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Novel biomarkers for alcohol-associated liver disease and their implications across clinical settings

Kaanthi Rama1orcid, Vinay Jahagirdar1orcid, Francisco Idalsoaga2,3orcid, Hanna Blaney1orcid, S. Fisher Rhoads1orcid, Luis Antonio Díaz2,4orcid, Marco Arrese2orcid, Juan Pablo Arab1,2orcid
Clinical and Molecular Hepatology 2026;32(2):443-463.
Published online: November 25, 2025

1Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA, USA

2Departamento De Gastroenterología, Escuela De Medicina, Pontificia Universidad Católica De Chile, Santiago, Chile

3Division of Gastroenterology and Hepatology, Western University & London Health Sciences Centre, London, Canada

4MASLD Research Center, Division of Gastroenterology and Hepatology, University of California, San Diego, San Diego, CA, USA

Corresponding author : Juan Pablo Arab Stravitz-Sanyal Institute for Liver Disease and Metabolic Health, Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, 1200 E. Broad St, 14th floor, PO Box 980341, Richmond, VA 23298, USA Tel: +1-804-828-4060, Fax: +1-804-828-5348, E-mail: JuanPablo.Arab@vcuhealth.org

Editor: Do Seon Song, The Catholic University of Korea, Korea

• Received: August 15, 2025   • Revised: November 14, 2025   • Accepted: November 16, 2025

