Systemic Oxidative Stress Correlates with Sarcopenia and Pruritus Severity in Primary Biliary Cholangitis (PBC): Two Independent Relationships Simultaneously Impacting the Quality of Life—Is the Low Absorption of Cholestasis-Promoted Vitamin D a Puzzle Piece?

Abstract

Background: Unlike other chronic liver disorders, in primary biliary cholangitis (PBC), systemic oxidative stress (SOS) worsens along with liver disease progression status (DPS), influencing muscle metabolism, muscle quantity (MQ), and itch pathways. Synergistically, cholestasis contributes to reduced vitamin D absorption, with a negative impact on MM and SOS. Despite this evidence, the prevalence of sarcopenia in PBC, and the SOS-MQ relationship comparing PBC with other CLDs, has never been investigated. Moreover, the relationship between vitamin D and MQ-SOS, and the correlation between SOS and pruritus severity, remains unexplored in PBC. Methods: A total of 40 MASLD, 52 chronic HBV infections, 50 chronic HCV infections, and 41 ursodeoxycholic acid/antioxidant-naïve PBC patients were enrolled. Biochemical, nutritional, and liver stiffness (LSM) data were collected, and sarcopenia was assessed after a normalizing 3-month dietetic–physical exercise regimen. The d-ROMs/BAP tests evaluated SOS. The validated “PBC-40 questionnaire” estimated pruritus and quality of life (QoL). Results: Unlike other CLDs, in PBC patients, sarcopenia was more prevalent in initial mild fibrosis (PBC: 57.10% vs. MASLD: 30.76%, HBV: 22.60%, HCV: 20.70%, all p < 0.0001), and SOS significantly correlated with MQ (dROMs-ASM/h², p: 0.0002; BAP-ASM/h²: p: 0.0092). PBC patients presented lower vitamin D levels and a significant correlation of these with SOS and MQ (all p < 0.0001). SOS also correlated with pruritus severity (dROMs, R: 0.835; BAP, R: −0.775, p < 0.0001). QoL impairment was significantly more represented in PBC individuals with sarcopenia, SOS imbalance, and relevant pruritus (p: 0.0228). Conclusions: In PBC, SOS correlates with MQ impairment and pruritus severity, configuring two independent relationships simultaneously impacting QoL.

1. Introduction

Primary biliary cholangitis (PBC) is a cholestatic autoimmune chronic liver disease (CLD) characterized by progressive destruction of the intrahepatic bile ducts, resulting in cholestasis, cirrhosis, and liver failure [1]. Epidemiologically, PBC primarily affects women in the 35–70-year-old range [1,2].

Chronic inflammation, determining the classical “chronic non-suppurative destructive cholangitis”, leads to intrahepatic biliary epithelial cell (iBEC) necrosis and promotes local oxidative stress [3,4].

In recent years, systemic oxidative stress (SOS) has been correlated with the severity of cholestasis and liver fibrosis, emerging as a leitmotiv in PBC, rather than other CLDs [5]. Reactive oxygen metabolites (dROMs) and biological antioxidant potential (BAP) are validated complementary tests to assess SOS levels [5]. Moreover, SOS imbalance, defined as a disequilibrium between the systemic mechanisms promoting the production of reactive oxygen species (ROS) and physiological antioxidant defenses [6], has been revealed to perfectly reflect the disease progression status (DPS) in PBC [5]. Relevantly, emerging findings support the impact of SOS on muscle metabolism and muscle quantity in different animal and human models, potentially contributing to the onset of sarcopenia [7,8].

Sarcopenia is a progressive and generalized loss of muscle mass and function (muscle strength and physical performance) [9], causing increased disability which dramatically worsens outcomes in several pathological settings [10,11,12], including advanced chronic liver disease (ACLD) [13,14].

The prevalence and relevance of this disorder in the PBC context have been only recently systematically investigated [15]. Sarcopenia has been highlighted as an independent risk factor for adverse clinical events, especially in PBC-ACLD patients [15]. However, the prevalence of sarcopenia, according to DPS, as well as the relationship between SOS levels and muscle quantity, remains unexplored in PBC.

The prognosis of PBC patients is affected by frequently experiencing malnutrition, resulting from cholestasis-related micronutrient malabsorption (e.g., liposoluble vitamins) [16,17,18,19]. Consistent with this, reduced vitamin D levels contribute to osteoporosis onset and worsening, with relatively serious repercussions even on short-term outcomes [19,20,21]. Previous research has progressively clarified several other vitamin D physiological functions [20,22]: it also emerged as a precious systemic antioxidant immunomodulator [23,24,25], as well as an inducer of myogenic differentiation in skeletal muscle-derived stem cells [26]. Based on this, it has appeared conceivable to hypothesize a physiopathological role of vitamin D deficiency in simultaneously influencing SOS and muscle quantity (contributing to sarcopenia onset) in PBC. Although vitamin D levels have been largely related to disease progression in PBC [27], the relationship between vitamin D levels and muscle quantity, as well as vitamin D levels and SOS, has never been directly evaluated.

Quality of life (QoL) of PBC patients is severely affected by cholestasis-related pruritus [28,29,30]. Although its pathogenesis remains only partially elucidated, emerging evidence supports the role of SOS in inducing and worsening pruritus via several peripheral and central itch pathogenetic pathways [31].

Considering this background, we primarily aimed to investigate the relationship between SOS and sarcopenia, as well as between SOS and pruritus severity, evaluating the relative repercussions on the QoL in PBC patients. Finally, as an ancillary outcome, the present research explored the relationship between vitamin D levels and MQ, as well as between vitamin D levels and SOS.

2. Materials and Methods

2.1. Experimental Design

In this observational study, we enrolled patients receiving a first diagnosis of PBC, as well as individuals with metabolic dysfunction–associated steatotic liver disease (MASLD) and ongoing Hepatitis B virus (HBV) and Hepatitis C virus (HCV) chronic infection.

