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

Primary Biliary Cholangitis (PBC) represents a chronic inflammatory cholestatic autoimmune disease and a prominent cause of liver cirrhosis, whose pathogenesis remains still partially obscure. In this scenario, the local and systemic impact of oxidative stress on biliary epithelial cells (BECs), contributing to apoptosis, premature senescence, destruction, and loss of ductal cells, configure a promising new “tail” of the pathogenetic mosaic. Moreover, it could represent the fil rouge linking the liver disease progression and extrahepatic complications onset.
Biological antioxidant potential (BAP) and Reactive Oxygen Metabolites (dROMs) evaluations are two complementary routinely-used validated tests largely adopted to assess the systemic oxidative stress balance, never applied before in the clinical PBC context.

Aims & Methods

We aimed to evaluate the systemic oxidative balance of PBC patients and explore its correlation with different stages of liver disease progression. In this observational study, from January 2020 to January 2023, we consecutively enrolled 21 patients receiving a first serological diagnosis of PBC and 10 healthy subjects. All the PBC patients started ursodeoxycholic acid (UDCA) treatment after the enrolment and none underwent to a previous UDCA-based and/or systemic antioxidants therapeutic regimen. For each patient, clinical, biochemical, and anthropometric parameters as well as a 5 ml venous blood sample were collected. A transient-elastometry-based non-invasive assessment of liver disease progression defined the hepatic fibrosis degree [Liver Stiffness Measurement (LSM) (kPa)] and steatosis severity [Controlled Attenuation Parameter (CAP) (db/m)]. The systemic oxidative balance was evaluated by using colorimetric determination of d-ROMs and BAP test. According to manufacturer instructions, we considered d-ROMs > 340 CARR-U and/or BAPs < 2000 µmol iron/L as related to oxidative stress imbalance.

Results

d-ROMs levels appeared higher in PBC patients in comparison to healthy subjects (p <0.0001); contrariwise, lower BAP levels were observed in PBC than in healthy individuals (p <0.0001). Consistently, dROMs and BAP levels showed respectively a direct and inverse correlation with both liver fibrosis (dROMs, R:0.96; BAP, R: -0.84) and steatosis (dROM, R:0.80; BAP, R: -0.68) severity (p<0.0001 all). A progressively increasing trend of d-ROMs values (F0-F2 vs F3: p<0.0001; F3 vs F4: p=0.002) and reduction of BAP levels (F0-F2 vs F3: p<0.0001; F3 vs F4 p=0.04) according to the worsening of liver fibrosis was also highlighted. Finally, the prevalence of oxidative stress imbalance was significantly different between mild fibrosis (F0-F2) and advanced-fibrosis (F3-F4)-affected patients.

Conclusion

PBC patients are characterized by a disequilibrium of the oxidative balance whose correlation with liver disease progression remarks its importance as relevant pathogenetic mechanisms which cannot be underestimated.

References

-Lleo A, Leung PSC, Hirschfield GM, Gershwin EM. The Pathogenesis of Primary Biliary Cholangitis: A Comprehensive Review. Semin Liver Dis. 2020 Feb;40(1):34-48. Doi: 10.1055/s-0039-1697617. Epub 2019 Sep 19. PMID: 31537031.
-Motoko Sasaki; Hiroko Ikeda; Hironori Haga; Toshiaki Manabe; Yasuni Nakanuma (2005). Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss. 205(4), 451–459. Doi:10.1002/path.1729
-Sorrentino P, Terracciano L, D’Angelo S, Ferbo U, Bracigliano A, Tarantino L, Perrella A, Perrella O, De Chiara G, Panico L, De Stefano N, Lepore M, Mariolina, Vecchione R. Oxidative stress and steatosis are cofactors of liver injury in primary biliary cirrhosis. J Gastroenterol. 2010 Oct;45(10):1053-62. Doi: 10.1007/s00535-010-0249-x. PMID: 20393861.
-Sasaki M, Ikeda H, Nakanuma Y. Activation of ATM signaling pathway is involved in the oxidative stress-induced expression of mito-inhibitory p21WAF1/Cip1 in chronic non-suppurative destructive cholangitis in primary biliary cirrhosis: an immunohistochemical study. J Autoimmun. 2008 Aug;31(1):73-8. Doi: 10.1016/j.jaut.2008.03.005. Epub 2008 May 5. PMID: 18456456.

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

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

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.