Copyright © 2026 by The Korean Association for the Study of the Liver

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • Alcohol-associated liver disease (ALD) is a leading cause of preventable cirrhosis, hepatocellular carcinoma (HCC), and liver-related mortality, yet current laboratory and imaging tools detect only late-stage disease. This narrative review synthesizes emerging evidence on novel biomarkers that capture the multidimensional pathophysiology of ALD and discusses their utility for routine clinical practice. Traditional serum-based liver fibrosis markers (e.g., cytokeratin-18 fragments, Pro-C3, the enhanced liver fibrosis test) improve non-invasive staging risk beyond aminotransferases, while elastography techniques, such as vibration-controlled transient elastography and magnetic resonance elastography, can also quantify liver stiffness with high precision. Among novel mechanistic biomarkers, genetic polymorphisms in PNPLA3, TM6SF2, MBOAT7, HSD17B13, and polygenic risk scores define lifetime risk, whereas sex-specific hormonal milieus also modify susceptibility and progression. Moreover, gut dysbiosis signatures, including reduced Faecalibacterium prausnitzii, Akkermansia muciniphila, and a lower Firmicutes/Bacteroidetes ratio, and their metabolites (short-chain fatty acids, and bile acids, trimethylamine N-oxide) correlate with liver inflammation and fibrosis. Endocrine imbalances of cortisol, testosterone, and thyroid hormones further stratify metabolic vulnerability. Ultimately, multi-omics platforms (i.e., transcriptomics, lipidomics, proteomics, metabolomics, and epigenomics) can reveal distinct molecular signatures that predict steatohepatitis, fibrogenesis, and early HCC. Integrating these biomarkers enables phase-specific enrichment strategies, earlier intervention windows, adaptive dose-finding, and mechanismbased endpoints in ALD trials. Remaining challenges include assay standardization, validation across diverse cohorts, and incorporation into regulatory frameworks. Future work could evaluate cost-effectiveness and feasibility in routine clinical practice. Widespread adoption promises earlier diagnosis, personalized risk reduction, and more efficient drug development for this globally prevalent disorder.
  • Keywords: Alcohol-associated liver disease; Alcoholic cirrhosis; Alcoholic liver disease, fatty liver; Biomarkers; Clinical trials, precision medicine
Liver stiffness by transient elastography reliably detects advanced fibrosis/cirrhosis in alcohol-associated liver disease (ALD); interpretation should account for inflammation (e.g., aspartate aminotransferase [AST]).
Serum fibrosis panels like enhanced liver fibrosis (ELF) have comparable high accuracy for advanced fibrosis and outperform APRI/fibrosis-4 (FIB-4).
Magnetic resonance imaging (MRI)-based methods are most accurate for quantifying steatosis; controlled attenuation parameter (CAP) (with TE) provides acceptable pointof-care detection and often outperforms ultrasound.
Injury/inflammation markers like cytokeratin-18 (CK-18) fragments rise in alcohol-associated hepatitis (AH) and advanced disease, though not AH-specific; cytokines (e.g., interleukin [IL]-1β, tumor necrosis factor [TNF], IL-8) are elevated but need validation for prognostic use.
Emerging classes: Extracellular vesicle proteins and circulating miRNAs show promise for diagnosis/prognosis in ALD [1,2].
Integrated, mechanism-to-clinic framework spanning genetics, sex hormones, microbiome, and endocrine axes, linked to diagnosis, staging, prognosis, and monitoring across ALD.
Novel fibrosis and cell-death markers in one place (e.g., Pro-C3/ADAPT, CK-18) with up-to-date outcome data and how to combine them with TE/ELF in practice.
Multi-omics roadmap (transcriptomic, lipidomic, proteomic, metabolomic, epigenomic) for trial enrichment and mechanism-based endpoints, plus implementation hurdles (standardization, validation, cost-effectiveness).
Actionable visuals/tables that map biomarkers to clinical use and provide a consolidated ALD-specific atlas to guide adoption when advanced imaging is limited.
Alcohol is a major contributor to the global burden of disease, including chronic liver disease and hepatocellular carcinoma (HCC) [3-5]. ALD is a leading cause of preventable liver-related morbidity and mortality globally, accounting for over 1.8 million deaths in 2021 [6]. The ALD spectrum ranges from asymptomatic steatosis to steatohepatitis, fibrosis, cirrhosis, and HCC [7]. Notably, AH is a severe acute phenotype with high short-term mortality (20–30% at 1 month in severe cases) that can occur regardless of the presence of cirrhosis [8]. Also, 10–20% of patients with ALD progress to cirrhosis, with the highest risk in those with at least one AH episode [9]. Once cirrhosis develops, the risk of decompensation and liver-related mortality increases substantially [10]. However, progression to advanced stages of ALD is preventable, depending on alcohol consumption and, to a lesser degree, on the presence of cardiometabolic risk factors, including type 2 diabetes mellitus and obesity [2,11]. Nonmodifiable factors, including sex and genetic predisposition, can also affect liver fibrosis progression [7].
Given that globally approximately 52.2% of men and 35.4% of women are current drinkers, of which 38% engage in heavy episodic drinking or binge-drinking [3], risk stratification is essential to identify individuals at higher risk of developing clinically relevant ALD and liver-related events requiring therapeutic intervention. ALD severity strongly correlates with the degree of alcohol consumption and, consequently, detection of ethanol exposure is an es-sential aspect of the assessment of individuals with ALD [10,12]. Liver fibrosis is another major risk factor for mortality due to ALD, and several biomarkers are routinely employed to detect fibrosis and subsequently stratify risk [1,13]. However, traditional markers often fail to detect early-stage disease or accurately stratify patients based on disease severity. Patients with ALD are 12 times more likely to be diagnosed at an advanced stage of disease than patients with viral hepatitis, resulting in delayed interventions and personalized management [14].
Since current biomarkers for ALD exhibit substantial limitations and caveats in performance and affordability, a variety of novel biomarkers have been developed to overcome these barriers, but their validation requires a clear understanding of ALD pathophysiology. In this review, we examine traditional and novel biomarkers for ALD, focusing on how genetics, sex, microbiome, and endocrine function influence disease development (Fig. 1). We also discuss the implications of these findings for clinical practice, emphasizing the incorporation of serum-, imaging-, and omics-based biomarkers to advance precision medicine in ALD. Our scope is exclusive to biomarkers for diagnosing, staging, and progression or regression in ALD. Biomarkers of alcohol consumption have not been focused on; nonetheless, they remain critical for detecting and quantifying alcohol use and guiding clinical management [12].
Literature search and study selection
This review is a narrative (non-systematic) review. We searched PubMed/MEDLINE for English-language publica-tions from January 1, 2000 to September 30, 2025 (with a final targeted update on October 20, 2025) using combinations of the following terms: “alcohol-associated liver disease” OR “alcoholic hepatitis” OR “ALD” AND “biomarker” OR “diagnostic” OR “prognostic” OR “risk stratification” OR specific markers (e.g., “FGF21”, “CK-18”, “cytokeratin-18”, “ELF”, “VCTE”, “elastography”). We also screened reference lists of key articles and recent guidelines to identify additional relevant studies. Inclusion prioritized original human studies and high-quality reviews addressing diagnostic or prognostic performance of circulating, imagingbased, or composite biomarkers in ALD/AH, with emphasis on studies reporting effect sizes (AUC, sensitivity/specificity, OR/HR) or clinical endpoints (e.g., severity grades, decompensation, mortality, transplant-free survival). Exclusions were non-English articles, case reports without analytic performance data, animal-only studies, and conference abstracts without full data. Because a systematic review was not performed, this approach may introduce selection and publication bias, favoring better-known biomarkers and studies with positive findings. To mitigate this, we sought convergent evidence across study designs, prioritized recent multi-center or prospective cohorts when available, and explicitly noted areas where evidence is inconsistent or limited.
Indirect serum biomarkers
Traditional indirect serum markers of liver injury, such as AST and alanine aminotransferase (ALT), have been widely used to detect liver injury in ALD. Elevated levels of AST and ALT might indicate hepatocyte injury and inflammation, providing important information about disease activity and severity. In ALD, the transaminases are typically elevated, and the AST/ALT ratio is often >2. This pattern is attributed not only to greater mitochondrial injury (as AST is present in both cytosol and mitochondria, while ALT is predominantly cytosolic), but also to alcohol-induced pyridoxal 5’-phosphate (vitamin B6) deficiency, which disproportionately reduces ALT activity [15]. An AST/ALT ratio greater than 1 is a predictor of cirrhosis, with a sensitivity of 81.3% and specificity of 55.3% [16]. Moreover, a ratio greater than 1.5 is suggestive of AH in the appropriate clinical scenario (abrupt onset of jaundice, malaise, decompensated liver disease, and coagulopathy) [17].
Gamma-glutamyl transferase (GGT) and alkaline phosphatase might also be elevated due to cholestasis and represent common abnormalities detected in ALD evaluation. GGT, in particular, might be disproportionately elevated in ALD, even in the absence of relevant hepatocellular damage or cholestasis, due to alcohol-induced microsomal enzyme induction and oxidative stress pathways that upregulate GGT expression [18]. Mean corpuscular volume (MCV) is often elevated in individuals with chronic alcohol use, which can be attributed to the direct toxic effect on erythropoiesis, altered red cell metabolism, and vitamin B12 deficiency [19-21]. Its diagnostic value is limited by low sensitivity and specificity, as MCV is influenced by age, sex, liver disease, and coexisting conditions such as megaloblastic anemia and microcytosis [21]. Therefore, MCV should be interpreted in conjunction with other biomarkers such as GGT to enhance diagnostic accuracy [22]. Lastly, platelet count, an important component of the FIB-4 score to assess liver fibrosis, might be reduced in ALD not only due to advanced fibrosis and portal hypertension, but also from alcohol-induced bone marrow suppression and splenic sequestration [23,24]. Despite their extensive use, these markers lack specificity and might be influenced by factors unrelated to ALD, limiting their utility for precise risk stratification and prognostication.