PBC, HBV, and HCV were serologically diagnosed following the current clinical practice guidelines (CPGs) [32,33,34]. MASLD was diagnosed based on the recently proposed Delphi-consensus diagnostic criteria [35].

All the recruited patients received a controlled dietary/exercise regimen prepared by a certified nutritionist over three months. During this time, dedicated professional nutritional diary software documented individual food intake weekly (detailed report below), and the validated “International Physical Activity Questionnaire Short Form” (IPAQ-SF) assessed physical exercise habits (Supplementary File S1).

After this period, upon specialist visits, demographic, anthropometric, physical activity (relative to the previous 3-month controlled regimen), and clinical data, including pruritus severity and QoL (limitedly to PBC patients, by using specific items, opportunely extracted from the PBC-40 questionnaire [36]—detailed report below—and in Supplementary File S2), were collected. Contextually, body composition with sarcopenia assessment using bioelectrical impedance analyses (BIAs) (BIALight Dietosystem DS MEDICA srl Portable impedance meter in single frequency, Milan, Italy) and the liver fibrosis stage using liver stiffness measurements (LSMs) (FibroScan^® [version 502 (Echosens, Paris, France)]) were also evaluated [37,38].

Finally, for each patient, a 5 mL venous blood sample was collected to assess SOS and routine biochemical variables (including total bilirubin).

SOS was evaluated by two complementary validated colorimetric tests: Reactive Oxygen Metabolites^® (d-ROMs) and the Biological Antioxidant Potential^® (BAP) test.

Total bilirubin levels >2 mg/dL identified severe cholestasis according to CPGs [32].

The experimental design is illustrated in Figure 1.

Investigating the relationship between SOS and sarcopenia severity among different CLD etiologies represented a primary outcome of this research. The other main outcomes were to evaluate the relationship between SOS and pruritus severity, and the simultaneous impact of sarcopenia–SOS–pruritus on QoL in PBC.

The ancillary outcomes were (a) the assessment of vitamin D serum levels among various CLD etiologies; (b) the assessment of vitamin D serum levels in PBC patients according to the severity of cholestasis; and (c) the relationship between vitamin D levels and muscle quantity (MQ), as well as between vitamin D levels and SOS severity in a PBC setting.

2.2. Patients

This research was approved by the ethical committee of the University of Campania “Luigi Vanvitelli” in Naples (protocol n. 339/2022) and conducted in accordance with the Declaration of Helsinki (1975).

After signing the informed consent, between March 2022 and May 2024, we consecutively enrolled 41 patients receiving a first diagnosis of PBC and 142 individuals newly diagnosed with CLDs of other etiologies, including ongoing HBV (n: 52), ongoing HCV (n: 50), and MASLD (n: 40) (Figure 1).

The diagnosis of PBC was determined serologically following the criteria reported in the current clinical practice guidelines (CPGs): (1) chronic (at least six months) cholestasis (elevated ALP or GGT levels) and the presence of anti-mitochondrial antibodies (AMAs) (titer > 1:40); (2) chronic cholestasis associated with specific antinuclear antibodies (ANAs) [sp100 or gp210, detected by immunofluorescence (nuclear dots or perinuclear rims) or enzyme-linked immunosorbent assay (ELISA)] defining the “PBC-AMA-negative” entity [32].

Evidence of icterus and/or total bilirubin levels >2 mg/dL, in the absence of other potential pre-, intra-, and post-hepatic causes of jaundice, was used to discriminate moderate to severe cholestasis, according to CPGs [32].

HBV and HCV infections were defined based on serological and virological evidence of chronic (lasting at least six months) ongoing infection: based on the current CPGs, the detection of an HCV antibody (HCVAb) with HCV-RNA positivity and HBsAg (HBV surface antigen) positivity with HBV-DNA viral load >10⁷ international units (IUs)/mL defined chronic HCV and HBV infection, respectively [33,34].

Finally, MASLD was determined according to the recently proposed Delphi-consensus diagnostic criteria when hepatic steatosis, detected by imaging, was concomitant with either the presence of specific cardiometabolic risk factors such as overweight or obesity [body mass index (BMI) > 25 kg/m²] [35].

The enrollment was conducted at the Hepato-Gastroenterology Division of the University of Campania “Luigi Vanvitelli” between March 2022 and May 2024.

Age ranged between 18 and 80 years and a newly received CLD (PBC, HBV, HCV, or MASLD) diagnosis represented the inclusion criteria. Exclusion criteria encompassed smoking (past and/or current), alcoholic liver disorder (ALD), the presence of extra-hepatic chronic inflammatory diseases, acute or chronic kidney disease, rheumatoid arthritis, systemic lupus erythematosus, or other major systemic inflammatory diseases or malignancies, ongoing infections, alcohol or drug abuse history, previous neoplasms (including hepatocellular carcinoma—HCC), use of antioxidant-based regimens or hepatoprotective drugs within 12 months before enrollment, decompensated ACLD (Child–Pugh B or C) at the time of enrollment or in the preceding 12 months as well as endoscopic or radiological signs of clinically significant portal hypertension (CSPH) [39], and psychological/psychiatric conditions that could compromise the informed consent. All PBC patients began treatment with UDCA after serum sample collection, and none received a prior (in the previous 12 months) UDCA-based regimen and/or systemic antioxidant therapy. Anthropometric measurements included BMI, calculated as weight divided by the square of height (kg/m²). A clinical evaluation involving a comprehensive medical history, including substance abuse, comorbidities, and current therapies, along with the assessment of nutritional and lifestyle factors (comprising physical activity, dietary habits, and alcohol), was performed. In addition, 5 mL venous blood samples for each participant were collected for laboratory assessments.

2.3. Controlled Regimen for Diet, Nutritional, and Lifestyle-Related Assessments

During the initial three months of this study, all the enrolled patients were subjected to a low-calorie diet (20–25% less than the caloric intake required to preserve their current weight), with a precise rate of protein (10 and 20%), fats (20 and 30%, with saturated ones being under 10%), and carbohydrates (50–60%, sucrose less than 5%). A qualified nutritionist prepared the dietary plans to satisfy the wishes, tastes, and needs of each patient. The food intake (Kcal/die) relative to a complete week, with both working days and weekends, and the calories assumed at each main meal, as well as the list of dietary components, were recorded for each patient (details in Supplementary File S3).

