Non-invasive tests for non-alcoholic fatty liver disease (NAFLD) are needed for assessing disease stage, prognosis and treatment efficacy. Extracellular matrix biomarkers, such as PRO-C3, are useful as biomarkers of advanced liver fibrosis. However, non-invasive biomarkers of early-stage NAFLD, characterized by pericellular fibrosis, are lacking. Here, we measured serological biomarkers of type IV and VIII collagens reflecting the remodeling of the pericellular basement membrane to explore the effect of bariatric surgery on pericellular fibrosis in patients with early NAFLD. Seventy patients with severe obesity underwent bariatric surgery. The cohort consisted of 61 % females who had a mean age of 44. Patients had a median NAFLD activity score of 3 and mild-to-moderate fibrosis F0 (3 %), F1 (86 %), and F2 (11 %). Blood samples were taken at baseline, three, six and 12 months after surgery. At 12 months, 40 patients had a follow-up liver biopsy. The biomarkers PRO-C3, PRO-C4, C4M, and PRO-C8 were measured using indirect competitive ELISAs. Twelve months after surgery patients had significantly lower levels of ALT, GGT, HbA1c, fasting glucose, and CRP. The pericellular fibrosis biomarkers, C4M, PRO-C4, and PRO-C8 decreased by 24 %, 18 % and 44 %, respectively (p < 0.0001), while the interstitial matrix fibrosis marker PRO-C3 remained unchanged. Furthermore, baseline C4M was associated with histologically assessed hepatocyte ballooning and lobular inflammation in patients with (p = 0.032) and without (p = 0.032) steatosis, respectively. Biomarkers of pericellular fibrosis decrease in early-stage NAFLD after patients undergo bariatric surgery and potentially reflect an improvement in liver histology.

Heat shock protein (HSP)47 is a collagen-specific chaperone, essential for the correct formation of fibrillar procollagens. Collagen accumulation in the extracellular matrix (ECM) is a hallmark of fibrogenesis. The expression of HSP47 is proportional to the rate of collagen formation. Thus, HSP47 is a potential drug target for fibrotic diseases. We hypothesized that a C-terminal fragment of HSP47 (HSP47-C) could be quantified serologically and related to liver fibrosis stage. For this, a novel competitive enzyme-linked immunosorbent assay (ELISA) was developed. An ELISA employing a monoclonal antibody targeting HSP47-C was developed and technically validated. The assay was evaluated in serum from a cross-sectional biopsy-controlled study of 281 patients with alcohol-related liver disease (ALD) and 50 gender, age and BMI matched healthy controls (HC). All liver biopsies from ALD patients were scored by one pathologist according to fibrosis stage (F0-4). The HSP47-C assay was technically robust and specific for the target sequence. HSP47-C was 39% higher in ALD patients (median 17.7 ng/mL, IQR 12.4-24.0 ng/mL) compared to HC (median 12.7 ng/mL, IQR 9.4-15.7 ng/mL, p < 0.0001). In addition, HSP47-C was elevated in patients with severe fibrosis (F3-4, median 22.8 ng/mL, IQR 17.5-33.3 ng/mL) compared to none-to-moderate fibrosis (F0-2, median 16.5 ng/mL, IQR 11.8-22.5 ng/mL) with an AUROC of 0.72 (p < 0.0001). HSP47-C also correlated with other liver disease parameters, albumin, bilirubin and aspartate transaminase. We developed a competitive ELISA for serological detection of HSP47-C. The study supports HSP47 as a potential marker of liver fibrosis in ALD.

Background: Identification of progressive liver disease necessitates the finding of novel non-invasive methods to identify and monitor patients in need of early intervention. Investigating patients with early-liver injury may help identify unique biomarkers. Early-liver injury is characterized by remodeling of the hepatocyte basement membrane (BM) of the extracellular matrix. Thus, we quantified biomarkers targeting two distinct neo-epitopes of the major BM collagen, type IV collagen (PRO-C4 and C4M), in patients spanning the non-alcoholic fatty liver disease (NAFLD) spectrum. Methods: We evaluated PRO-C4 and C4M in a cross-sectional study with 97 patients with NAFLD confirmed on histology. Serological levels of PRO-C4 and C4M were quantified using validated competitive enzyme-linked immunosorbent assays (ELISA). Using the fatty liver inhibition of progression (FLIP) algorithm, we stratified patients into two groups: non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). Biomarker levels were investigated in the two groups in patients stratified by the NAFLD activity score (NAS). In both groups, biomarker measurements were analyzed in relation to histological scorings of steatosis, inflammation, ballooning, and fibrosis. Results: Patients had a body mass index (BMI) of 30.9 ± 5.6 kg/m², age of 53 ± 13 years and a NAS range of 1–8. Upon stratification by FLIP, the NASH patients had higher platelets, ALT, and AST levels than the NAFL group. Both PRO-C4 (p = 0.0125) and C4M (p = 0.003) increased with increasing NAS solely within the NAFL group; however, a large variability was present in the NASH group. Furthermore, both markers were significantly associated with lobular inflammation (p = 0.020 and p = 0.048) and steatosis (p = 0.004 and p = 0.015) in patients with NAFL. Conclusions: This study found that type IV collagen turnover increased with the increase in NAS in patients with NAFL; however, this was not the case in patients with NASH. These findings support the assessments of the BM turnover using biomarkers in patients with early-disease development. These biomarkers may be used to track specific processes involved in the early pathobiology of NAFL.

Background: Early diagnosis of metabolic dysfunction–associated steatotic liver disease (MASLD) fibrosis may prevent liver-related morbidity and mortality. We investigated the effectiveness of multiple 2-step approaches for detecting at-risk advanced MASLD fibrosis, defined as a hierarchical clinical score comprising liver histology and VCTE depending on availability, in primary and hospital outpatient care. Methods: Patients at risk for MASLD were recruited from general practices and regional and tertiary internal medicine clinics, after excluding other liver diseases. Simultaneous measurement of non-invasive tests (NITs), including fibrosis-4 score (FIB4), metabolic dysfunction–associated fibrosis-5 score (MAF5), NAFLD fibrosis score (NFS), enhanced liver fibrosis (ELF) test, procollagen type III N-terminal propeptide (PRO-C3), procollagen type VI N-terminal propeptide (PRO-C6), and vibration-controlled transient elastography (VCTE), allowed testing of 2-step referral pathways. Referral patterns were compared with regular care between 2016 and 2024 using a predefined reference standard. Results: Among 595 participants, 9.4% were at-risk of advanced MASLD fibrosis. A care pathway consisting of FIB4 (≥1.30) followed by VCTE yielded the greatest improvement, increasing the correct referral rate 5.0-fold (95% CI: 2.4–10.1) compared with regular care. However, care pathways incorporating MAF5 (≥1.0) or NFS (≥−1.455) followed by VCTE demonstrated markedly higher sensitivity at comparable increases in detection (both >4.5-fold). Among other second-step NITs, PRO-C3 outperformed ELF, PRO-C6, and ADAPT, resulting in a 2.2-fold (95% CI: 1.1–4.4) increase in correct referrals when used after FIB4 (≥1.30). Conclusion: Two-step care pathways substantially improve the detection of at-risk advanced MASLD fibrosis across care settings. Pathways using FIB4, MAF5, or NFS followed by VCTE offer the best performance overall, while PRO-C3 is the most effective alternative blood-based second-step test. Export