A study looking at veterans with nonalcoholic fatty liver disease (NAFLD) found that serial changes in FIB-4 are associated with risk of progression to cirrhosis and HCC, and increasing FIB-4 over time signals higher risk for adverse outcomes [25]. There is limited evidence on the optimal frequency and longitudinal use of FIB-4 for ongoing monitoring in ALD, and further research is needed to define best practices for serial assessment. For now, it can be used to stratify risk and guide referral and management decisions in ALD.
Markers of alcohol consumption
Phosphatidylethanol (PEth) is a highly specific and sensitive biomarker for recent alcohol consumption and is increasingly used as both a diagnostic and prognostic marker in ALD. Its levels correlate with the amount of alcohol consumed, and values ≥200 ng/mL are indicative of regular high alcohol intake, which is typical in ALD [26]. PEth outperforms traditional indirect biomarkers (e.g., AST/ALT ratio, GGT, MCV) in sensitivity and specificity for detecting harmful drinking and distinguishing ALD from metabolic-associated steatotic liver disease (MASLD) and mixed etiologies (MetALD) [27]. PEth enables objective subclassification of steatotic liver disease, reducing misclassification due to underreporting of alcohol use. Studies show that adding PEth to self-reporting increases detection of ALD and MetALD by 3- to 4-fold (cut-offs ≥200 and ≥35 ng/mL, respectively), revealing substantial underreporting in clinical populations [28,29]. Moreover, elevated PEth levels are independently associated with increased risk of major adverse liver outcomes, including progression to cirrhosis and liver-related mortality, even after adjusting for confounders such as age, sex, and fibrosis stage [29].
Alcohol represents a reversible driver of prognosis in ALD. In a meta-analysis covering around 19,000 patients, abstinence was associated with substantially better overall survival and lower risk of hepatic decompensation, with benefits persisting even in decompensated alcohol associated cirrhosis [30]. Yet only about half of patients are abstinent, and most included cohorts ascertained drinking by self-report, relatives’ reports, routine biochemistry, or clinician judgment. None confirmed abstinence using direct alcohol metabolites, raising concern for misclassification. Incorporating objective alcohol biomarkers like PEth into routine care would more accurately classify exposure, sharpen prognostication and re-compensation assessment, and ensure that observed survival gains are truly attributable to abstinence. Sharing biomarker trends with patients provides concrete, immediate feedback that links verified abstinence to improved outcomes, thereby reinforcing motivation alongside counseling and pharmacotherapy.
Serum biomarkers of fibrosis and cell injury
The progression of ALD from simple steatosis to advanced fibrosis and cirrhosis underscores the need for reliable biomarkers capable of assessing fibrosis stage and guiding therapeutic decisions. In recent years, an array of novel serum-based biomarkers targeting key pathogenic pathways involved in hepatic fibrogenesis has emerged, offering enhanced sensitivity, specificity, and prognostic value compared to traditional markers. FIB-4, while less specific, is widely accessible and useful for stratifying patients in large-scale trials and real-world settings, with a sensitivity of 70% and a specificity of 97% for detecting advanced fibrosis [31,32]. More specific serum biomarkers include the ELF, Pro-C3, and CK-18 fragments, among others. These biomarkers might reduce reliance on liver biopsy, reducing costs and facilitating decision-making in clinical practice [33].
The ELF test is a direct fibrosis biomarker that includes hyaluronic acid, procollagen III N-terminal peptide (PIIINP), and tissue inhibitor of metalloproteinase-1, reflecting the dynamic interplay between extracellular matrix deposition and degradation in fibrotic liver tissue [16]. The ELF score has been validated in ALD as a sensitive and non-invasive indicator of hepatic fibrosis and has been shown to predict liver-related outcomes and disease progression [34]. In ALD, ELF has an area under the receiver operating characteristics (AUROC) of 0.92 for the diagnosis of advanced fibrosis and 0.94 for cirrhosis, with a sensitivity of 79% and a specificity of 91% [35,36]. Given the strong correlation with histological fibrosis, the ELF test could be considered as a laboratory reflex test using the same blood sample in individuals with abnormal FIB-4 values, facilitating non-invasive risk stratification and monitoring antifibrotic therapy [35,37-39].
Pro-C3, a neoepitope of PIIINP generated during active fibrogenesis, has demonstrated a higher correlation with histologic fibrosis severity than traditional indirect markers [40,41]. Pro-C3 is a direct marker of collagen formation, showing its potential utility in staging fibrosis and monitoring antifibrotic therapy. It is a part of composite scores like ADAPT, improving risk stratification and predicting liver-related events. Pro-C3 levels can be affected by fibrotic processes outside the liver and might lack sensitivity in early fibrotic stages [42].
CK-18 fragments, especially M30 (apoptotic cleavage product) and M65 (total cell death marker), reflect hepatocyte apoptosis and necrosis and are elevated in patients with ALD, particularly those with AH [43]. Elevated CK-18 levels have been proposed as a tool that correlates with histological severity and prognosis [44]. However, CK-18 levels can overlap with other liver diseases involving apoptosis and necrosis, limiting diagnostic specificity in etiologies with mixed liver injury [45]. By quantifying the extent of fibrosis, these biomarkers facilitate risk stratification and therapeutic decision-making, guiding the selection of appropriate interventions and monitoring treatment response over time.
Imaging-based markers
Ultrasound imaging serves as a useful, inexpensive tool for qualitative assessment of liver echogenicity and visualization of hepatic parenchymal patterns, with increased echogenicity indicative of hepatic steatosis. While ultrasound lacks sensitivity for detecting mild-to-moderate steatosis and is operator-dependent, it remains widely utilized in clinical practice due to its cost-effectiveness, portability, and lack of ionizing radiation [46]. Ultrasonography with two-dimensional shear wave elastography (2D-SWE) can help quantify liver stiffness by measuring the velocity of shear waves generated within the liver, providing a real-time, operator-adjustable map of tissue elasticity. 2D-SWE has a sensitivity of 88% and a specificity of 95% for advanced fibrosis in ALD, making it a valuable tool for ruling out advanced fibrosis and cirrhosis, and for monitoring disease progression. This technique enables concurrent assessment of liver parenchymal characteristics and fibrosis stage, and its integration into standard ultrasound makes it accessible and practical for routine clinical use [36,47]. A large prospective study in patients with ALD found that a 2D-SWE cut-off of 10.2 kPa best identified significant fibrosis (Ishak ≥3), while 16.4 kPa was optimal for cirrhosis (Ishak ≥5), with AUROC values exceeding 0.92 for both endpoints [48]. Another multicenter study using the Canon Aplio i800 system reported similar thresholds: 9.8 kPa for severe fibrosis and 13.1 kPa for cirrhosis [49].
Vibration-controlled transient elastography (VCTE) represents a non-invasive technique for assessing hepatic fibrosis by measuring liver stiffness using ultrasound-based elastography. It provides quantitative measurements of liver stiffness, reflecting the degree of fibrosis, with higher values indicating more advanced disease stages, facilitating risk stratification, therapeutic decision-making, and prognostication in individuals with ALD [50]. VCTE offers high accuracy for staging fibrosis in ALD with validated cutoffs of 12.1 kPa for ≥F3 and 18.6 for F4 (AUROCs of 0.90 and 0.91, respectively); however, these values should be interpreted in the context of aminotransferases and bilirubin levels as hepatic inflammation and cholestasis elevate LSM independent of fibrosis severity [1,47,51]. In ALD, VCTE values below 8–10 kPa have high utility in ruling out advanced fibrosis with pooled sensitivities of 92–94% at these cutoffs. Specificities range from 89% to 95% for advanced fibrosis at thresholds of 12–15 kPa, supporting their use to confirm disease after excluding other causes of increased liver stiffness [52]. CAP is also included in the VCTE and quantifies steatosis, providing complementary information to liver stiffness in ALD [53].
VCTE’s accuracy in alcohol-related cirrhosis is affected by hepatic inflammation, steatohepatitis, and elevated AST or bilirubin, which can lead to overestimation of liver stiffness and fibrosis stage [1]. The optimal cutoffs for fibrosis stages are not fully standardized, with most guidelines recommending a threshold of 12.5 kPa for cirrhosis, but specificity is moderate (0.71), leading to false positives, especially in low-prevalence settings [54,55]. VCTE is less reliable in acute AH and may be confounded by ongoing alcohol use, as liver stiffness can decrease after abstinence [1,56].
VCTE is recommended for initial diagnosis, risk stratification, and longitudinal monitoring of fibrosis progression or regression in compensated alcohol-related liver disease [57]. Serial liver stiffness measurements (LSM) can predict decompensation and mortality, and dynamic changes (≥20% increase or decrease) are clinically meaningful for prognosis and management [58]. Annual screening may be reasonable for ongoing alcohol users at low risk, though optimal intervals are not established. Spleen stiffness measurement (SSM) via VCTE or other elastography methods is emerging as a surrogate for portal hypertension severity and response to therapy. SSM correlates well with hepatic venous pressure gradient and may outperform LSM for monitoring portal hypertension and treatment response, with thresholds >40–45 kPa indicating clinically significant portal hypertension [59].