To simultaneously assess the time (hours/week) spent on moderate physical exercise and sitting periods (item 7) (hours/day) during the 3-month controlled diet, the validated “International Physical Activity Questionnaire Short Form” (IPAQ-SF) was adopted [40] (Supplementary File S1).

2.4. Biochemical Assessments

The biochemical data assessed were aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total bilirubin (TB), platelet count (PLT), albumin, and International Normalized Ratio (INR). GGT levels were enzymatically evaluated by adopting commercially available kits (R&D Systems, Minneapolis, MN, USA); a colorimetric assay kit (Amplite 13801/13803 and Thermo Fisher Scientific EIAGLUC, Rome, Italy) was used to assess AST and ALT. The 25-hydroxy vitamin D test kit (Mount Sinai) was adopted to rapidly estimate the vitamin D serum levels (ng/mL).

2.5. Body Composition and Sarcopenia Assessment

Body composition, with multicompartment parameters, was analyzed by using a multifrequency bioelectrical impedance analysis (BIA) system (InBody, Seoul, Republic of Korea) (details in Supplementary File S3). The BIA system calculated fat-free mass (FFM), fat mass (FM), body cell mass (BCM), and extracellular cell mass (ECM), expressed both as a percentage and in kilograms (Kg), as well as total body water (TBW), skeletal muscle mass (SMM) (Kg), SMM index (SMMI), and the impedance index (details in Supplementary File S3). To estimate appendicular skeletal muscle mass (ASM), we used the following sex-specific validated equations: (a) ASM-Men = 0.197 × (impedance index) + 0.179 × (weight) − 0.019; (b) ASM-Women = 0.221 × (impedance index) + 0.117 × (weight) + 0.881 [41]. Finally, ASM/h² was obtained by dividing the ASM by height squared (m²) (Kg/m²).

The following parameters were considered to define sarcopenia: (a) muscle quantity (estimated with ASM/h²), (b) muscle strength (defined by the hand-grip test and the chair-stand test), and (c) physical performance [defined by the timed up and go (TUG) test, the gait speed test, and the Short Physical Performance Battery (SPPB)] (details in Supplementary File S3) [42]. According to the revised criteria proposed by the European Working Group on Sarcopenia in Older People (EWGSOP2), sarcopenia was diagnosed when a reduction in muscle strength was detected alongside either an MQ impairment or a decline in physical performance [42].

Impaired muscle strength was defined by an HGT < 27 kg in males (<16 kg in females) and a chair-stand test > 15 s. Physical performance reduction was indicated by an SPBB < 8, a gait speed test < 0.8 m/second, and a TUG ≥ 20 s. MQ impairment was defined by an ASM/h² < 7 Kg/m² in males (<5.5 Kg/m² in females).

2.6. Systemic Oxidative Stress Assessment

The systemic pro-oxidant and antioxidant statuses were, respectively, evaluated using the d-ROM and BAP assays, following the manufacturer’s instructions (Diacron International, Grosseto, Italy). The d-ROM test indirectly detects the hydroperoxide concentration. Reference values from 20 to 24 mg/dL H₂O₂ were defined as the normal range. The intra- and inter-assay coefficients of variation are 0.3–6.6% and 0.3–5.1%, respectively (details in Supplementary File S3). Conversely, the BAP test, detailed in Supplementary File S3, measures the antioxidant blood capacity. According to the manufacturer, d-ROMs ≥ 27.20 mg H₂O₂/dL and BAP ≤ 2200 µmol iron/L represent threshold values indicative of SOS impairment [43].

2.7. Liver Stiffness Measurement Assessment

The LSM was performed using FibroScan^® [version 502 (Echosens, Paris, France)] with M and XL probes [37]. An XL probe was utilized when BMI was >30 or when the ultrasound-measured distance between the skin and liver capsule was >2.5 cm. Ten valid measurements, defined as successful LSM, were performed by an experienced physician using FibroScan^®. The quality of measurements was assessed according to the criteria proposed by Boursier, based on the interquartile range (IQR): “very reliable” (IQR/M ≤ 0:1), “reliable” (0:1 < IQR/M ≤ 0:3 or IQR/M > 0:3 with LS median < 7:1 kilopascal), or “poorly reliable” (IQR/M > 0:3 with LS median ≥ 7:1 kPa) [37,38].

According to the recent World Federation for Ultrasound in Medicine and Biology (WFUMB) guidelines, considering that several factors (e.g., inflammation/biliary obstruction and BMI > 30 Kg/m²) may influence LSM independently of effective hepatic fibrosis, the interpretation of results was based on the disease etiology, adopting specific validated cut-offs [44]. The following LSM cut-off scores were applied to identify the different liver fibrosis stages in PBC patients: (a) F0–F2 ≤ 9.8 kPa; (b) F3: 9.9–17.3 kPa; and (c) F4 ≥ 17.8 kPa. Advanced fibrosis (AF) (F3–F4) was defined by LSM values ≥ 9.9 kPa [45]. For MASLD, the following LSM cut-off scores were used to identify the different liver fibrosis stages according to the Metavir score: (a) F0–F2 ≤ 9.6 kPa; (b) F3: 9.7–13.5 kPa; and (c) F4 ≥ 13.6 kPa. AF (F3–F4) was defined as LSM values ≥ 9.7 kPa [46]. In patients with HBV, the following LSM cut-off scores were used to identify the different liver fibrosis stages: (a) F0–F2 ≤ 8.1 kPa; (b) F3: 8.2–11 kPa; and (c) F4 ≥ 11.1 kPa. AF (F3–F4) was defined by LSM values ≥ 8.2 kPa [47]. The following LSM cut-off scores were used to identify the different liver fibrosis stages for HCV patients: (a) F0–F2 ≤ 9.6 kPa; (b) F3: 9.7–14.6 kPa; and (c) F4 ≥ 14.7 kPa. AF (F3–F4) was defined by LSM values ≥ 9.7 kPa [48]. For each CLD etiology, F0–F2 defined “initial–mild” fibrosis, while F3–F4 suggested “AF”.

