Introduction

Volatile organic compounds (VOCs) derived from gut dietary non-starch polysaccharides fermentation become detectability in biological fluids due to the impairment of the gut permeability (“leaky-gut syndrome”) occurring in several pathological contexts such as insulin-resistance (IR)-related chronic disorders. The urinary VOCs profile, configuring a specific disease biosignature, has been proposed as a useful non-invasive diagnostic tool. In Metabolic dysfunction Associated Fatty Liver Disease (MAFLD) affected patients, IR and Diabetes Mellitus Type 2 (T2DM) crucially promote the progression, according to the disease's natural history, from simple steatosis (SS) to steatohepatitis (NASH) and advanced fibrosis. However, despite the crucial role exerted by IR, MAFLD patients may be characterized by IR stigmata without T2DM as well as T2DM subjects may not be affected by hepatic steatosis.

Aims & Methods

In the present study, the differences in the urinary VOCs profile of T2DM, MAFLD, and MAFLD/T2DM patients were investigated and compared to define the MAFLD/T2DM group’s characteristics and to find out new features occurring when these two conditions coexist, otherwise not detectable in each pathological state. We consecutively enrolled T2DM, MAFLD, and MAFLD/T2DM patients. For each participant, anthropometrical, biochemical (including insulin levels assessment), and clinical data were recorded. A sample of morning urine after overnight fasting was also collected. The samples were immediately frozen and stored until the analysis. Headspace solid-phase microextraction (HS-SPME), coupled with gas chromatography-mass spectrometry (GC-MS), was used to identify VOCs in urine samples. Subsequently, VOCs previously isolated were profiled via a mass selective detector. VOCs classification was performed by matching mass spectra within the available database libraries and comparing their retention indices (RI) with literature data.

Results

13 T2DM, 13 MAFLD, and 13 MAFLD/T2DM patients were enrolled. All patients presenting hyperinsulinemia (insulin >24 microU/ml). VOCs profiles obtained by using acid pH extraction did not show statistical differences among the analyzed groups, while VOCs acquired in alkaline conditions revealed differences among the three groups. The multivariate and univariate data analysis showed 21 metabolites levels statistically significantly different in the urine of MAFLD/T2DM compared to T2DM (p=0.021) and MAFLD (p=0.031) subjects. Among these metabolites, 1-pentanol (A16), Pyridine (N5), Dimethyl sulfone (S3), and 1-piperidine carboxaldehyde (N19) were higher in MAFLD/T2DM subjects compared to T2DM patients; 3-heptanone (K10) and 3-octane (K13) were lower in MAFLD/T2DM subjects than MAFLD patients; 2-hexanone (K6) presented lower levels in MAFLD/T2DM patients in comparison to T2DM subjects.

Conclusion

VOCs’profiling could non-invasively discriminate insulin-resistant T2DM, MAFLD, and MAFLD/T2DM patients. Moreover, the highlighted different VOCs' signatures suggest specific pathogenetic mechanisms in MAFLD and T2DM to explore and remark MAFLD and T2DM coexistence as not the mere sum of two diseases.

References

1. Arasaradnam, R.P., McFarlane, M., Daulton, E., Westenbrink, E., O'Connell, N., Wurie, S., Nwokolo, C.U., Bardhan, K.D., Savage, R.S., & Covington, J.A. (2015). Non-invasive distinction of non-alcoholic fatty liver disease using urinary volatile organic compound analysis: early results. Journal of Gastrointestinal and Liver Diseases, 24(2):197-201. https://doi.org/10.15403/jgld.2014.1121.242.ury.
2. Bannaga, A.S., Tyagi, H., Daulton, E., Covington, J.A., & Arasaradnam, R.P. (2021). Exploratory study using urinary Volatile Organic Compounds for the detection of Hepatocellular Carcinoma. Molecules, 22;26(9):2447. https://doi.org/10.3390/molecules26092447.
3. Chen, X., Liu, W., Wang, L., Lin, D., Nie, L., He, K., Guo, Z., Zhu, F., Feng, W., Liu, W., Yuan, J., Yang, X., Spencer, P., & Liu, J. (2020). Diabetes mellitus is associated with elevated urinary pyrrole markers of γ-diketones known to cause axonal neuropathy. BMJ Open Diabetes Research & Care, 8(1):e001575. https://doi.org/10.1136/bmjdrc-2020-001575.
4. Cozzolino, R., De Giulio, B., Marena, P., Martignetti,A., Günther, K., Lauria, F., Russo, P., Stocchero, M., & Siani, A. (2017). Urinary volatile organic compounds in overweight compared to normal-weight children: results from the Italian I.Family cohort. Scientific Reports 7, 15636. https://doi.org/10.1038/s41598-017-15957-7.

