatic encephalopathy (Protocol for a Cochrane Review). The Cochrane Library 1997, Issue 1. Original review (2003): doi.org/10.1002/14651858.CD001939 Review update (2015 Feb): doi.org/10.1002/14651858.CD001939.pub2 Review update (2015 Sep) doi.org/10.1002/14651858.CD001939.pub3 Review update (2017): doi.org/10.1002/14651858.CD001939.pub4.

INTRODUCTION AND OBJECTIVES:Risk stratification is important in the management of metabolic dysfunction-associated steatotic liver disease (MASLD) to prioritize monitoring and treatment resources. We evaluated whether non-invasive tests (NITs) can predict major adverse liver outcomes (MALOs) and diagnose advanced fibrosis in patients with MASLD. PATIENTS AND METHODS:A prospective cohort of 270 patients with MASLD (median age 56 years; 44% female; 41% type 2 diabetes) was evaluated using nine NITs: FIB-4, LSM, Agile3+, Agile4, FAST, ADAPT, ELF, PROC3, and C1M. Cox proportional hazards models and ROC analyses were used to assess prognostic performance for MALOs and diagnosis of advanced fibrosis, respectively. RESULTS:Over a four-year follow-up period, 25 patients experienced MALOs. All NITs were associated with MALOs, though confidence intervals were wide given the limited number of events. Most NITs demonstrated positive associations with risk (HR range 1.82-5.39; p < 0.05), whereas C1M was inversely associated (HR 0.63, 95% CI 0.49-0.82; p < 0.001), indicating reduced risk. As a secondary objective, we evaluated whether each NIT could diagnose advanced fibrosis. FIB-4 and LSM effectively ruled out advanced fibrosis, while ELF and the Agile scores showed the strongest overall discriminative performance. For confirming advanced fibrosis, ELF (81%/76%) and Agile3+ (85%/79%) achieved the best sensitivity-specificity balance. CONCLUSIONS:Our findings suggest that NITs may hold both diagnostic and prognostic value, helping to identify advanced fibrosis and stratify future risk of MALOs in MASLD which is a clinically heterogenous but clinically relevant outcome. Additional research is needed to evaluate their performance in primary care settings. IMPACT AND IMPLICATIONS:In MASLD, NITs have the potential for identification of at-risk patients. Identifying NITs that predict beneficial as well as detrimental outcomes could also add value to monitoring strategies although further evidence is needed to evaluate this potential.

