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Background/purpose of the Study: There is a need to find a standardized and low-risk diagnostic tool that can non-invasively detect non-alcoholic steatohepatitis (NASH). Surface enhanced Raman spectroscopy (SERS), which is a technique combining Raman spectroscopy (RS) with nanotechnology, has recently received considerable attention due to its potential for improving medical diagnostics. We aimed to investigate combining SERS and neural network approaches, using a liver biopsy dataset to develop and validate a new diagnostic model for non-invasively identifying NASH., Methods: Silver nanoparticles as the SERS-active nanostructures were mixed with blood serum to enhance the Raman scattering signals. The spectral data set was used to train the NASH classification model by a neural network primarily consisting of a fully connected residual module., Results: Data on 261 Chinese individuals with biopsy-proven NAFLD were included and a prediction model for NASH was built based on SERS spectra and neural network approaches. The model yielded an AUROC of 0.83 (95% confidence interval [CI] 0.70-0.92) in the validation set, which was better than AUROCs of both serum CK-18-M30 levels (AUROC 0.63, 95% CI 0.48-0.76, p = 0.044) and the HAIR score (AUROC 0.65, 95% CI 0.51-0.77, p = 0.040). Subgroup analyses showed that the model performed well in different patient subgroups., Conclusions: Fully connected neural network-based serum SERS analysis is a rapid and practical tool for the non-invasive identification of NASH. The online calculator website for the estimated risk of NASH is freely available to healthcare providers and researchers ( http://www.pan-chess.cn/calculator/RAMAN_score )., (© 2022. Asian Pacific Association for the Study of the Liver.)

Background and Aims: With metabolic dysfunction-associated fatty liver disease (MAFLD) incidence and prevalence sharply increasing globally, there is an urgent need for non-invasive diagnostic tests to accurately screen high-risk MAFLD patients for liver inflammation and fibrosis. We aimed to develop a novel sequential algorithm based on N-terminal propeptide of type 3 collagen (PRO-C3) for disease risk stratification in patients with MAFLD., Methods: A derivation and independent validation cohort of 327 and 142 patients with biopsy-confirmed MAFLD were studied. We compared the diagnostic performances of various non-invasive scores in different disease states, and a novel sequential algorithm was constructed by combining the best performing non-invasive scores., Results: For patients with high-risk progressive steatohepatitis (i.e., steatohepatitis + NAFLD activity score ≥ 4 + F ≥ 2), the AUROC of FAST score was 0.801 (95% confidence interval (CI): 0.739-0.863), and the negative predictive value (NPV) was 0.951. For advanced fibrosis (≥ F3) and cirrhosis (F4), the AUROCs of ADAPT and Agile 4 were 0.879 (95%CI 0.825-0.933) and 0.943 (95%CI 0.892-0.994), and the NPV were 0.972 and 0.992. Sequential algorithm of ADAPT + Agile 4 combination was better than other combinations for risk stratification of patients with severe fibrosis (AUROC = 0.88), with similar results in the validation cohort. Meanwhile, in all subgroup analyses (stratifying by sex, age, diabetes, NAS, BMI and ALT), ADAPT + Agile 4 had a good diagnostic performance., Conclusions: The new sequential algorithm reliably identifies liver inflammation and fibrosis in MAFLD, making it easier to exclude low-risk patients and recommending high-risk MAFLD patients for clinical trials and emerging pharmacotherapies., (© 2022. Asian Pacific Association for the Study of the Liver.)