Introduction

Phase Angle (PA) represents a useful bioimpedance analysis-assessable marker of sarcopenia, accurately predicting liver-related hospitalizations in advanced chronic liver disease (ACLD), correlating with C-reactive protein levels. Clinically significant portal hypertension (CSPH) and systemic inflammation constitute the main hepatic decompensation drivers. A relevant proportion of Metabolic dysfunction-associated steatotic liver disease (MASLD) patients present sarcopenia (configuring the paradox of “sarcopenic obesity”), as well as compensated ACLD (cACLD)-MASLD individuals more rapidly progress to decompensation (dACLD) rather than other etiologies. However, the clinical applications of PA have never been specifically investigated in the setting of MASLD.

Aims & Methods

Considering the background, in MASLD, we primarily aimed to evaluate the trend of PA values across the different liver disease progression stages, investigating the relationship with the hepatic decompensation drivers; in dACLD-MASLD, we aimed to estimate the accuracy of the novel PA/C-reactive protein (CRP) index (PAC-I) in predicting 3-month further liver-related events (LREs)-determining hospitalizations.
In this observational study, between December 2023 and January 2025, MASLD patients were consecutively enrolled and stratified according to current LSM and eventual previous LREs in < F3 fibrosis (AF) (n.50), cACLD (n.50), and dACLD (n.50) patients. At baseline, anthropometrical, biochemical, nutritional, and clinical data [including the endoscopic assessment of CSPH and the relative severity (F1, F2, F3) of esophageal varices (OEVs)] were collected. Bioimpedance analysis was adopted to assess body composition parameters. Sarcopenia was diagnosed according to the European Working Group on Sarcopenia (EGWSOP2) criteria. MASLD-dACLD patients were followed up, and further acute decompensation (AD)-LREs-determining hospitalizations [severe ascites, severe hepatic encephalopathy (HE), OEV bleeding, and any acute bacterial infections] in the subsequent 3 months were recorded.

Results

In MASLD patients, PA (°) values inversely correlated with LSM [R: -0.711; p<0.0001], progressively decreasing according to the worsening of liver disease stages, emerging as statistically significantly lower in dACLD compared with cACLD individuals (p<0.0001). In dACLD patients, PA inversely correlated with CRP levels [R: -0.945; p:0.001]. Focusing on CSPH, PA values were significantly lower in > F2-OEV-affected individuals (p<0.0001). Multivariate analysis revealed CSPH (aHR 1.221, p<0.0001), CRP (aHR: 1.112, p:0.002), and PA (aHR: 1.178, p:0.003) as the variables significantly associated with 3-month further LREs’ occurrence in dACLD patients.
ROCy analysis revealed an elevated accuracy and superiority compared to PA alone (AUC PA:0.717; AUC PAC-I: 0.893; p:0.003) of PAC-I in predicting 3-month further LREs’ occurrence, identifying PAC-I < 2.35 as the best cut-off. Consistently, dACLD patients showing baseline PAC-I < 2.35 presented a higher risk [HR: 2.287, p: 0.032] of further LREs.

Conclusion

In MASLD, PA correlates with the main driver of progression, and the PAC-I represents a useful noninvasive tool in predicting short-term liver-related hospitalizations in dACLD patients.