LSM is prognostically valuable in both ALD and viral-related compensated advanced chronic liver disease. Its predictive interpretation in the grey zone (typically LSM 13–24 kPa) is more susceptible to confounding in ALD than in viral cirrhosis. In ALD, LSM is a strong predictor of decompensation and mortality. However, its accuracy can be confounded by active inflammation and steatohepatitis, which may transiently elevate LSM values independent of fibrosis severity. For example, AST >200 IU/L or elevated bilirubin can overestimate fibrosis in ALD, and LSM may decrease after abstinence, reflecting reduced inflammation rather than true fibrosis regression [36]. In viral-related cirrhosis (e.g., hepatitis B or C), LSM also predicts decompensation and HCC, but the relationship is more stable, as inflammation tends to be less variable outside of flares or antiviral therapy. In hepatitis C, LSM values can decrease rapidly after successful antiviral therapy, but this may reflect reduced inflammation rather than fibrosis regression, so pre-treatment LSM is more reliable for staging [47].
Computed tomography (CT) imaging plays a crucial role in evaluating advanced complications of ALD, such as portal hypertension, varices, and HCC, and in evaluating disease severity and prognosis in AH [60,61]. Contrast-enhanced CT scans allow for detailed visualization of hepatic vasculature, portal vein patency, and portosystemic collaterals, facilitating the diagnosis and management of portal hypertension-related complications, including variceal bleeding and ascites [62]. Contrast-enhanced dynamic imaging protocols and multi-phase CTs enable the detection and characterization of hepatic lesions, including HCC, allowing for the early detection of HCC in individuals with ALD, guiding timely intervention and improving patient outcomes. However, cumulative radiation exposure leading to increased cancer risk remains a limitation for repeated use of CT in longitudinal follow-up, and alternative non-ionizing modalities such as MRI may be preferable when serial imaging is required [63,64].
MRI represents the state-of-the-art imaging modalities for comprehensive assessment of ALD, offering high spatial resolution, multi-parametric tissue characterization, and quantitative assessment of liver stiffness and fat content [65]. MRI-based techniques, such as proton density fat fraction (PDFF) mapping, provide voxel-wise measurements of fat content throughout the liver, allowing for precise localization of steatotic lesions and differentiation from other liver pathologies [66,67]. MRI-PDFF is also reliable in obesity and comparable to a liver biopsy. MRI-PDFF plays a central role in non-invasive quantification and monitoring of hepatic steatosis in ALD, with high diagnostic accuracy and prognostic relevance, but does not directly assess inflammation or fibrosis [47,68-70]. However, some evidence suggests that a ≥30% relative reduction in MRI-PDFF correlates with a higher odds of ≥1 stage improvement in fibrosis [71].
Magnetic resonance elastography (MRE) uses external mechanical vibrations to non-invasively assess liver stiffness, providing quantitative measurements of tissue stiffness and fibrosis. MRE has a sensitivity of 96% and specificity of 95.4% for detecting advanced fibrosis in ALD [36]. When combined with MRI-based fat quantification techniques like PDFF mapping, it can provide comprehensive evaluations of both hepatic fibrosis and steatosis in individuals with ALD. This information guides therapeutic decisions and prognosis. However, these findings have primarily been extrapolated from studies on MASLD and require confirmation in patients with ALD [72].
Besides traditional methods, a new group of biomarkers related to genetic polymorphisms, the endocrine system, gut microbiota, and omics technologies has shown promising results (Fig. 2). Although most of these techniques have not been incorporated into routine practice yet, they could provide a more comprehensive assessment of liver fibrosis risk and progression in ALD.
Genetic susceptibility and single nucleotide polymorphisms
Genetic predisposition plays a critical role in determining the individual risk of developing ALD. Polymorphisms in genes encoding alcohol-metabolizing enzymes, such as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), substantially influence rates of alcohol metabolism and subsequent acetaldehyde accumulation, a toxic byproduct implicated in liver injury [73]. Variants in ADH1B and ALDH2, which alter alcohol clearance and acetaldehyde detoxification, respectively, are associated with ALD risk, with certain populations exhibiting heightened susceptibility due to specific allele frequencies [74]. Additionally, genetic variations in genes involved in hepatic lipid metabolism, oxidative stress response, inflammation, and fibrogenesis contribute to individual differences in ALD susceptibility and severity. Polymorphisms in genes encoding cytokines, such as TNF-α and interleukins, modulate the inflammatory milieu within the liver, and can influence disease progression and response to therapy [75].
Genetic modifiers, including single nucleotide polymor phisms (SNPs), copy number variations, and epigenetic modifications, interact with environmental triggers, such as chronic alcohol consumption, to modulate disease trajectory and treatment response [76]. Variants in patatin-like phospholipase domain-containing 3 (PNPLA3 rs738409 C>G), transmembrane 6 superfamily member 2 (TM6SF2 rs58542926 C>T), glucokinase regulator (GCKR rs1260326 T>C), Membrane-Bound O-Acyltransferase Domain-Containing 7 (MBOAT7 rs641738 C>T), and Serpin Family A Member 1 (SERPINA1 rs17580), among other genetic polymorphisms, have been associated with an increased risk of hepatic steatosis, cirrhosis, and HCC across the steatotic liver disease spectrum, including ALD.7 In contrast, a lossof-function variant in the hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) can play a role in protecting from liver disease progression in ALD [77].
The PNPLA3 rs738409 p.I148M variant remains the most impactful common genetic determinant of ALD [78]. This highrisk polymorphism can interact synergistically with alcohol consumption and obesity, carrying an estimated 10-year cirrhosis risk of up to 8% [79]. To capture a broader inherited genetic risk, a weighted polygenic risk score (PRS) or simple genetic risk score (GRS) has been developed, integrating the cumulative effect of multiple genetic variants [80]. For example, a GRS can be calculated by summing the number of risk alleles across selected loci, each weighted by its effect size loci such as TM6SF2, MBOAT7, GCKR, PNPLA3, minus the protective HSD17B13 variant [81,82]. A PRS incorporating 20 SNPs significantly improved risk prediction for alcohol-associated cirrhosis compared to clinical risk factors alone, with those in the highest PRS decile having up to a fourfold increased risk compared to the lowest decile [83]. A GRS based on three specific SNPs (PNPLA3:rs738409, SUGP1-TM6SF2:rs10401969, HSD17B13:rs6834314) combined with diabetes status strongly predicted the development of alcohol-related cirrhosis in heavy drinkers from three large cohorts [84]. In a biopsy-controlled, cross-sectional study of 325 patients with excessive alcohol use, presence of risk alleles in PNPLA3 (rs738409-G) and TM6SF2 (rs58542926-T) was independently associated with more advanced fibrosis [85]. In MASLD, higher PRS or GRS values reflect a greater predisposition to advanced liver disease, faster fibrosis progression, and higher liver stiffness per decade [86]. However, it is necessary to validate these findings in individuals with ALD. Although short-term additive values of PRS are limited over tools like FIB-4, it could be beneficial for long-term outcomes, including the development of HCC and in identifying patients for early intervention [87,88].
In advanced liver disease, genetic polymorphisms in genes encoding components of the renin-angiotensin-aldosterone system have been implicated in the development of portal hypertension and variceal bleeding [89], highlighting the role of genetic modifiers in disease severity and prognosis. Moreover, genetic variants might influence individual responses to pharmacotherapies and lifestyle interventions, necessitating tailored treatment approaches based on genetic profiling. By integrating genetic information into risk stratification algorithms and treatment protocols, the accuracy and effectiveness of ALD management can be improved. Patients with ALDH2*2 and ADH1C*1 showed greater reduction in drinking, while the ADH1B*3 allele was associated with worse outcomes, though it has a limited role in predicting liver fibrosis [90]. On the other hand, CYP2E1 directly encodes an enzyme generating toxic metabolites, modestly increasing risk of progressive ALD, hepatocellular injury, oxidative stress, steatohepatitis, and ultimately fibrosis and cirrhosis [91]. Genetic variants can predict patient response to medications for alcohol use disorder. The μ-opioid receptor gene polymorphism OPRM1 rs1799971 enhances naltrexone efficiency, with Asp40 allele carriers showing greater reductions in alcohol use [92]. Variants in ADK1B, ALDH2, and CYP2E1 exhibit inter-individual differences in alcohol metabolism and drug toxicity, influencing efficacy and tolerability to naltrexone and disulfiram [93]. Recognizing the role of genetic polymorphisms in modulating ALD susceptibility, progression, and therapeutic response highlights the need to include genetic information in patient care.
Liver disease and sex
The interplay between sex-specific biological, behavioral, and sociocultural factors profoundly influences ALD susceptibility, disease progression, clinical presentation, and treatment outcomes. Historically, ALD has been more prevalent and severe among men [94], reflecting differences in alcohol consumption patterns, metabolism, and societal norms, with higher rates of hazardous drinking and alcohol dependence placing them at increased risk for developing ALD [95]. However, evolving societal and economic norms, along with targeted marketing strategies, have led to a narrowing of the gender gap in alcohol consumption patterns, placing women at a heightened risk for ALD [96]. Incidence of ALD in women, particularly young women, is rising [97]. Thus, the landscape of ALD is evolving, with an alarming rise in alcohol consumption and related liver diseases among women observed in recent years [98].