Background/purpose of the Study: Although low skeletal muscle mass is associated with non-alcoholic fatty liver disease (NAFLD), it is currently uncertain whether there are associations between weight-adjusted appendicular skeletal muscle (ASM%), severity of histological features of NAFLD, and the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism. Our aim was to test for a possible influence of the PNPLA3 rs738409 variant on the association between ASM% and severity of NAFLD histological features., Methods: We enrolled 401 Chinese male with biopsy-proven NAFLD. Using a bioelectrical-impedance body composition analyzer (BIA, Inbody 720, Japan Inc., Tokyo), we calculated the ASM% as the percentage of total appendicular skeletal muscle mass (ASM, kg)/total body mass (kg) × 100., Results: Compared to those with high ASM%, patients with low ASM% (≤ 30.6, i.e., the median value of distribution of the whole sample) had a greater severity of individual histological features of NAFLD. These patients also had a higher risk of severe steatosis and non-alcoholic steatohepatitis (NASH) (adjusted-odds ratio [OR] 2.34, 95% CI 1.39-3.93, and adjusted-OR 2.22, 95% CI 1.30-3.77) even after adjusting for age, body mass index, diabetes, and serum creatinine levels. Carriage of the G allele of PNPLA3 rs738409 plus low ASM% was associated with a higher risk of severe steatosis and presence of liver fibrosis (OR 3.02, 95% CI 1.46-6.26, p = 0.003 and OR 2.18, 95% CI 1.03-4.60, p = 0.041 respectively), and there was a non-significant but borderline increased risk of NASH (OR 2.00, 95% CI 0.98-4.06, p = 0.056)., Conclusions: Low ASM% and the presence of a G allele within PNPLA3 rs738409 is associated with more severe histological features of NAFLD., (© 2022. Asian Pacific Association for the Study of the Liver.)

Background and Aim: Copper is an essential trace element involved in oxidative stress reactions and energy metabolism. While nonalcoholic fatty liver disease (NAFLD) is closely related to metabolic dysfunction, the role of copper in the development of simple steatosis (NAFL) and nonalcoholic steatohepatitis (NASH) is still unclear. We aimed to compare serum copper levels between patients with simple steatosis and those with NASH., Methods and Results: We studied 102 patients with biopsy-proven NASH (cases) and 102 NAFL controls, who were matched for age, sex, and residential city. Multivariable conditional logistic analysis was performed to explore associations between serum copper levels and the presence of NASH. Serum copper levels were significantly lower in patients with NASH than in those with matched NAFL controls (15.53 ± 2.41 μmol/l vs. 16.34 ± 3.23 μmol/l; P = 0.029). This intergroup difference in serum copper levels was more pronounced in men than in women. The per unit, per SD, and per doubling of serum copper levels were associated, respectively, with an approximately 20, 40, and 90% decrease in risk of having NASH, even after adjustment for potential confounding factors., Conclusion: Lower serum copper concentrations are significantly associated with higher prevalence of NASH among biopsied-proven NAFLD patients, particularly in men., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background and Aims: Non-alcoholic fatty liver disease (NAFLD) is a usual chronic liver disease and lacks non-invasive biomarkers for the clinical diagnosis and prognosis. Extracellular vesicles (EVs), a group of heterogeneous small membrane-bound vesicles, carry proteins and nucleic acids as promising biomarkers for clinical applications, but it has not been well explored on their lipid compositions related to NAFLD studies. Here, we investigate the lipid molecular function of urinary EVs and their potential as biomarkers for non-alcoholic steatohepatitis (NASH) detection., Methods: This work includes 43 patients with non-alcoholic fatty liver (NAFL) and 40 patients with NASH. The EVs of urine were isolated and purified using the EXODUS method. The EV lipidomics was performed by LC-MS/MS. We then systematically compare the EV lipidomic profiles of NAFL and NASH patients and reveal the lipid signatures of NASH with the assistance of machine learning., Results: By lipidomic profiling of urinary EVs, we identify 422 lipids mainly including sterol lipids, fatty acyl lipids, glycerides, glycerophospholipids, and sphingolipids. Via the machine learning and random forest modeling, we obtain a biomarker panel composed of 4 lipid molecules including FFA (18:0), LPC (22:6/0:0), FFA (18:1), and PI (16:0/18:1), that can distinguish NASH with an AUC of 92.3%. These lipid molecules are closely associated with the occurrence and development of NASH., Conclusion: The lack of non-invasive means for diagnosing NASH causes increasing morbidity. We investigate the NAFLD biomarkers from the insights of urinary EVs, and systematically compare the EV lipidomic profiles of NAFL and NASH, which holds the promise to expand the current knowledge of disease pathogenesis and evaluate their role as non-invasive biomarkers for NASH diagnosis and progression., (© 2022. The Author(s).)