References

1. Espirito Santo Silva DD, Waitzberg DL, Passos de Jesus R, Oliveira LPM, Torrinhas RS, Belarmino G. Phase angle as a marker for sarcopenia in cirrhosis. Clin Nutr ESPEN. 2019 Aug;32:56-60. doi: 10.1016/j.clnesp.2019.05.003. Epub 2019 May 31. PMID: 31221291.
2. Saueressig C, Glasenapp JH, Luft VC, Alves FD, Ferreira PK, Hammes TO, Dall'Alba V. Phase Angle Is an Independent Predictor of 6-Month Mortality in Patients With Decompensated Cirrhosis: A Prospective Cohort Study. Nutr Clin Pract. 2020 Dec;35(6):1061-1069. doi: 10.1002/ncp.10584. Epub 2020 Oct 15. PMID: 33058222.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver illness in Western countries. Bisphenol A (BPA) is a well-known endocrine disrupter, widely produced worldwide primarily as a monomer for polycarbonate plastics and plenty cumulated in the human body. Its exposure is epidemiologically related to type 2 diabetes mellitus and cardiovascular and liver diseases. Trained Immunity (TI) represents a novel concept of immunological memory that promotes a response of innate immune cells, triggered by a second antigenic stimulus, and determines a long-term reversible pro-inflammatory phenotype. Although further studies are needed to clarify the role of BPA in the alteration of immune mechanisms, current evidence suggests its implication in the immunometabolic switch and in the remodeling of epigenetic marks which could result in a different immune reactivity.

Aims & Methods

We aimed to explore whether BPA affects TI reactivity in NASH patients due to a possible hyperelicitation of pro-inflammatory pathways.
Serum samples of healthy individuals (n.25) and histologically proven NASH patients (n. 10) were subjected to liquid-liquid extraction of bisphenol A with methanol (1:1, v/v), then to solid-phase extraction cartridge for clean-up and concentration. The analysis of sample extracts was carried out by the HPLC system coupled to a triple quadrupole mass spectrometer. In parallel, for each subject, a peripheral venous blood sample was collected (20 mL). Consequently, monocytes were isolated through a density gradient centrifugation and then stimulated with BPA for 24 h (1 nM, 10 nM, 20 nM) and lipopolysaccharide (LPS) (10 ng/mL) for 24 h (at day 0 and day 6 respectively). In order to assess the pro-inflammatory (tumor necrosis factor TNF-α, interleukin IL-6) and anti-inflammatory response (IL-10) ELISA assays were performed.

Results

The mean values of BPA quantification in serum samples of healthy individuals were 0.1398 ± 0.0495 ng/mL in comparison to NASH patients 7.79 ± 2.34 (P < 0.0001).
The induction of TI mediated by BPA (1 nM, 10, nM, and 20 nM) emerged higher in NASH patients compared to healthy individuals (p<0.05). The hyperelicitation of TI mechanisms in monocytes has been expressed by the high levels of pro-inflammatory cytokines (TNF-α and IL-6) in NASH patients compared to the healthy subjects, in each concentration chosen for the stimulation. Additionally, our data also showed an increased concentration of IL-10 in NASH patients, probably due to a compensative phenomenon for the excessive inflammatory process.

Conclusion

Considering the emerging role of BPA as a strong stimulator of innate immune training in healthy subjects and NASH patients, it is conceivable to address a key role of BPA in the induction of TI as a priming antigen and in the establishment of a pro-inflammatory phenotype potentially involved in the pathogenesis of the disease.

References

• Netea M.G. et al. Trained immunity: A program of innate immune memory in health and disease.Science 2016. 352(6284):aaf1098.
• Dallio, M. et al. Role of bisphenol A as environmental factor in the promotion of non-alcoholic fatty liver disease: in vitro and clinical study. Alimentary Pharmacology Therapheutics 2017.
• Dallio M. et al. Immunity as cornerstone of Non-Alcoholic Fatty Liver Disease: The Contribution of Oxidative Stress in the Disease Progression. International Journal of Molecular Sciences 2021. 22(1):436.