2.8. Evaluation of Pruritus and QoL in PBC Patients

To assess pruritus and evaluate the disease impact on QoL, PBC patients received a questionnaire opportunely extracted from the PBC-40 questionnaire [36] (Supplementary File S2). Specifically, domain 3 of the PBC-40 questionnaire was used to evaluate pruritus, and the impact on QoL was estimated using domain 8 and domain 9, both of which explore the potential effects of PBC on social life (details in Supplementary File S3). Potentially ranging from a minimum of 7 to a maximum of 35, a total score of at least 21 defined an impairment of QoL.

2.9. Statistical Analysis

Mean and standard deviations were used to describe continuous data, while n (%) defined categorical variables. To evaluate if parametric or non-parametric analysis should be applied, the Kolmogorov–Smirnov test for normality was executed.

Mann–Whitney tests and t-tests for independent groups, the Kruskal–Wallis test, or ANOVA tests with post hoc Tukey analysis, in the case of non-normal or normal distribution, respectively, were used to compare the continuous variables.

The chi-square test was performed to assess statistically significant frequency distribution differences between the cohorts. To evaluate the relationship (R) between continuous variables, multilinear regression analysis was executed. Statistical significance was established as p < 0.05 in a two-tailed test with a 95% confidence interval (C.I.). GraphPad Prism vs.9.1 (GraphPad Software, Boston, MA, USA) was used to perform the analysis.

The sample size was calculated using a chi-square test comparing two independent proportions, singularly predicting a 20% difference in the prevalence of subjects affected by sarcopenia in the PBC group compared to other CLD etiologies (significance: 0.05, type II error: 0.1; power: 0.9) (STATA14 for MacOS), and resulted in n.40 for the groups.

3. Results

3.1. Characteristics and Liver Disease Progression Status (DPS) of the Study Population

3.1.1. Anthropometric, Demographic, and Clinical Features of the Study Population

The present observational study enrolled 41 PBC, 40 MASLD, 52 HBV, and 50 HCV patients.

Table 1. Demographic, anthropometric, biochemical, and liver DPS of the study population.

Demographic Data
	PBC (a) (n: 41)	MASLD (b) (n: 40)	HBV (c) (n: 52)	HCV (d) (n: 50)	p*/** (a)(b)	p*/** (a)(c)	p*/** (a)(d)
Gender—male (number and %)	2 (0.048%)	20 (50%)	18 (34.62%)	21 (42%)	<0.0001	<0.0001	<0.0001
Gender—female (number and %)	39 (95.12%)	20 (50%)	34 (65.38%)	29 (58%)
Age (mean ± SD)	58.61 ± 11.26	54.30 ± 11.21	52.40 ± 8.22	56.44 ± 7.79	n.s.	n.s.	n.s.
Anthropometric variables
Variables (mean ± SD)	PBC (a) (n: 41)	MASLD (b) (n: 40)	HBV (c) (n: 52)	HCV (d) (n: 50)	p* (a)(b)	p* (a)(c)	p* (a)(d)
BMI (Kg/m2)	28.51 ± 3.96	32.3 ± 2.21	27.56 ± 2.61	26.58 ± 3.41	0.03	n.s.	n.s.
Weight (Kg)	73.68 ± 13.08	82.75 ± 14.06	67.58 ± 12.01	72.43 ± 11.05	0.04	n.s.	n.s.
Height (cm)	158.7 ± 10.71	153.7 ± 12.41	161.3 ± 11.64	159.2 ± 9.62	n.s.	n.s.	n.s.
Biochemical data
Variables (mean ± SD)	PBC (a) (n: 41)	MASLD (b) (n: 40)	HBV (c) (n: 52)	HCV (d) (n: 50)	p* (a)(b)	p* (a)(c)	p* (a)(d)
AST (IU/L)	31.39 ± 13.20	34.38 ± 36.62	42.65 ± 4.46	40.68 ± 5.56	n.s.	0.002	0.02
ALT (IU/L)	32.68 ± 16.55	31.98 ± 19.39	46.88 ± 3.97	46.08 ± 5.36	n.s.	0.008	0.006
GGT (IU/L)	194.5 ± 69.65	86.93 ± 23.80	87.15 ± 6.11	93.60 ± 10.27	<0.0001	<0.0001	<0.0001
ALP (IU/L)	192.1 ± 94.99	81.63 ± 44.94	134.9 ± 15.89	105.3 ± 8.81	<0.0001	<0.0001	<0.0001
PLT (103/mm3)	179.4 ± 18.3	174.8 ± 97.99	175 ± 12.36	170.3 ± 14.25	n.s.	n.s.	n.s.
Total bilirubin (mg/dL)	2.06 ± 0.68	1.09 ± 0.79	1.08 ± 0.65	1.14 ± 0.92	0.006	0.002	0.002
Albumin (g/L)	3.89 ± 0.42	4.21 ± 0.54	3.99 ± 0.61	3.85 ± 0.22	n.s.	n.s.	n.s.
INR	1.18 ± 0.29	1.08 ± 0.56	0.96 ± 0.37	1.01 ± 0.54	n.s.	n.s.	n.s.
Non-invasive tool evaluating liver disease progression status
Variables (mean ± SD)	PBC (a) (n: 41)	MASLD (b) (n: 40)	HBV (c) (n: 52)	HCV (d) (n: 50)	p* (a)(b)	p* (a)(c)	p* (a)(d)
LSM (kPa)	10.28 ± 6.02	10.47 ± 6.45	9.94 ± 4.63	9.88 ± 5.05	n.s.	n.s.	n.s.
CAP (dB/m)	275.1 ± 25.83	274.2 ± 21.65	274.8 ± 29.01	278 ± 28.63	n.s.	n.s.	n.s.