Women are biologically predisposed to accelerated progression of ALD and increased vulnerability to alcohol-related liver injury compared to men. They also experience a greater degree of liver injury than men at lower levels of alcohol consumption [57]. This can be attributed to differences in alcohol metabolism, liver physiology, hormonal fluctuations, body composition, effects on liver function, and immune responses [99]. Estrogen and progesterone, the primary female sex hormones, have both beneficial and harmful effects on the liver, modulating hepatic inflammation, fibrogenesis, and regeneration. Estrogen protects the liver by reducing oxidative stress and inhibiting the activation of hepatic stellate cells. However, hormonal changes during menstruation, pregnancy, and menopause might exacerbate liver injury and promote fibrosis progression in women with ALD, especially in premenopausal women [100,101]. Estrogen receptor α binds to a specific enhancer near PNPLA3, leading to an increase in PNPLA3 mRNA and protein expression in hepatocytes with estradiol and tamoxifen treatment. This regulatory mechanism might contribute to hepatic steatosis observed in patients on tamoxifen in carriers of the PNPLA3 I148M variant [101]. Women with obesity show a higher PNPLA3 expression than men, emphasizing the role of estrogen in liver disease progression [101].
Conversely, androgens such as testosterone have varying effects on hepatic metabolism and inflammation, influencing both the vulnerability to and severity of ALD in men. Testosterone exerts beneficial effects on liver function by reducing fat accumulation, promoting hepatocyte regeneration, and suppressing the production of pro-inflammatory cytokines. An increase in testosterone levels is often observed in the majority of individuals after liver transplantation [102]. However, excessive androgen exposure, as seen in endocrine disorders or anabolic steroid abuse, might exacerbate liver injury and promote the development of AH and alcohol-associated cirrhosis in men [103].
There is a notable discrepancy in liver enzymes with normal ALT levels of 29–33 IU/L in males and 19–25 IU/L in females. Levels above the upper normal limit have been linked to increased liver-related mortality, reinforcing the need for sex-specific thresholds [104]. Similarly, in a crosssectional study of around 400,000 individuals, the upper normal limit for AST in men was found to be 32 IU/L and 26 IU/L in women. Hence, disparities in AST/ALT ratios are also observed between sexes, with women showing higher ratios, which can complicate the interpretation of ALD with a cut-off of >2 [105]. This emphasizes the importance of understanding sex-based differences in alcohol consumption and metabolism, hormonal regulation, and genetic susceptibility in interpreting biomarkers.
Microbiome and endothelial gut barrier
The human gut microbiome is a complex ecosystem made up of trillions of microorganisms that live in the gastrointestinal tract. Its vital role in regulating host metabolism, immune function, and maintaining homeostasis has been well established [106]. Emerging evidence suggests that dysbiosis, characterized by alterations in the composition and function of gut microbes, contributes to the onset and progression of various liver diseases [107,108]. In ALD, both microbiota and their metabolites, such as short-chain fatty acids (SCFAs), ethanol, bile acids, and trimethylamine Noxide (TMAO), modulate hepatic inflammation, oxidative stress, and fibrogenesis [109].
SCFAs, primarily acetate, propionate, and butyrate, exert anti-inflammatory and immunomodulatory effects by engaging with host receptors such as G protein-coupled receptors (GPCRs) and histone deacetylases. They promote regulatory T cell (Tregs) differentiation and inhibit pro-inflammatory cytokine production, mitigating hepatic inflammation and injury in ALD [110]. Conversely, TMAO, a metabolite derived from dietary choline and carnitine, has been implicated in hepatic inflammation and fibrosis through activation of toll-like receptors (TLRs) and inflammasome pathways [111]. Ethanol metabolism by gut bacteria produces acetaldehyde, a toxic intermediate that contributes to hepatic oxidative stress, lipid peroxidation, and pro-inflammatory cytokine release, exacerbating ALD progression and leading to increased hepatocellular injury, disruption of gutliver, axis and progression towards steatosis and fibrosis. Acetaldehyde also disrupts the gut barrier through disassembling intestinal tight junctions [112]. Bile acids, synthesized in the liver and metabolized by gut bacteria, serve as signaling molecules that regulate lipid absorption, glucose metabolism, and immune responses. Dysbiosis alters bile acid composition and signalling, promoting cholestatic injury, hepatocyte apoptosis, and fibrogenesis in ALD [113].
Decreased abundance of Faecalibacterium prausnitzii, Akkermansia muciniphila and Bacteroidetes alongside increases in Proteobacteria and Enterobacteriaceae, correlate with ALD severity and progression [114]. Patients with alcohol use disorder exhibit lower abundance of multiplebutyrate producing families, including Ruminococcaceae, Lachnospiraceae, Butyricicoccaceae and Oscillospiraceae and have a decreased Firmicutes/Bacteroidetes ratio (F/B ratio), which is frequently used as a marker of microbiome balance [115]. As liver injury progresses to fibrosis, gut microbiota alterations progress alongside liver injury, characterized by an expansion of endotoxin-producing bacteria and decline of beneficial autochthonous taxa [116].
Immune-microbiome interactions and hepatic fibrogenesis
The gut-liver axis serves as a dynamic interface for immune-microbiome interactions that shape ALD progression and fibrogenesis. Dysbiosis disrupts intestinal barrier integrity by damaging tight junctions in intestinal epithelial cells thereby increasing gut permeability leading to translocation of microbial products, such as lipopolysaccharides, into the portal circulation, triggering hepatic inflammation and fibrogenesis by activating TLR4 signaling pathways. Activation of hepatic Kupffer cells and stellate cells promotes pro-inflammatory cytokine production, extracellular matrix deposition, and collagen synthesis, driving the development of liver fibrosis in ALD [110,117].
The gut microbiome regulates the balance between proinflammatory and anti-inflammatory immune responses by modulating mucosal immune cell populations, including Tregs, Th17 cells, and innate lymphoid cells. Dysbiosis-induced changes in the composition and function of immune cells disrupt immune tolerance mechanisms, leading to chronic inflammation and tissue damage in ALD [118]. Additionally, microbial-derived metabolites, such as secondary bile acids and SCFAs, regulate the activation and differentiation of immune cells by interacting with host receptors, such as the farnesoid X receptor (FXR) and GPCRs, linking gut dysbiosis and hepatic immune dysregulation in ALD [119]. Chronic alcohol consumption upregulates the expression of hepatic bile acid efflux transporters, including multidrug resistance-associated proteins 3 and 4, organic solute transporter αβ and apical sodium-dependent bile acids (ASBT). Additionally, alcohol inhibits FXR activity in the liver and intestine, promoting bile acid synthesis [120].
Understanding the intricate interplay between microbiome signaling pathways and ALD pathogenesis holds promise for the development of novel therapeutic strategies targeting the gut-liver axis. Preclinical [121] and clinical studies investigating the efficacy of probiotics, antibiotics, and microbial metabolite modulators in ameliorating ALD severity and progression have shown benefits to restore gut microbial homeostasis and mitigate hepatic inflammation and fibrogenesis [122]. Therapeutically, targeting bile acid metabolism and gut-liver axis is a promising strategy. Modulation of bile acid transport with agents like ASBT inhibitors has demonstrated reduced inflammation, attenuation of liver injury, and restoration of bile acid homeostasis [120]. Further, in mice, supplementation with Faecalibacterium prausnitzii and Akkermansia muciniphila improved the gut barrier [123]. Decrease in F/B ratio might be used as a biomarker for intestinal dysbiosis.
Recent evidence demonstrates that specific gut microbial signatures correlate with ALD severity and may predict treatment response. For example, a prospective study in severe alcoholic hepatitis found that lower baseline abundance of Prevotella was associated with poor response to rifaximin, and restoration of certain taxa (e.g., Prevotella, Prevotellaceae) after treatment correlated with clinical improvement, suggesting these taxa may serve as predictive biomarkers for therapy response [124]. Similarly, dysbiosis characterized by increased Veillonella and decreased butyrate-producing bacteria is consistently associated with more advanced disease and worse outcomes [115]. Fecal microbiota transplantation (FMT) and probiotics have shown clinical benefit in pilot studies, with FMT associated with lower mortality in severe alcoholic hepatitis compared to historical controls, supporting the utility of microbiome modulation as both a therapeutic and prognostic tool [125]. However, most studies to date are cross-sectional or smallscale, and causality is not fully established [126]. Machine learning models using microbiome profiles have achieved high diagnostic accuracy for ALD, but prospective validation for predicting progression or treatment response is still emerging [115]. While the microbiome is implicated in ALD pathogenesis and may serve as a biomarker, robust prospective studies are needed to confirm its predictive value in clinical practice.
Endocrine system
Chronic alcohol consumption disrupts endocrine signaling pathways that regulate metabolism, inflammation, and tissue repair. Alcohol-induced alterations in hypothalamicpituitary-adrenal axis function lead to cortisol dysregulation, insulin resistance, and hepatic steatosis in ALD. Elevated cortisol levels, along with altered expression of glucocorticoid-responsive genes (e.g., FKBP5, PER1) have been associated with immune dysregulation and metabolic imbalance in ALD. These components are under investigation as potential biomarkers for disease activity and might aid in identifying patients who are at risk of more severe hepatic injury [127]. Alcohol-mediated suppression of gonadotropin-releasing hormone secretion disrupts reproductive hormone levels, resulting in hypogonadism and impaired sexual function in both men and women with ALD [128].
Alcohol-induced thyroid dysregulation represents another under-recognized yet important component of ALD. Alcohol impairs peripheral conversion of thyroxine (T4) to active triiodothyronine (T3), resulting in subclinical hypothyroidism [129]. Thyroid hormones regulate key metabolic pathways involved in fatty acid oxidation, gluconeogenesis, and mitochondrial biogenesis, suggesting a potential link to hepatic steatosis in ALD [130]. Serum TSH, free T3/T4 levels can serve as accessible biomarkers of thyroid status in ALD, while intrahepatic markers such as type 1 deiodinase (DIO1), type 3 deiodinase (DIO3), and thyroid hormone receptor-β (THR-β) are of growing interest, particularly with THR-β agonists, including resmetirom, as therapeutic agents.