Introduction

Metabolic Dysfunction–Associated Steatotic Liver Disease (MASLD) is currently the leading cause of chronic liver disease worldwide, affecting more than 65% of patients with Type 2 Diabetes Mellitus (T2DM). The aim of our study was to determine the prevalence of MASLD among T2DM patients, with or without coexisting significant fibrosis, to assess its association with diabetic complications, and to identify the factors linked to MASLD in our population.

Aims & Methods

We conducted a retrospective, descriptive, and analytical study including T2DM patients admitted to the Endocrinology Department in 2019. MASLD diagnosis was based on the presence of hepatic steatosis detected by abdominal ultrasound or a Hepatic Steatosis Index (HSI) >36. Fibrosis was assessed using the Fibrosis-4 (FIB-4) index (with a threshold of ≥1.3 or <1.3). Chronic Kidney Disease (CKD) associated with T2DM was defined by an estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m² and/or dipstick-positive proteinuria (>1+). Diabetic Peripheral Neuropathic Pain (DPNP) was assessed using the Douleur Neuropathique 4 (DN4) questionnaire, and Diabetic Retinopathy was diagnosed via dilated eye examination.

Results

Among the 202 patients included, the median age was 57 years (±12 years) with a marked female predominance (sex ratio = 2.6). Regarding comorbidities, 58.4% of patients had hypertension and 34.2% had dyslipidemia. The median Body Mass Index (BMI) was 27 kg/m² (±5.62), with overweight or obesity present in 61.4% of the cohort. The median duration of diabetes was 10 years (±7.8 years). Impaired renal function (eGFR <60 ml/min/1.73 m²) was found in 9.9% of patients, and proteinuria in 6.9%. Diabetic neuropathy (DN4 score ≥4) was present in 7.9% of patients, while diabetic retinopathy was diagnosed in 37.6% of cases.
The prevalence of MASLD in our cohort was 73.3%. The median FIB-4 score was 0.91 (±0.63), with 25.7% of patients presenting a FIB-4 >1.3. Statistical analysis identified several factors significantly associated with the presence of MASLD: female sex (p=0.004), dyslipidemia (p=0.004), BMI (p<0.001), alanine aminotransferase (ALT) levels (p<0.001), low-density lipoprotein cholesterol (LDL-C) levels (p=0.006), and triglyceride levels (p=0.013). In particular, higher BMI was strongly associated with MASLD (Odds Ratio [OR] = 1.970; 95% Confidence Interval [CI]: 1.580–2.460; p<0.001), as were elevated ALT levels (OR = 1.115; 95% CI: 1.053–1.18; p<0.001).
However, after adjustment, LDL-C levels (p=0.163), triglycerides (p=0.402), dyslipidemia (p=0.329), and sex (p=0.360) were no longer significantly associated with MASLD. Finally, a significantly higher proportion of CKD was observed in patients with MASLD (22.3%) compared to those without MASLD (9.2%, p=0.036), although this association was not maintained in the subgroup with advanced fibrosis (FIB-4 >1.3).

Conclusion

Our study confirmed the high prevalence of MASLD among patients with T2DM. Early screening is therefore essential, particularly in female patients and in those presenting with chronic kidney disease or other associated cardiometabolic risk factors such as obesity and dyslipidemia.

References

Younossi ZM, Golabi P, Price JK, Owrangi S, Gundu-Rao N, Satchi R, Paik JM. The Global Epidemiology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis Among Patients With Type 2 Diabetes. Clin Gastroenterol Hepatol. 2024 Oct;22(10):1999-2010.e8. doi: 10.1016/j.cgh.2024.03.006. Epub 2024 Mar 21. PMID: 38521116.