DPS: disease progression status; IU: international unit; BMI: body mass index; AST: aspartate aminotransferase; ALT: alanine aminotransferase; GGT: gamma-glutamyl transferase; ALP: alkaline phosphatase; PLT: platelet count; INR: International Normalized Ratio; LSM: liver stiffness measurement; PBC: primary biliary cholangitis; SD: standard deviation; MASLD: metabolic dysfunction–associated steatotic liver disease; HBV: chronic Hepatitis B infection; HCV: chronic Hepatitis C infection. ** Mann–Whitney U test. * Chi-square test. Statistically significant differences (p < 0.05) are reported in bold; n.s.: not statistically significant.

reports the demographic, anthropometric, and biochemical data, as well as liver DPS (LSM-assessed disease severity staging) of the study population.

Of 41 PBC patients, 21 (51.21%) presented severe cholestasis, and 9 (21.95%) were AMA-negative. As expected, the female frequency distribution was significantly higher in PBC patients (95.12%) in comparison with all other CLD etiologies (MASLD: 50%; HBV: 34.62%; HCV: 42%) (chi-square, p < 0.0001).

PBC patients also presented the typical features of biochemical cholestasis, showing more elevated levels of GGT and ALP compared to other CLDs (all p < 0.0001), whereas BMI (p: 0.03) and weight (p: 0.04) appeared significantly higher in MASLD individuals (Table 1).

3.1.2. Liver Disease Progression Status (DPS) of the Study Population

Concerning LSM-assessed liver DPS, 28 (68.29%) PBC, 26 (65%) MASLD, 31 (59.61%) HBV, and 29 (58%) HCV patients presented an initial mild/moderate fibrosis (F0–F2), whereas AF (F3–F4) was observed in 13 (31.71%) PBC, 14 (35%) MASLD, 21 (40.39%) HBV, and 21 (42%) HCV individuals.

Globally, considering the hepatic disease progression status of the study population, no statistically significant differences in terms of AF prevalence distribution were revealed comparing PBC with other CLD (MASLD, HBV, and HCV) patients, as well as MASLD with HBV, HCV with HBV, and MASLD with HCV (chi-square comparisons, all p > 0.05).

Finally, AF was similarly revealed in PBC AMA-positive patients (10 = 31.25%) and AMA-negative individuals (3 = 33.33%).

3.2. Nutritional Assessment: Evaluation of Dietary Habits, Physical Exercise, and Body Composition

Concerning dietary habits, the nutritional assessment evidenced no significant differences in quantitative (i.e., total calorie intake) and qualitative (i.e., intake of macro- and micronutrient pattern, including vitamin D daily assumption) daily food intake during the 3-month controlled dietary regimen in PBC patients compared with other CLDs, as well as in PBC individuals with severe cholestasis compared with PBC subjects with moderate cholestasis (Supplementary Table S1).

On the same lines, regarding physical exercise relative to the 3-month controlled regimen period, no statistically significant differences, in terms of hours/week spent on the activity and the percentage of patients participating in “active physical exercise”, were evidenced in PBC individuals compared to other CLDs (Supplementary Table S1).

Finally, focusing on body composition variations, compared with other CLDs, PBC patients showed a significantly reduced FFM (Kg) (PBC vs. MASLD, p: 0.003; PBC vs. HCV and PBC vs. HCV, p: 0.001), FFM (%) (PBC vs. MASLD, p: 0.041; PBC vs. HBV and PBC vs. HCV, p > 0.001), SMMI (Kg/m²) (PBC vs. MASLD, p: 0.046; PBC vs. HBV and PBC vs. HCV, p: 0.002), and ASM/h² (Kg/m²) (PBC vs. MASLD, PBC vs. HBV, and PBC vs. HCV, all p: 0.001), as well as decreased HGT (Kg)-defined muscle strength [PBC vs. MASLD, PBC vs. HBV, and PBC vs. HCV, all p < 0.0001] (

Table 2. Body composition evaluations and sarcopenia assessment of the study population.

BIA-Assessed Parameters
Variables (Mean ± SD)	PBC (a) (n: 41)	MASLD (b) (n: 40)	HBV (c) (n: 52)	HCV (d) (n: 50)	p* (a)(b)	p* (a)(c)	p* (a)(d)
FFM (Kg)	54.88 ± 9.28	58.30 ± 8.72	62.94 ± 8.77	63.52 ± 7.34	0.003	0.001	0.001
FFM (%)	68.59 ± 3.93	70.14 ± 5.99	80.08 ± 3.81	79.70 ± 4.39	0.041	>0.001	>0.001
FM (Kg)	68.72 ± 3.37	74.97 ± 3.16	70.58 ± 3.08	67.22 ± 4.13	0.002	n.s.	n.s.
FM (%)	32.41 ± 3.93	39.86 ± 5.99	30.93 ± 3.08	31.30 ± 4.39	0.001	n.s.	n.s.
BCM (Kg)	26.35 ± 4.07	25.58 ± 5.30	26.55 ± 4.19	27.08 ± 3.51	n.s.	n.s.	n.s.
BCM (%)	34.46 ± 2.96	32.03 ± 4.69	34.30 ± 3.71	35.32 ± 2.68	n.s.	n.s.	n.s.
ECM (Kg)	31.53 ± 6.63	30.73 ± 6.15	31.39 ± 6.52	31.40 ± 5.08	n.s.	n.s.	n.s.
ECM (%)	33.13 ± 4.88	32.11 ± 5.27	33.78 ± 5.09	32.27 ± 4.61	n.s.	n.s.	n.s.
TBW	40.88 ± 9.28	39.26 ± 8.62	41.95 ± 6.41	40.37 ± 5.36	n.s.	n.s.	n.s.
SMM (Kg)	20.01 ± 4.17	23.21 ± 5.923	28.93 ± 4.08	30.12 ± 3.71	0.003	>0.001	>0.001
SMMI (Kg/m2)	7.08 ± 1.42	8.98 ± 2.21	10.19 ± 1.01	10.46 ± 1.13	0.046	0.002	0.002
DIMENSIONS (I-II-III) DEFINING “SARCOPENIA”
Muscle Quantity (I)
Variables (mean ± SD)	PBC (a) (n: 41)	MASLD (b) (n: 40)	HBV (c) (n: 52)	HCV (d) (n: 50)	p* (a)(b)	p* (a)(c)	p* (a)(d)
ASM/h2 (Kg/m2)	6.39 ± 0.68	7.52 ± 0.89	7.46 ± 1.04	7.28 ± 1.58	0.001	0.001	0.001
Muscle Strength (II)
HGT (Kg)	22.13 ± 2.68	27.51 ± 4.58	28.52 ± 1.86	31.78 ± 1.09	<0.001	<0.001	<0.001
Chair stand (s)	16.88 ± 1.62	15.84 ± 2.89	15.01 ± 2.24	15.18 ± 0.71	n.s.	n.s.	n.s.
Physical Performance (III)
SPBB (total score)	5.15 ± 1.63	5.27 ± 1.06	6.42 ± 0.93	6.51 ± 0.95	n.s.	n.s.	n.s.
TUG (s)	16.15 ± 1.22	15.11 ± 3.13	16.37 ± 2.24	15.33 ± 2.19	n.s.	n.s.	n.s.
Gait speed test (m/s)	0.66 ± 0.16	0.72 ± 0.19	0.71 ± 0.05	0.72 ± 0.14	n.s.	n.s.	n.s.

