Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic progressive hepatopathy in children, and the identification of non-invasive biomarkers is urgently needed. Growth differentiation factor 15 (GDF15) was associated with MASLD in adults. In this study, we investigated the circulating and hepatic levels of GDF15 and their association with liver damage in pediatric MASLD and in a murine model. This observational study included 158 children with biopsy-proven MASLD. Patients with MASLD were categorized into two groups based on steatohepatitis (MASH) presence and evaluated for GDF15 circulating levels, while GDF15 hepatic levels were assessed only in a subset of patients. Children with MASLD exhibited higher levels of circulating GDF15 compared to the controls. Moreover, the MASH subgroup had significantly higher values of GDF15 compared to the Not-MASH subgroup. The GDF15 levels in the MASH subgroup showed a positive correlation with fibrosis. Finally, the hepatic expression of the GDF15 gene correlated with GDF15 circulating levels and with the hepatic expression of the COL1A1 and COL3A1 genes in 15 children with MASLD. In conclusion, our study demonstrated that GDF15 levels are associated with liver damage, reinforcing the potential role of GDF15 as a biomarker for MASLD-related fibrosis in children.

The increasing prevalence of metabolic dysfunction-associated fatty liver disease (MASLD) in children requires robust, non-invasive biomarkers to enable accurate disease staging and risk stratification. Elevated serum levels of homocysteine (Hcy) have emerged as potential risk factors for cardiometabolic disease in adults, including MASLD. In this observational retrospective study, we investigated the role of serum Hcy levels as a potential biomarker for disease severity and liver fibrosis in a pediatric cohort of 182 children with MASLD. In 89 patients, liver biopsy allowed the classification into metabolic dysfunction-associated steatohepatitis (MASH). Associations between Hcy, metabolic parameters, fibrosis scores, and histological features were examined, and the diagnostic performance of Hcy for liver fibrosis was evaluated using ROC analysis. Multivariate analyses identified elevated Hcy levels as independently associated with HOMA-IR (β = 0.55; p = 0.049), TG/HDL ratio (β = 3.23; p = 0.002), and liver fibrosis (β = 2.59; p = 0.04). Hcy showed a predictive accuracy of 81% for fibrosis. However, the combined diagnostic models of Hcy with non-invasive fibrotic scores (i.e., APRI and FIB-4) or TG/HDL ratio showed only a modest accuracy (AUC = 0.62–0.69). In conclusion, our data suggest that Hcy is associated with fibrosis and cardiometabolic risk. However, these results should be interpreted as exploratory and do not establish homocysteine as a diagnostic biomarker.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an increasingly prevalent condition in both adults and children. Dysregulated telomere maintenance has been proposed as a mechanism underlying disease progression, although pediatric evidence remains limited and controversial. This study aimed to investigate the relationship between telomere length (TL) and hepato-metabolic features in children with MASLD. A total of 212 pediatric patients with biopsy-proven MASLD and 40 controls were enrolled. Telomere length in leukocytes (LTL) and liver tissue (HTL) was measured using quantitative polymerase chain reaction, and telomerase reverse transcriptase (TERT) mRNA and protein expression were also evaluated. Associations between TL and clinical, metabolic, and perinatal variables were analyzed. Children with MASLD showed significantly shorter LTL and HTL compared to controls. Shorter LTL was observed in more advanced steatohepatitis (MASH) and was associated with fibrosis severity. TERT expression was reduced in patients. LTL was also associated with perinatal factors, including preterm birth and low birthweight. Multivariable analysis identified MASH, fibrosis, and small-for-gestational-age status as independently associated with shorter LTL. In conclusion, LTL is associated with disease severity in pediatric MASLD, particularly fibrosis. These findings support a potential role of telomere dynamics in disease progression, although causal relationships require confirmation in longitudinal studies.

Short-chain fatty acids (SCFAs) are key biomarkers of gut microbiota and intestinal health, yet their analysis is challenging due to high volatility, hydrophilicity, and the complexity of the faecal matrix. This study introduces a novel, green, and fully automated workflow for monitoring SCFAs in faecal water samples. It combines microwave-assisted derivatisation, vacuum-assisted cooling multiple solid-phase microextraction (Vac-HS-cooling-MSPME), and fast gas chromatography–mass spectrometry (GC-MS). On-sample derivatisation with 2,3,4,5,6-pentafluorobenzyl bromide was optimised via design of experiments, reducing reaction time from 3 h to just 2 min at 100 °C while preserving high yields and selectivity. Integration of Vac-HS-cooling-MSPME with a fast GC column (5 m × 0.1 mm ID × 0.4 µm) increased sample throughput from two to approximately six samples per hour, with total chromatographic times of 5.24 min. MSPME specifically improved sensitivity for low-response analytes such as pentafluorobenzyl formate. The method demonstrated excellent linearity (R² > 0.998), low limits of detection (0.002–0.09 mg/L), good intra- and inter-day precision, accuracies exceeding 85%, and consistent recovery for each analyte (86–90%). The AGREE greenness score improved from 0.65 to 0.68, confirming alignment with green analytical chemistry principles. This system offers a high-throughput, sensitive, and eco-friendly tool for SCFA quantification, confirmed by the application on real paediatric faecal water samples.

The increasing prevalence of metabolic dysfunction-associated fatty liver disease (MASLD) in children requires robust, non-invasive biomarkers to enable accurate disease staging and risk stratification. Elevated serum levels of homocysteine (Hcy) have emerged as potential risk factors of cardiometabolic disease including MASLD in adults. In this observational retrospective study, we investigated the role of serum Hcy levels as a potential biomarker for disease severity and liver fibrosis in a pediatric cohort of 182 children with MASLD. Liver biopsies in 89 patients, allowed the classification into metabolic dysfunction-associated steatohepatitis (MASH). Associations between Hcy, metabolic parameters, fibrosis scores, and histological features were examined, and the Hcy diagnostic performance for liver fibrosis evaluated through ROC analysis. Multivariate analyses identified elevated Hcy levels as independently associated with HOMA-IR (β=0.55; p=0.049), TG/HDL ratio (β=3.23; p=0.002) and liver fibrosis (β=2.59; p=0.04). HCy showed excellent predictive accuracy for fibrosis (AUC= 0.0809) superior to that of other fibrosis scores. Finally, combined diagnostic models of Hcy with APRI, FIB-4, or TG/HDL ratio showed only modest accuracy, (AUC=0.62-0.69). Our data demonstrated that serum Hcy is a strong independent predictor of fibrosis in pediatric MASLD patients, supporting its role as a non-invasive biomarker to identify patients at risk of severe liver complications.