Introduction

A plethora of findings suggests the correlation between Metabolic-(Dysfunction)Associated Fatty Liver Disease (MAFLD) and long-term incorrect lifestyle behavior. The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV2) pandemic lockdown represented a unique event forcing the entire population to suddenly change their lifestyle, favoring a drastic reduction in physical activity and an improper diet, with consequences on metabolic homeostasis never experienced before. In this study, we aimed to investigate the effects of the SARS-CoV-2 confinement and related lifestyle changes on MAFLD evolution.

Aims & Methods

A cohort of 222 MAFLD patients was followed two years before, two years during, and one year after the pandemic. At four time points [January 2018: T0; January 2020: T1; January 2022: T2; January 2023: T3], anthropometrical, biochemical, nutritional, bioelectrical impedance analysis (BIA) data, non-invasive tools for disease staging and hepatocellular carcinoma (HCC) occurrence were collected.

Results

At T2 an increase in fats and carbohydrate intake consistently with impaired weekly physical activity was demonstrated, associated with an increase in body mass index and waist-to-hip ratio (p<0.0001 for all), as well as a worsening in levels of total cholesterol, low/high-density lipoprotein, triglycerides, glucose, insulin, homeostatic model assessment for insulin resistance and transaminases (insulin: p=0.0003, glucose: p=0.0007; p<0.0001 for the others). Non-invasive tools assessments revealed a NAFLD fibrosis score, Liver Stiffness Measurement (LSM), and Controlled Attenuation Parameter impairment at T2 (p<0.0001 all). In comparison with T0 and T1, the BIA evidenced at T2 an increase in Fat Mass (FM) and a reduction of Free Fat Mass (FFM), Body Cell Mass (BCM), and Extracellular Mass (ECM). During the confinement period, the overall HCC and Milan-out staged HCC occurrence revealed HR:2.521, p=0.01, and HR:13.78, p=0.0009 respectively. Contrariwise to the modifications of the biochemical parameters, a significant association between body composition parameters modification during the study evaluations with HCC overall and HCC Milan-out criteria occurrence was highlighted (ΔFFM pre- vs during pandemic, OR: 0.704; ΔFM pre- vs during-pandemic, OR: 1.531; p <0.0001).
Receiving operator curve (ROC) analysis identified respectively -0.92 Kg (AUC:0.86) and -1.90 Kg (AUC:0.90) for FFM and +1.35 Kg (AUC:0.87) and +1.51 Kg (AUC:0.94) for FM as the best cut-off (BCO) variations (pre-pandemic vs pandemic) in the prediction of HCC overall and HCC Milan-out staged occurrence during the confinement. Time-to-time analysis revealed a higher incidence of HCC overall occurrence along the pandemic period in MAFLD patients presenting the following body modification composition “combined pattern” (BMCC) [BCO FFM decrease + BCO FM increase] in comparison to individuals presenting only one of two variations (p:0.025). At T3, compared to the pandemic period, simultaneously with improvement in physical activity as well as a decrease in fats and carbohydrate intake, the insulin levels, CAP, and FM were significantly decreased (p<0.001). Relevantly, a higher incidence of HCC overall occurrence even after the pandemic period in BMCC-MAFLD patients (p:0.048) was observed.

Conclusion

The confinement-related lifestyle changes impacted MAFLD evolution via body composition modifications, negatively influencing the HCC occurrence even after the pandemic period and the restoration of pre-existing life habits, as we are still observing the consequences of the confinement-induced "damages".

References

1. Taheri E, Bostick RM, Hatami B, et al. Dietary and Lifestyle Inflammation Scores Are Inversely Associated with Metabolic-Associated Fatty Liver Disease among Iranian Adults: A Nested Case-Control Study. J Nutr 2022;152:559-567.
2. Dallio M, Romeo M, Gravina AG, et al. Nutrigenomics and Nutrigenetics in Metabolic- (Dysfunction) Associated Fatty Liver Disease: Novel Insights and Future Perspectives. Nutrients 2021;13.
3. Ariya M, Koohpayeh F, Ghaemi A, et al. Assessment of the association between body composition and risk of non-alcoholic fatty liver. PLoS One 2021;16:e0249223.
4. Fujii H, Nakamura N, Fukumoto S, et al. Lifestyle changes during the coronavirus disease 2019 pandemic impact metabolic dysfunction-associated fatty liver disease. Liver Int 2022;42:995-1004.