Endocrine biomarkers such as serum cortisol, testosterone, estradiol, TSH, and free T3/T4 in patients with ALD might provide inexpensive, non-invasive tools for risk stratification of patients in both inpatient and outpatient settings. Testosterone and thyroid hormone testing might assist in identifying patients with cirrhosis at risk of sarcopenia, fatigue, and metabolic decompensation [2,131]. Monitoring DIO1/DIO3 expression could enhance diagnostic accuracy for thyroid-mediated liver dysfunction and stratify patients who benefit from THR-β targeted therapies or thyroid hormone supplementation. Incorporating these markers could enable mechanism-based endpoints and dose selection in early-phase ALD trials, though further validation across diverse ALD populations is needed [132].
Transcriptomics
Transcriptomic profiling enables the systematic analysis of gene expression patterns and regulatory networks underlying ALD pathogenesis and progression. High-throughput RNA sequencing and microarray technologies allow for genome-wide characterization of mRNA transcripts, noncoding RNAs (e.g., microRNAs), and alternative splicing events associated with ALD-associated phenotypes [133]. Dysregulated gene expression signatures associated with hepatic inflammation, fibrogenesis, and carcinogenesis in ALD. Changes in certain microRNAs found in the bloodstream, such as miR-122 and miR-155, have shown potential as non-invasive biomarkers for liver injury and fibrosis in ALD [134]. By deciphering the transcriptional landscape of ALD, researchers can identify biomarkers predictive of disease progression, treatment response, and clinical outcomes, paving the way for precision medicine approaches in ALD management.
Lipidomics
Alcohol disrupts hepatic lipid synthesis and transport, and patients with ALD have been shown to have specific lipid profiles [135]. Sphingomyelins have a considerable inverse correlation to fibrosis stage and are helpful as negative predictors for complications of fibrosis [136]. Sphingomyelins are lowest in patients with ALD without metabolic risk factors. This is attributed to alcohol-induced upregulation of acid sphingomyelinase activity in response to pro-inflammatory cytokines, resulting in enhanced hydrolysis of sphingomyelins into ceramides. The accumulation of ceramides induces lipotoxic stress, promotes mitochondrial dysfunction, and impairs insulin signaling via inhibition of AMPK and Akt pathways, mechanisms that are implicated in both ALD and MASLD [137-139]. Animal models have demonstrated that hepatic ceramide accumulation exacerbates steatosis, while liver-specific ceramidase overexpression alleviates alcohol-induced steatosis and improves insulin sensitivity [140]. Exploring the sphingolipid-mediated axis might have a beneficial role in the diagnosis and management of ALD.
Proteomics
Proteomic profiling enables the comprehensive analysis of protein expression, post-translational modifications, and protein-protein interactions implicated in ALD pathogenesis and progression. Mass spectrometry-based proteomic techniques facilitate the identification and quantification of proteins dysregulated in ALD, offering insights into disease mechanisms and biomarker discovery. Proteomic studies have identified protein biomarkers associated with hepatic steatosis, inflammation, fibrosis, and carcinogenesis in ALD, providing diagnostic and prognostic information beyond traditional markers. These biomarkers include cytokeratins, heat shock proteins, C-reactive protein, interleukins, matrix metalloproteinases, and tissue inhibitors of metalloproteinases [141]. Specific plasma proteins (such as beta-2 microglobulin, insulin-like growth factor acid-labile subunit, insulin-like growth factor binding protein 3, thrombin, and hepatocyte growth factor α) have demonstrated high diagnostic accuracy for severe ALD and cirrhosis, often outperforming traditional fibrosis indices [142-145]. These signatures can also help subtype patients with overlapping metabolic and alcohol-related liver disease for personalized risk assessment [144].
Metabolomics
Metabolomic profiling enables the systematic analysis of small molecule metabolites and metabolic pathways dysregulated in ALD, providing insights into disease pathophysiology and identifying biomarkers reflective of disease status and progression. Nuclear magnetic resonance spectroscopy and mass spectrometry-based metabolomic platforms enable comprehensive characterization of the metabolome in ALD. Metabolomic studies have identified metabolic signatures associated with hepatic steatosis, oxidative stress, mitochondrial dysfunction, and dysregulated lipid metabolism in ALD [146]. These findings offer valuable insights into the mechanisms of disease and potential targets for therapy. Promising biomarkers for the progression of ALD and response to treatment include acylcarnitines, phosphatidylcholines, and bile acids [56]. Elevated glycineand taurine-conjugated bile acids (e.g., glycocholic acid, taurocholic acid) and altered amino acids (e.g., glutamic acid, L-phenylalanine, L-tyrosine) are strongly associated with disease progression and correlate with established clinical markers such as liver enzymes, bilirubin, and fibrosis scores [147-149].
Epigenomics
Epigenomic profiling enables the characterization of epigenetic modifications, such as DNA methylation, histone modifications, and chromatin remodeling, that are implicated in ALD pathogenesis and progression. Genome-wide DNA methylation profiling and chromatin immunoprecipitation sequencing enable comprehensive analysis of epigenetic alterations associated with ALD phenotypes. Epigenomic studies have identified aberrant DNA methylation patterns, histone modifications, and chromatin accessibility profiles associated with hepatic inflammation, fibrogenesis, and carcinogenesis in ALD [150]. Hypomethylation of long interspersed nucleotide elements and the ALDH gene is more pronounced in patients with alcohol use disorder and cirrhosis compared to those without cirrhosis, suggesting that DNA methylation status could serve as a biomarker for advanced disease and risk stratification [151,152].
Most omics, microbiome, and endocrine markers in ALD remain exploratory, with significant limitations in their clinical applicability. Limited sample sizes and cohort heterogeneity are common in omics-based studies, which often use small, single-center cohorts that may not represent the broader ALD population. This restricts the generalizability and external validity of findings [147,153]. Confounding by comorbidities such as obesity, viral hepatitis, and metabolic syndrome can obscure the specific contributions of alcohol to liver disease and biomarker profiles, making it difficult to attribute observed changes solely to ALD. Assay standardization is a major barrier: many omics platforms use semiquantitative or untargeted approaches, resulting in high analytical variability and poor reproducibility across studies and populations [154]. This lack of standardization impedes regulatory approval and clinical translation, as candidate biomarkers often fail to demonstrate consistent performance in external validation cohorts. Microbiome and metabolomic markers show promise for understanding ALD pathogenesis and severity, but most findings are preliminary. For example, specific microbial taxa and metabolites have been associated with ALD progression, but these associations require validation in larger, diverse cohorts and with standardized methodologies [154]. No omics-based or microbiome-derived biomarkers have yet reached routine clinical use in ALD. Clinicians should interpret these data with caution given the methodological caveats.
Recent advances in biomarker research provide opportunities to improve early detection and risk stratification through accessible, non-invasive tools (Table 1). Integrating markers of fibrosis, metabolic dysfunction, genetic predisposition and microbiome alterations into clinical care may allow for earlier identification of at-risk individuals and more tailored management strategies. The use of validated biomarkers in routine practice has the potential to improve outcomes and reduce the burden of ALD complications. While serum-based biomarkers hold great promise for improving ALD diagnosis, risk stratification, and therapeutic monitoring, several challenges need to be addressed to fully integrate them into clinical practice. Standardizing assay protocols, validating diverse patient populations, and integrating biomarkers into clinical algorithms are essential for translating biomarker discoveries into actionable insights for patient care.
Longitudinal studies assessing prognostic value and cost-effectiveness of serum biomarkers in real-world clinical settings are also necessary. These studies provide evidence-based guidelines and facilitate the widespread adoption of serum biomarkers in ALD management. Collaborative efforts involving multidisciplinary teams of researchers, clinicians, and industry partners are crucial for overcoming existing barriers and advancing biomarker in-tegration in ALD care. Initiatives, such as multi-ethnic consortia and data-sharing platforms, are needed for aggregating diverse genetic datasets and improving statistical power to detect genetic associations with ALD phenotypes.
Omics technologies and translational research offer incredible opportunities to revolutionize the diagnosis and treatment of ALD. This will substantially influence the design of clinical trials and patient care. As these biomarkers continue to be validated and integrated into clinical practice, there is immense potential for early detection, improved outcomes, and an enhanced quality of life for individuals affected by ALD.
In conclusion, advances in biomarker discovery are transforming the approach to ALD, enabling earlier diagnosis, mechanistic disease classification, and precision therapy. Integration of serum fibrosis indices, advanced imaging, genetic and epigenetic profiling, microbiome and endocrine signatures, and multi-omics platforms may substitute one-dimensional staging with a systems-level view of disease pathology. Such integration can identify highrisk individuals earlier, guide adaptive biomarker-based trials, and accelerate targeted therapy development, shifting the field from late-stage intervention to proactive, personalized prevention and treatment.