BIA: bioelectrical impedance analysis; FFM: fat-free mass; FM: fat mass; BCM: body cell mass; ECM: extracellular cell mass; TBW: total body water; SMM: skeletal muscle mass; SMMI: skeletal muscle mass index; ASM: appendicular skeletal mass; h²: the square of the height; HGT: Hand-grip test; SD: standard deviation; SPBB: Short Physical Performance Battery; TUG: timed up and go test. * Mann–Whitney U test. Statistically significant differences (p < 0.05) are reported in bold; n.s.: not statistically significant.

).

Furthermore, the sub-analysis comparing PBC AMA-positive with PBC AMA-negative patients revealed no significant differences in the above-evidenced parameters [FFM (Kg), 53.78 ± 7.11 vs. 53.62 ± 7.04, p: 0.078; FFM (%) 67.39 ± 3.13 vs. 67.22 ± 3.01, p: 0.089; SMMI (Kg/m²), 7.02 ± 1.31 vs. 7.11 ± 1.28, p: 0.088; ASM/h2 (Kg/m²) 6.32 ± 0.57 vs. 6.41 ± 0.52, p: 0.093; and HGT (Kg), 23.11 ± 2.18 vs. 22.09 ± 2.01, p: 0.083].

The detailed report of BIA-assessed parameters, as well as the tests evaluating the three dimensions (I—muscle quantity; II—muscle strength; III—physical performance) of sarcopenia of the study population, are completely shown in Table 2.

3.3. Systemic Oxidative Stress and Liver DPS in PBC vs. Other CLDs

Globally, no significant differences in dROM and BAP levels among the study population emerged, appearing with SOS levels similar in PBC and other CLD etiologies [dROMs (mg H₂O₂/dL), PBC: 32.64 ± 5.20, MASLD: 32.91 ± 8.375, CHB: 30.831 ± 4.112, and CHC: 33.76 ± 7.889; all comparison, p > 0.05] [BAP (micromol/L), PBC: 1823 ± 105.7, MASLD: 1697 ± 928.3, CHB: 1961 ± 275.5, and CHC: 1516 ± 687.8, all comparison, p > 0.05]. The sub-analysis of SOS in PBC AMA-negative patients revealed no significant differences compared with PBC AMA-positive individuals [PBC AMA-positive vs. PBC AMA-negative, dROMs (mg H₂O₂/dL): 32.21 ± 5.20 vs. 31.97 ± 4.20, p: 0.892; BAP (micromol/L): 1811 ± 102.3 vs. 1819 ± 104.2, p: 0.937].

Moreover, exclusively (MASLD, GGT, R-squared: 0.146, p: 0.087; ALP, R-squared: 0.134, p: 0.814; CHB, GGT, R-squared: 0.052, p: 0.105; ALP, R-squared: 0.045, p: 0.880; CHC, GGT, R-squared: 0.114, p: 0.460; ALP, R-squared: 0.156, p: 0.931) in PBC patients, a significant positive correlation between dROMs (mg H₂O₂/dL) and GGT (u/l) (R-squared: 0.834; C.I.95%: 0.481–0.977; p: 0.0003), as well as dROMs (mg H₂O₂/dL) and ALP (u/l) (R-squared: 0.728; C.I.95%: 0.692–0.954; p: 0.0094), was revealed, suggesting a relationship linking the severity of cholestasis with the impairment of SOS balance in the driving of disease worsening.

Consistent with this, after stratifying for fibrosis severity, a statistically significant parallel trend of oxidative stress and DPS (i.e., progressive increase in dROM levels simultaneously with a BAP decrease) was observed exclusively in PBC patients (Supplementary Figure S1).

3.4. Sarcopenia and Liver DPS in PBC vs. Other CLDs

Overall, a significantly higher prevalence of sarcopenia in PBC individuals [PBC: 25 (60.97%)] was highlighted in comparison with other CLDs [PBC vs. MASLD: 18 (45%), vs. HBV: 20 (38.46%), vs. HCV: 20 (40%), all p: 0.001]. The evaluation concerning the prevalence of sarcopenia according to liver DPS across PBC patients and the various CLDs was subsequently performed.

Similarly to other CLDs (MASLD, HBV, and HCV; chi-square, all p < 0.0001), the prevalence of sarcopenia was significantly higher in AF stages compared with initial mild/moderate fibrosis (F0–F2) even in PBC (chi-square, p: 0.03), thus progressively increasing in parallel with the worsening of liver DPS in all the liver disease etiologies (Figure 2A–D).