Introduction

Malnutrition, sarcopenia, and frailty constitute the three vertices of a nutrition-status-determined physio-pathological triangle constantly featuring and dramatically burdening the prognosis of advanced chronic liver disease (ACLD) patients.
Dysgeusia is defined by a distortion of the sense of taste whose prevalence, relationship with the progression of the disease, and the direct contribution to malnutrition (and consequently, sarcopenia-frailty) in ACLD have never been systematically explored.

Aims & Methods

This observational study aimed to explore the prevalence of dysgeusia in a cohort of ACLD patients and evaluate the impact of this disorder on appetite levels and nutritional status (malnutrition, sarcopenia, and frailty), as well as to prospectively estimate the repercussions of dysgeusia-induced impaired nutritional status on liver-related events (LREs) occurrence. For this purpose, 40 controls, 50 chronic-liver disease-affected individuals staging initial-mild fibrosis (<F3), and 150 ACLD patients of various etiologies were enrolled. Anthropometrical, biochemical, clinical, and nutritional [European Prospective Investigation into Cancer and Nutrition (EPIC) questionnaire] data were collected. The opportunely modified Chemotherapy-induced Taste Alteration Scale (CITAS) questionnaire was used to assess the dysgeusia. Child-Pugh (CP) and Liver Frailty Index (LFI) calculations, Liver Stiffness Measurement (LSM), and Bioelectrical Impedance Analysis (BIA) were performed. Skeletal Muscle Mass (SMM) index (SMMI) (SMM/height²) < 7 kg/m² (male) and < 5.7 kg/m² (female) defined sarcopenia. ACLD patients were 3 years followed up semiannually and LREs (including ascites, hepatic encephalopathy, and bleeding) were collected.

Results

The prevalence of dysgeusia was significantly higher in ACLD (68%) in comparison to <F3 (8%) individuals and controls (5%) and in decompensated ACLD (dACLD) than compensated (cACLD) (54% vs 6%) patients (all p<0.0001). In line, CITAS-dysgeusia severity positively correlated with CP (R:0.81), as well as with the levels of appetite impairment (R:0.93) (both p<0.0001). Logistic regression analysis, appropriately weighted for factors notoriously impacting food intake in ACLD patients (low-sodic diet, drugs/supplements taken, physical activity frequency, occurrence of encephalopathy and ascites episodes in the previous 2 weeks), revealed the level of impairment of the appetite as a factor significantly associated with dysgeusia [HR: 2.12; p:0.02]. Compared to no-dysgeusia-affected dACLD (p:0.03), dysgeusia-affected dACLD patients (dys-dACLD) showed a decreased daily protein intake, reduced Free Fat Mass % (p<0.0001), and SMMI (p<0.0001: p:0.04), in contrast with increased LFI (p<0.0001). Consistently, dys-dACLD patients showed a higher prevalence (59% vs 39%) of sarcopenia (p:0.0002). The Kaplan-Meier Log-Rank Test analysis on LREs occurrence showed HR:3.53 [95% C.I: 1.58-4.92] for dys-dACLD individuals.

Conclusion

The prevalence of dysgeusia, increasing in parallel to the worsening of liver disease, promoted sarcopenia-frailty pictures, negatively influencing ACLD patients' outcomes.

References

1. Jafari A, Alaee A, Ghods K. The etiologies and considerations of dysgeusia: A review of literature. Journal of oral biosciences. 2021;63(4):319-26.
2. Sieber CC. Malnutrition and sarcopenia. Aging clinical and experimental research. 2019;31(6):793-8.
3. Tandon P, Montano-Loza AJ, Lai JC, Dasarathy S, Merli M. Sarcopenia and frailty in decompensated cirrhosis. Journal of hepatology. 2021;75 Suppl 1(Suppl 1):S147-s62.