RATIONALE:Hepatic encephalopathy is a common complication of cirrhosis. Its development is associated with increased morbidity and mortality. Its exact pathogenesis is unknown, but ammonia, produced by bacterial action in the intestine, plays a key role. Antibiotics modulate the gut flora and may reduce intestinal ammonia production. Aminoglycosides such as neomycin, paromomycin, and ribostamycin have been used to treat hepatic encephalopathy, as have other antibiotics such as vancomycin and metronidazole. OBJECTIVES:To assess the beneficial and harmful effects of aminoglycosides, vancomycin, and metronidazole versus placebo, no intervention, other antibiotics, or other active pharmacological interventions, for the prevention and treatment of hepatic encephalopathy in people with cirrhosis. SEARCH METHODS:We searched the Cochrane Hepato-Biliary Group Controlled Trials Register, CENTRAL, MEDLINE, Embase, and three other databases to 15 April 2025. We also searched online trials registries for ongoing and unpublished trials, undertook manual searches of meeting and conference proceedings, checked bibliographies of relevant articles, and corresponded with investigators and pharmaceutical companies. ELIGIBILITY CRITERIA:We included randomised clinical trials (RCTs) involving participants with cirrhosis and hepatic encephalopathy, or who were at risk of developing hepatic encephalopathy, comparing aminoglycosides, vancomycin, or metronidazole to (1) placebo or no intervention; or (2) other pharmacological agents, including non-absorbable disaccharides, other antibiotics, or other potentially beneficial agents (e.g. branched-chain amino acids, L-ornithine L-aspartate, nitazoxanide (a broad-spectrum antiparasitic/antiviral agent), and nicotinohydroxamic acid (a potent urease inhibitor). We included trials irrespective of publication status, outcomes reported, language, or blinding. We excluded trials involving people with hepatic encephalopathy associated with acute liver failure or with non-cirrhotic portal hypertension. OUTCOMES:The critical outcomes were all-cause mortality, hepatic encephalopathy, and serious adverse events. The important outcomes were non-serious adverse events and health-related quality of life (HRQoL). Our primary time point was the maximum length of follow-up. RISK OF BIAS:We used Cochrane's original risk of bias tool (RoB 1) to assess the risk of bias. SYNTHESIS METHODS:We used standard Cochrane methods. We undertook random-effects meta-analyses to calculate risk ratios (RRs) or standardised mean differences (SMDs), with 95% confidence intervals (CIs). We assessed heterogeneity with the I2 statistic, and the certainty of evidence with the GRADE framework. INCLUDED STUDIES:We included 24 RCTs, involving 1405 participants experiencing 1418 hepatic encephalopathy events. Twenty-three trials evaluated the treatment of hepatic encephalopathy and one, the secondary prevention of hepatic encephalopathy; we analysed these trials jointly. The trials assessed three aminoglycosides: neomycin (15 trials), paromomycin (three trials), and ribostamycin (one trial), as well as vancomycin (two trials), and metronidazole (three trials). Overall, 670 participants received these pharmacotherapies while 735 participants received a placebo or other potentially beneficial agents. We classified 22 of the 24 trials to be at an overall high risk of bias based on domain-level assessments. SYNTHESIS OF RESULTS:The certainty of evidence for all comparisons was low to very low, mainly due to risk of bias, imprecision, and heterogeneity. Twenty-three of the 24 trials, involving 1383 participants, reported all-cause mortality data. Aminoglycosides may increase mortality slightly compared to other potentially active agents (RR 1.64, 95% CI 1.03 to 2.62; I² = 0%; 3 studies, 166 participants). The evidence was very uncertain about whether aminoglycosides versus a placebo (RR 1.02, 95% CI 0.62 to 1.69; I² = 0%; 3 studies, 137 participants), non-absorbable disaccharides (RR 1.21, 95% CI 0.57 to 2.59; I² not applicable; 4 studies, 266 participants), or other antibiotics (RR 1.00, 95% CI 0.24 to 4.23; I² = 83%; 8 studies, 496 participants) result in a difference in mortality risk. The evidence was also very uncertain when comparing vancomycin to non-absorbable disaccharides (RR 0.94, 95% CI 0.26 to 3.40; I² not applicable; 2 studies, 72 participants), and metronidazole to other active agents (RR 0.97, 95% CI 0.14 to 6.66; I² = 0%; 3 studies, 242 participants). Nineteen trials involving 1281 participants reported data on hepatic encephalopathy. There may be little to no difference in the effects of aminoglycosides versus non-absorbable disaccharides (RR 0.84, 95% CI 0.67 to 1.05; I² = 0%; 3 studies, 251 participants), aminoglycosides versus other potentially active agents (RR 1.21, 95% CI 0.79 to 1.85; I² = 0%; 3 studies, 166 participants), and metronidazole versus other active agents (RR 1.50, 95% CI 0.89 to 2.54; I² = 48%; 2 studies, 208 participants). The evidence is very uncertain about the effect of aminoglycosides versus placebo, other antibiotics, and vancomycin versus non-absorbable disaccharides. Twenty trials, involving 1186 participants, reported a total of 328 serious adverse events. Aminoglycosides may slightly increase the risk of serious adverse events compared with other potentially active agents (RR 1.60, 95% CI 1.03 to 2.47; I² = 0%; 3 studies, 166 participants). The evidence is very uncertain when comparing aminoglycosides to placebo and other antibiotics, or when comparing vancomycin to non-absorbable disaccharides. Eighteen trials, involving 922 participants, reported a total of 96 non-serious adverse events. There may be a slight increase in the risk of adverse events when comparing aminoglycosides to placebo (RR 2.80, 95% CI 1.11 to 7.04; I² not applicable; 2 studies, 98 participants), and to other antibiotics (RR 3.24, 95% CI 1.08 to 9.70; I² = 0%; 8 studies, 251 participants). The evidence is very uncertain about the effects of aminoglycosides versus non-absorbable disaccharides or other active agents, and metronidazole versus other active agents. Only one trial assessed HRQoL, but reported the data in a form that precluded meta-analysis. Eight trials received support from pharmaceutical companies while six did not. Ten trials did not provide this information. AUTHORS' CONCLUSIONS:Due to low- or very low-certainty evidence, we do not know if aminoglycosides benefit hepatic encephalopathy compared to placebo or other potentially active agents. There may be a slight increase in the risks of mortality and serious adverse events with aminoglycosides compared to other agents, and of non-serious adverse events when compared to placebo and other antibiotics. We do not know if vancomycin or metronidazole improve clinically relevant outcomes. Only one trial assessed health-related quality of life. FUNDING:This Cochrane review received no specific funding. REGISTRATION:https://doi.org/10.1002/14651858.CD012734.

Hyperglucagonemia is a hallmark of metabolic diseases including type 2 diabetes and metabolic dysfunction-associated steatotic liver disease (MASLD), yet the underlying mechanisms are unclear. This study aimed to characterize the liver's role in glucagon clearance and to elucidate whether enzymatic degradation or receptor-mediated uptake is the dominant clearance mechanism-particularly in the context of obesity-associated hepatic steatosis. Using an in situ perfused single-pass mouse liver model, hepatic glucagon clearance/disappearance was quantified directly in lean and diet-induced obese (DIO) male C57BL/6JRj mice. Glucagon disappearance across the liver was measured via immunoassays and mass spectrometry. To dissect the mechanism of clearance, glucagon was coinfused with either enzyme inhibitors [for dipeptidyl peptidase 4 (DPP-4) and neprilysin (NEP)] or a glucagon receptor antagonist. In lean mice, the liver extracted 20% of inflowing portal venous glucagon (at levels corresponding to postprandial conditions), an effect abolished by glucagon receptor antagonism but unaffected by enzyme inhibition. Mass spectrometry confirmed that nearly all glucagon remained intact, supporting receptor-mediated internalization as the primary clearance mechanism. In DIO mice with hepatic steatosis determined as elevated hepatic triglyceride content, glucagon clearance was markedly reduced. The glucagon receptor was downregulated in livers of DIO mice. These findings identify the liver as an active site of glucagon clearance, potentially through receptor-mediated mechanisms. Obesity-related hepatic steatosis disrupts this process and may contribute to hyperglucagonemia in metabolic disease. Targeting hepatic glucagon clearance may offer a novel approach to normalize glucagon levels and improve metabolic control.NEW & NOTEWORTHY We identify the liver as an active glucagon-clearing organ: ∼20% of portal glucagon is extracted under physiological conditions via glucagon receptor-dependent uptake, not enzymatic cleavage. DPP-4 and neprilysin inhibition does not alter hepatic glucagon disappearance, and mass spectrometry confirms glucagon remains largely intact. Strikingly, diet-induced hepatic steatosis nearly abolishes glucagon extraction, coinciding with reduced hepatic GCGR expression, suggesting impaired receptor-mediated clearance may contribute to hyperglucagonemia in MASLD.