Sarcopenic obesity is regarded as a risk factor for the progression and development of non-alcoholic fatty liver disease (NAFLD). Since male sex is a risk factor for NAFLD and skeletal muscle mass markedly varies between the sexes, we examined whether sex influences the association between appendicular skeletal muscle mass to visceral fat area ratio (SVR), that is, an index of skeletal muscle mass combined with abdominal obesity, and the histological severity of NAFLD. The SVR was measured by bioelectrical impedance in a cohort of 613 (M/F = 443/170) Chinese middle-aged individuals with biopsy-proven NAFLD. Multivariable logistic regression and subgroup analyses were used to test the association between SVR and the severity of NAFLD (i.e. non-alcoholic steatohepatitis (NASH) or NASH with the presence of any stage of liver fibrosis). NASH was identified by a NAFLD activity score ≥5, with a minimum score of 1 for each of its categories. The presence of fibrosis was classified as having a histological stage ≥1. The SVR was inversely associated with NASH in men (adjusted OR 0·62; 95 % CI 0·42, 0·92, P = 0·017 for NASH, adjusted OR 0·65; 95 % CI 0·43, 0·99, P = 0·043 for NASH with the presence of fibrosis), but not in women (1·47 (95 % CI 0·76, 2·83), P = 0·25 for NASH, and 1·45 (95 % CI 0·74, 2·83), P = 0·28 for NASH with the presence of fibrosis). There was a significant interaction for sex and SVR ( P interaction = 0·017 for NASH and P interaction = 0·033 for NASH with the presence of fibrosis). Our findings show that lower skeletal muscle mass combined with abdominal obesity is strongly associated with the presence of NASH only in men.

Background and Aims: Zinc is an essential trace element that plays an important role in maintaining health, and affecting gene expression, signal transduction and regulation of apoptosis. It is uncertain whether serum zinc levels are altered in patients with metabolic dysfunction-associated fatty liver disease (MAFLD). We aimed to investigate the association between serum zinc levels and the severity of hepatic necro-inflammation (HN) in patients with MAFLD., Methods and Results: Liver disease severity was graded histologically using the NAFLD activity score. HN was defined as the sum of ballooning and lobular inflammation. We used a smooth function regression model to analyze the relationship between serum zinc levels and HN. A total of 561 (76.5% men) patients with biopsy-confirmed MAFLD were enrolled. They had a mean age of 41.3 years, and a mean serum zinc level of 17.0 ± 4.1 μmol/L. Compared to those with mild hepatic necro-inflammation (MHN, grades 0-2; n = 286), patients with severe hepatic necro-inflammation (SHN, grades 3-5; n = 275) had lower serum zinc concentrations (16.3 ± 4.2 vs. 17.6 ± 4.0 μmol/L; p < 0.001). However, a threshold saturation effect analysis showed that there was an inflection in serum zinc levels at 24 μmol/L. After adjustment for potential confounders, serum zinc levels <24 μmol/L were inversely associated with SHN (adjusted-odds ratio 0.88, 95%CI 0.83-0.93; p < 0.001), whereas serum zinc levels >24 μmol/L were positively associated with SHN (adjusted-odds ratio 1.42, 95%CI: 1.03-1.97; p = 0.035)., Conclusions: There is a J-shaped relationship between serum zinc levels and the severity of hepatic necro-inflammation in patients with biopsy-proven MAFLD., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Background and Aims: There remains a need to develop a non-invasive, accurate and easy-to-use tool to identify patients with non-alcoholic steatohepatitis (NASH). Successful clinical and preclinical applications demonstrate the ability of quantitative ultrasound (QUS) techniques to improve medical diagnostics. We aimed to develop and validate a diagnostic tool, based on QUS analysis, for identifying NASH., Methods: A total of 259 Chinese individuals with biopsy-proven non-alcoholic fatty liver disease (NAFLD) were enrolled in the study. The histological spectrum of NAFLD was classified according to the NASH clinical research network scoring system. Radiofrequency (RF) data, raw data of iLivTouch, was acquired for further QUS analysis. The least absolute shrinkage and selection operator (LASSO) method was used to select the most useful predictive features., Results: Eighteen candidate RF parameters were reduced to two significant parameters by shrinking the regression coefficients with the LASSO method. We built a novel QUS score based on