Authors’ contributions

Authors confirm contribution to the article as follows: Kaanthi Rama: Investigation, Writing - Original Draft, Writing - Review and Editing; Vinay Jahagirdar: Investigation, Writing - Original Draft, Writing - Review and Editing; Francisco Idalsoaga: Writing - Review and Editing. Hanna Blaney: Writing - Review and Editing; S. Fisher Rhoads: Writing – Review and Editing; Luis Antonio Díaz: Conceptualization, Writing - Review and Editing; Marco Arrese: Investigation, Writing - Review and Editing; Juan Pablo Arab: Investigation, Writing - Review and Editing.

Conflicts of Interest

The authors have no conflicts to disclose.

Figure 1.
Clinical implications of novel biomarkers in alcohol-associated liver disease. Framework illustrating how emerging biomarkers can be integrated into patient care. Applications include early risk stratification through genetic polymorphisms and polygenic risk scores; the use of sex-specific diagnostic thresholds to account for differences in alcohol metabolism and hormonal modulation; noninvasive therapeutic monitoring with the fibrosis-4 index (FIB-4), enhanced liver fibrosis (ELF) test, and type III procollagen neo-epitope (Pro-C3); targeted therapy development based on gut–liver axis biomarkers and endocrine markers; precision trial design through omics-based phenotyping; and the adjunctive use of biomarkers when access to advanced imaging is limited. F/B ratio, Firmicutes/Bacteroidetes ratio; FXR, farnesoid X receptor; HCC, hepatocellular carcinoma; MRI, magnetic resonance imaging; PDFF, proton density fat fraction; SCFA, short-chain fatty acid; THR-β, thyroid hormone receptor-β; TMAO, trimethylamine-N-oxide.
cmh-2025-0921f1.jpg
Figure 2.
Pathophysiologic domains and representative biomarkers in alcohol-associated liver disease. Schematic overview of biologic systems contributing to disease pathogenesis and their associated biomarkers. Summary of genetic variants, gut microbiome alterations, endocrine dysregulation, and omics-based signatures relevant to ALD. Includes traditional markers (AST, ALT, GGT, ALP, platelets) and novel fibrosis or cell-death markers (ELF, FIB-4, Pro-C3, CK-18). ALD, alcohol-associated liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK-18, cytokeratin-18; ELF, enhanced liver fibrosis; FIB-4, fibrosis-4; GGT, gamma-glutamyl transferase; Pro-C3, procollagen type III N-terminal propeptide (type III collagen formation marker).
cmh-2025-0921f2.jpg
Table 1.
Summary of noninvasive biomarkers in alcohol-associated liver disease
Table 1.
S. No. Biomarker Type Target/function Strengths Limitations
1 AST/ALT Traditional Hepatocellular injury Widely available Low specificity
2 GGT Traditional Oxidative stress, enzyme induction, cholestasis Sensitive to alcohol Low specificity
3 MCV Traditional Macrocytosis, direct toxicity, B12 deficiency Cost effective Low sensitivity and specificity
4 FIB-4 score Novel Fibrosis estimation Non-invasive, widely used Limited utility in intermediate ranges, may miss early fibrosis
5 ELF test (HA, PIIINP, TIMP-1) Novel fibrosis marker ECM remodelling Non-invasive Cost, limited availability
6 Pro-C3 Fibrosis marker Collagen synthesis Strongly correlates with fibrosis Low specificity due to Extrahepatic expression
7 Cytokeratin-18 Cell death marker Apoptosis/necrosis Prognostic in alcohol-associated hepatitis Low specificity
8 PRS (PRF), PNPLA3, TM6SF2, HSD17B13 Genetic Steatosis, fibrosis risk Stratifies long-term fibrosis/HCC risk Not routinely available, resource limitation
9 miR-122, miR-155 Transcriptomics Inflammation, injury Emerging studies Not yet standardized for clinical practice
10 Cortisol, testosterone, TSH, DIO1/DIO3 Endocrine HPA disruption, Thyroid dysregulation Widely available, prognostic for sarcopenia and metabolic decline Influenced by non-hepatic conditions
11 SCFAs, TMAO, LCAs Gut microbiome Dysbiosis Reflects gut-liver axis No gold standard testing
12 Specific gut microbiota, F/B ratio Gut microbiome Dysbiosis Reflects gut-liver axis, can be used as a supplement No gold standard testing

ALT, alanine aminotransferase; AST, aspartate aminotransferase; DIO1/DIO3, iodothyronine deiodinase types 1 and 3; ECM, extracellular matrix; ELF, enhanced liver fibrosis; F/B ratio, Firmicutes/Bacteroidetes ratio; FIB-4, fibrosis-4 index; GGT, gamma-glutamyl transferase; HA, hyaluronic acid; HCC, hepatocellular carcinoma; HPA, hypothalamic-pituitary-adrenal (axis); HSD17B13, hydroxysteroid 17-beta dehydrogenase 13; LCAs, lithocholic acids (secondary bile acids); MCV, mean corpuscular volume; miR, microRNA; PIIINP, procollagen type III N-terminal peptide; PNPLA3, patatin-like phospholipase domain-containing protein 3; PRS (PRF), polygenic risk score; Pro-C3, procollagen type III N-terminal propeptide (type III collagen formation marker); SCFAs, short-chain fatty acids; TIMP-1, tissue inhibitor of metalloproteinases-1; TM6SF2, transmembrane 6 superfamily member 2; TMAO, trimethylamine N-oxide; TSH, thyroid-stimulating hormone.