However, notably, focusing exclusively on patients presenting an initial mild/moderate fibrosis (F0–F2), a significant proportion of PBC individuals were sarcopenic, and a higher prevalence of sarcopenia in F0–F2 PBC patients was reported in comparison to all other CLDs at equal liver DPSs (sarcopenia in F0–F2, PBC vs. MASLD, chi-square, p: 0.0135; PBC vs. HBV, p: 0.0006; PBC vs. HCV, p: 0.005) (Figure 2E). Finally, the prevalence of sarcopenia was not significantly different in F0–F2 PBC AMA-positive patients compared with F0–F2 PBC AMA-negative individuals (12 vs. 3, 60% vs. 50%, chi-square, p: 0.091).

Consistent with these findings, an inverse correlation between MQ (ASM/h², Kg/m²) and hepatic fibrosis severity (LSM, kPa) was revealed in all CLD etiologies, including PBC, appearing, nevertheless, stronger in MASLD (R: −0.436, p: 0.0049) and viral-related CLD (HBV+HCV) (R: −0.765, p < 0.0001) patients in comparison to PBC individuals (R: −0.395, p: 0.0104) (Figure 3).

3.5. Sarcopenia and Systemic Oxidative Stress in PBC vs. Other CLDs

Exclusively (MASLD, dROMs-ASM/h², R-squared: −0.145, p: 0.085; BAP-ASM/h², R-squared: 0.146, p: 0.812; HBV, dROMs-ASM/h², R-squared: −0.185, p: 0.082; BAP-ASM/h², R-squared: 0.178, p: 0.811; HCV, dROMs-ASM/h², R-squared: −0.191, p: 0.088; BAP-ASM/h², R-squared: 0.228, p: 0.078) in PBC patients, a significant inverse correlation between dROMs (mgH₂O₂/dL) and muscle quantity (ASM/h²) [R-squared: −0.783, C.I.95%: (−0.576)–(−0.892), p: 0.0002], as well as a positive correlation between BAP (micromol/L) and muscle quantity (ASM/h²) (R-squared: 0.748, C.I.95%: 0.651–0.984, p: 0.0092), was revealed, suggesting, limited to this CLD setting, a relationship linking the severity of sarcopenia with the impairment of systemic oxidative stress balance.

3.6. SOS, Sarcopenia, and Pruritus Global Impact on QoL in a PBC Setting: A Matter of Cholestasis

In the PBC setting, a higher prevalence of SOS imbalance (76.19%, chi-square test, p: 0.0278) and sarcopenia (80.95%, chi-square test, p: 0.0109) among patients with severe cholestasis compared with individuals with moderate cholestasis was revealed (Figure 4).

Moreover, compared with PBC patients with moderate cholestasis, PBC individuals affected by severe cholestasis presented higher PBC-40 mean scores, both concerning the itching (PBC-40 questionnaire, domain 3, items 8 to 10) (p < 0.0001) and impact on QoL (impaired QoL levels) in terms of social repercussions (PBC-40 questionnaire, domain 8, items 34 to 37, and domain 9, items 38 to 40) (p < 0.0001) (Figure 5A,B).

In line with the above-presented relationships, in PBC patients, relevantly, SOS also correlated with both pruritus severity (dROMs and PBC-40 questionnaire, domain 3, items 8 to 10, R: 0.835; BAP and PBC-40 questionnaire, domain 3, items: 8 to 10, R: −0.775, both p < 0.0001) (Figure 5C) and QoL-impairment levels (dROMs and PBC-40 questionnaire, domains 8 and 9, R: −0.703; BAP and PBC-40 questionnaire, domains 8 and 9, R: 0.743, both p < 0.0001) (Figure 5D).

Finally, a global QoL impairment (defined by the PBC-40 questionnaire, domain 8, items 34 to 37, and domain 9, items 38 to 40, total score ≥ 21) was evidenced in 21 PBC patients, appearing significantly more represented in PBC individuals simultaneously showing relevant pruritus, SOS imbalance, and sarcopenia (93.75%) in comparison to subjects presenting no relevant pruritus, no SOS imbalance, and no sarcopenia (50%) (chi-square, p: 0.0228) (Figure 5E).

3.7. Vitamin D Level Assessment and Relative Relationship with SOS and MQ in PBC vs. Other CLDs

Lower serum vitamin D levels (ng/mL) were observed in PBC patients in comparison to other CLDs (PBC vs. MASLD, HBV, HCV, all p < 0.0001), as well as in PBC patients with severe cholestasis compared with PBC subjects with moderate cholestasis (p < 0.0001) (Figure 6A,B).

Aligning with this, exclusively in PBC patients, vitamin D levels (ng/mL) significantly correlated with both SOS (BAP, R: 0.824; d-ROMs, R: −0.810; both p < 0.0001) (Figure 6C) and MQ (ASM/h², R: 0.766, p < 0.0001) (Figure 6D).

4. Discussion

Similar to other CLDs, in PBC, sarcopenia represents a common plague progressively burdening advanced liver disease stages [15].

In line with this, the present study confirmed the higher frequency distribution of sarcopenia in AF stages compared with initial mild/moderate fibrosis (F0–F2) for all CLD settings (MASLD, HBV, HCV). However, a significantly more elevated representation of sarcopenia in PBC individuals compared with other CLDs, as well as, relevantly, a higher prevalence of sarcopenia in F0–F2, was reported in PBC patients rather than all other CLD subjects showing equal fibrosis severity. This novel result appears in line with antecedent findings highlighting a significant proportion (46.4%) of non-cirrhotic PBC patients presenting sarcopenia in real life [49], without disdaining the association between LSM-assessed AF and sarcopenia previously evidenced by Yang et al. [15].