these two parameters, and this QUS score showed good discriminatory capacity and calibration for identifying NASH both in the training set (area under the ROC curve [AUROC]: 0.798, 95% confidence interval [CI] 0.731-0.865; Hosmer-Lemeshow test, P = .755) and in the validation set (AUROC: 0.816, 95% CI 0.725-0.906; Hosmer-Lemeshow test, P = .397). Subgroup analysis showed that the QUS score performed well in different subgroups., Conclusions: The QUS score, which was developed from QUS, provides a novel, non-invasive and practical way for identifying NASH., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Aim: Cytochrome P450 2E1 (CYP2E1) plays a role in lipid metabolism, and by increasing hepatic oxidative stress and inflammation, the upregulation of CYP2E1 is involved in development of nonalcoholic steatohepatitis (NASH). We aimed to explore the relationship between CYP2E1-333A>T (rs2070673) and the histological severity of nonalcoholic fatty liver disease (NAFLD)., Methods: We studied 438 patients with biopsy-proven NAFLD. NASH was defined as NAFLD Activity Score ≥ 5 with existence of steatosis, ballooning, and lobular inflammation. CYP2E1-333A>T (rs2070673) was genotyped by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Serum cytokines related to inflammation were measured by the Bio-plex 200 system to investigate possible mediating factors involved in the process., Results: The TA genotype of rs2070673 had a higher prevalence of moderate/severe lobular inflammation (27.6% vs 20.3% vs 13.3%, P < 0.01) and NASH (55.7% vs 42.4% vs 40.5%, P < 0.01) compared with the AA and TT genotypes, respectively. In multivariable regression modeling, the heterozygote state TA was associated with moderate/severe lobular inflammation (adjusted odds ratio: 2.31, 95% confidence interval 1.41-3.78, P < 0.01) or NASH (adjusted odds ratio: 1.82, 95% confidence interval 1.22-2.69, P < 0.01), independently of age, sex, common metabolic risk factors, and presence of liver fibrosis. Compared with no-NASH, NASH patients had significantly higher levels of serum interleukin-1 receptor antagonist, interleukin-18, and interferon-inducible protein-10 (IP-10), whereas only IP-10 was increased with the rs2070673 TA variant (P = 0.01). Mediation analysis showed that IP-10 was responsible for ~60% of the association between the rs2070672 and NASH., Conclusions: The TA allele of rs2070673 is strongly associated with lobular inflammation and NASH, and this effect appears to be largely mediated by serum IP-10 levels., (© 2021 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Rationale: Since non-invasive tests for prediction of liver fibrosis have a poor diagnostic performance for detecting low levels of fibrosis, it is important to explore the diagnostic capabilities of other non-invasive tests to diagnose low levels of fibrosis. We aimed to evaluate the performance of radiomics based on 18 F-fluorodeoxyglucose ( 18 F-FDG) positron emission tomography (PET) in predicting any liver fibrosis in individuals with biopsy-proven metabolic dysfunction-associated fatty liver disease (MAFLD). Methods: A total of 22 adults with biopsy-confirmed MAFLD, who underwent 18 F-FDG PET/CT, were enrolled in this study. Sixty radiomics features were extracted from whole liver region of interest in 18 F-FDG PET images. Subsequently, the minimum redundancy maximum relevance (mRMR) method was performed and a subset of two features mostly related to the output classes and low redundancy between them were selected according to an event per variable of 5. Logistic regression, Support Vector Machine, Naive Bayes, 5-Nearest Neighbor and linear discriminant analysis models were built based on selected features. The predictive performances were assessed by the receiver operator characteristic (ROC) curve analysis. Results: The mean (SD) age of the subjects was 38.5 (10.4) years and 17 subjects were men. 12 subjects had histological evidence of any liver fibrosis. The coarseness of neighborhood grey-level difference matrix (NGLDM) and long-run emphasis (LRE) of grey-level run length matrix (GLRLM) were selected to predict fibrosis. The logistic regression model performed best with an AUROC of 0.817 [95% confidence intervals, 0.595-0.947] for prediction of liver fibrosis. Conclusion: These preliminary data suggest that 18 F-FDG PET radiomics may have clinical utility in assessing early liver fibrosis in MAFLD., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)