ADH

alcohol dehydrogenase

AH

alcohol-associated hepatitis

ALD

alcohol-associated liver disease

ALDH

aldehyde dehydrogenase

ALT

alanine aminotransferase

ASBT

apical sodium-dependent bile acids

AST

aspartate aminotransferase

AUROC

area under the receiver operating characteristics

CAP

controlled attenuation parameter

ChIP-seq

chromatin immunoprecipitation sequencing

CNV

copy number variation

CT

computed tomography

DIO1

type 1 deiodinase

DIO3

type 3 deiodinase

ELF

enhanced liver fibrosis

ERα

estrogen receptor α

F/B ratio

Firmicutes/Bacteroidetes ratio

FIB-4

fibrosis-4

FXR

farnesoid X receptor

GGT

gammaglutamyl transferase

GnRH

gonadotropin-releasing hormone

GRS

genetic risk score

GPCR

G protein-coupled receptor

HCC

hepatocellular carcinoma

HDAC

histone deacetylase

HPA

hypothalamic-pituitary-adrenal

MASLD

metabolic dysfunction-associated steatotic liver disease

MCV

mean corpuscular volume

MRE

magnetic resonance elastography

MRI

magnetic resonance imaging

MRP3/4

multidrug resistance-associated proteins 3 and 4

OSTαβ

organic solute transporter αβ

PDFF

proton density fat fraction

PIIINP

procollagen III N-terminal peptide

PRS

polygenic risk score

RAAS

renin-angiotensin-aldosterone system

SCFA

short-chain fatty acid

SNP

single nucleotide polymorphism

TGF-β

transforming growth factor-β

THR-β

thyroid hormone receptor-β

TIMP-1

tissue inhibitor of metalloproteinase-1

TLR

toll-like receptor

TMAO

trimethylamine-N-oxide

TNF-α

tumor necrosis factor-alpha

Treg

regulatory T cell

VCTE

vibration-controlled transient elastography
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Novel biomarkers for alcohol-associated liver disease and their implications across clinical settings
Clin Mol Hepatol. 2026;32(2):443-463.   Published online November 25, 2025
DOI: https://doi.org/10.3350/cmh.2025.0921
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Novel biomarkers for alcohol-associated liver disease and their implications across clinical settings
Clin Mol Hepatol. 2026;32(2):443-463.   Published online November 25, 2025
DOI: https://doi.org/10.3350/cmh.2025.0921
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Novel biomarkers for alcohol-associated liver disease and their implications across clinical settings
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Figure 1. Clinical implications of novel biomarkers in alcohol-associated liver disease. Framework illustrating how emerging biomarkers can be integrated into patient care. Applications include early risk stratification through genetic polymorphisms and polygenic risk scores; the use of sex-specific diagnostic thresholds to account for differences in alcohol metabolism and hormonal modulation; noninvasive therapeutic monitoring with the fibrosis-4 index (FIB-4), enhanced liver fibrosis (ELF) test, and type III procollagen neo-epitope (Pro-C3); targeted therapy development based on gut–liver axis biomarkers and endocrine markers; precision trial design through omics-based phenotyping; and the adjunctive use of biomarkers when access to advanced imaging is limited. F/B ratio, Firmicutes/Bacteroidetes ratio; FXR, farnesoid X receptor; HCC, hepatocellular carcinoma; MRI, magnetic resonance imaging; PDFF, proton density fat fraction; SCFA, short-chain fatty acid; THR-β, thyroid hormone receptor-β; TMAO, trimethylamine-N-oxide.
Figure 2. Pathophysiologic domains and representative biomarkers in alcohol-associated liver disease. Schematic overview of biologic systems contributing to disease pathogenesis and their associated biomarkers. Summary of genetic variants, gut microbiome alterations, endocrine dysregulation, and omics-based signatures relevant to ALD. Includes traditional markers (AST, ALT, GGT, ALP, platelets) and novel fibrosis or cell-death markers (ELF, FIB-4, Pro-C3, CK-18). ALD, alcohol-associated liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK-18, cytokeratin-18; ELF, enhanced liver fibrosis; FIB-4, fibrosis-4; GGT, gamma-glutamyl transferase; Pro-C3, procollagen type III N-terminal propeptide (type III collagen formation marker).

Figure 1.

Figure 2.

Novel biomarkers for alcohol-associated liver disease and their implications across clinical settings
S. No. Biomarker Type Target/function Strengths Limitations
1 AST/ALT Traditional Hepatocellular injury Widely available Low specificity
2 GGT Traditional Oxidative stress, enzyme induction, cholestasis Sensitive to alcohol Low specificity
3 MCV Traditional Macrocytosis, direct toxicity, B12 deficiency Cost effective Low sensitivity and specificity
4 FIB-4 score Novel Fibrosis estimation Non-invasive, widely used Limited utility in intermediate ranges, may miss early fibrosis
5 ELF test (HA, PIIINP, TIMP-1) Novel fibrosis marker ECM remodelling Non-invasive Cost, limited availability
6 Pro-C3 Fibrosis marker Collagen synthesis Strongly correlates with fibrosis Low specificity due to Extrahepatic expression
7 Cytokeratin-18 Cell death marker Apoptosis/necrosis Prognostic in alcohol-associated hepatitis Low specificity
8 PRS (PRF), PNPLA3, TM6SF2, HSD17B13 Genetic Steatosis, fibrosis risk Stratifies long-term fibrosis/HCC risk Not routinely available, resource limitation
9 miR-122, miR-155 Transcriptomics Inflammation, injury Emerging studies Not yet standardized for clinical practice
10 Cortisol, testosterone, TSH, DIO1/DIO3 Endocrine HPA disruption, Thyroid dysregulation Widely available, prognostic for sarcopenia and metabolic decline Influenced by non-hepatic conditions
11 SCFAs, TMAO, LCAs Gut microbiome Dysbiosis Reflects gut-liver axis No gold standard testing
12 Specific gut microbiota, F/B ratio Gut microbiome Dysbiosis Reflects gut-liver axis, can be used as a supplement No gold standard testing
Table 1. Summary of noninvasive biomarkers in alcohol-associated liver disease

ALT, alanine aminotransferase; AST, aspartate aminotransferase; DIO1/DIO3, iodothyronine deiodinase types 1 and 3; ECM, extracellular matrix; ELF, enhanced liver fibrosis; F/B ratio, Firmicutes/Bacteroidetes ratio; FIB-4, fibrosis-4 index; GGT, gamma-glutamyl transferase; HA, hyaluronic acid; HCC, hepatocellular carcinoma; HPA, hypothalamic-pituitary-adrenal (axis); HSD17B13, hydroxysteroid 17-beta dehydrogenase 13; LCAs, lithocholic acids (secondary bile acids); MCV, mean corpuscular volume; miR, microRNA; PIIINP, procollagen type III N-terminal peptide; PNPLA3, patatin-like phospholipase domain-containing protein 3; PRS (PRF), polygenic risk score; Pro-C3, procollagen type III N-terminal propeptide (type III collagen formation marker); SCFAs, short-chain fatty acids; TIMP-1, tissue inhibitor of metalloproteinases-1; TM6SF2, transmembrane 6 superfamily member 2; TMAO, trimethylamine N-oxide; TSH, thyroid-stimulating hormone.

Table 1.

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