Unlike other CLD etiologies, SOS has been recently demonstrated to progressively increase along with hepatic fibrosis worsening exclusively in PBC [5]. Consistent with this, our study revealed an inverse correlation between MQ (ASM/h²) and hepatic fibrosis severity (LSM) in all CLD etiologies, including PBC, appearing, nevertheless, weaker in MASLD and viral-related CLD (HBV + HCV) patients in comparison to PBC individuals.

A possible explanation for this emerging clinical–epidemiological peculiarity is attributable to both the cholestatic and autoimmune nature of PBC, critically contradistinguishing this from other CLDs.

Cholestasis promotes severe malnutrition pictures via micronutrient malabsorption, including overall liposoluble vitamins and vitamin D in PBC patients [16,17,18,19]. Interestingly, besides the nutritional involvement and the already largely demonstrated correlation with disease progression in PBC [27], vitamin D also plays an additional double role by decisively contributing to systemic antioxidant mechanisms [23,24,25] and, by inducing myogenic differentiation in skeletal muscle-derived stem cells, crucially promoting myogenesis [26]. Aligning with this, in our cohort, lower serum vitamin D levels (ng/mL) were observed in PBC patients in comparison to other CLDs (PBC vs. MASLD, HBV, and HCV), as well as, after opportunely stratifying according to the severity of cholestasis [32], in PBC patients showing severe outcomes compared with subjects presenting moderate cholestasis. Moreover, further supporting this, exclusively in PBC patients, vitamin D levels significantly correlated with both SOS levels and muscle quantity.

As an autoimmune disease, considering the well-known mutual relationship linking immune dysregulation, systemic inflammation, and SOS [50,51,52], PBC appears to even be pathogenetically reciprocally imbricated with sarcopenia. In PBC patients, indeed, the dysregulated muscle protein homeostasis has been demonstrated to be considerably affected by circulating proinflammatory mediators [including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and others) [53] whose serum levels, in turn, have been reported to critically increase in SOS-related conditions, configuring a dramatic vicious circle [50,51,52]. Moreover, interestingly, especially in leaky-gut-related disorders, elevated lipopolysaccharide (LPS) serum levels, by determining systemic endotoxemia and activating muscle cells’ innate immunity Toll-like receptors, increase proteolysis and muscle catabolism, thus promoting MQ reduction and sarcopenia [54,55].

In the CLD context, CSPH represents a well-recognized factor contributing, by mainly altering gut permeability, to both nutrient-reduced absorption and systemic LPS translocation, configuring a critical pathogenetic moment in ACLD-related sarcopenia/malnutrition onset and worsening [56]. However, considering CSPH patients were opportunely and voluntarily ruled out from the recruitment, it appears pivotal to underline how this event did not influence the effects observed in our research and was purely, or at least, mostly attributable to cholestasis-promoting SOS. Consistent with this, in the PBC setting, a higher prevalence of SOS-I (76.19%) and sarcopenia (80.95%) among patients with severe cholestasis compared with individuals with moderate cholestasis was revealed, and a significant inverse correlation between dROMs (mgH₂O₂/dL) and MQ contemporarily with a positive correlation between BAP (micromol/L) and MQ emerged, suggesting a relationship linking sarcopenia with the impairment of SOS balance.

Pruritus severely burdens QoL, representing the other crucial clinical plague in PBC patients [28,29], as well as a hard therapeutic challenge without an available second-line therapy [57]. Although the pathogenesis is only partially clarified, the potential role of SOS in inducing and worsening pruritus via several peripheral and central itch pathogenetic complex pathways has been proposed [31]. Interestingly, in our study, also in line with the other above-presented relationships, in PBC patients, SOS correlated with both pruritus severity and QoL impairment levels.

Altogether, SOS, sarcopenia, and pruritus configure a rigid conditioning routine life and daily activities of PBC patients, with serious potential socioeconomic repercussions. Notably, concerning this, our research revealed an overall QoL impairment in 51.21% of PBC patients, appearing, as expected, significantly more represented in individuals simultaneously showing relevant pruritus, SOS-I, and sarcopenia (93.75%) in comparison to subjects presenting no relevant pruritus, no SOS-I, and no sarcopenia (50%).

Our study presents limitations: Firstly, despite adequate sample size representation, the present research represented a monocentric experience with a relatively small cohort.

Secondly, PBC was serologically diagnosed, and fibrosis severity was for all CLD etiologies estimated by LSM. However, following current CPGs, both these methods represent a valid way in routine clinical practice to diagnose and stage PBC and CLDs, reserving liver biopsy to a few limited cases [32].

Moreover, in our research, sarcopenia was assessed by BIA, respecting the novel EWGSOP2 proposed criteria [42]. Indeed, even though the MQ was not directly (by performing Computerized Tomography (CT) or Dual-Energy X-ray Absorptiometry) determined, this parameter was instantly and equally obtained by opportunely combining BIA-assessed variables [41].

Several studies have reported the influence of demographic features and lifestyle habits (including, diet, physical exercise, and smoking habits) on muscle mass, muscle performance, and oxidative status [58,59,60]. Focusing on demographic data, all patients enrolled were Caucasian, and no significant differences in terms of age were reported. Concerning sex distribution, in the PBC patients, the prevalence of female individuals was significantly higher in comparison to other CLDs. In addition, considering the epidemiology of PBC, and, in general, autoimmune disorders [1,2], this is not to be considered a selection bias, but rather a faithful portrait of what occurs in the real world.

Regarding lifestyle habits, to minimize the pressure of these variables on the observed outcomes, all data were collected after a normalizing 3-month equally prescribed dietetic–physical exercise regimen. This last methodological caution also allowed us to reduce, to a minimum, the inter-individual variability of vitamin D serum levels physiologically derived by heterogeneous diet regimens and endogenous production attitudes [61]. In addition, following other research in the same field [22,27], vitamin D absorption was not directly assessed, and its bioavailability was opportunely estimated by simultaneously collecting the daily assumption and the relative serum levels [62,63].

Eventually, given the evidence on PBC-AMA-negative pathogenesis is controversial, suggesting a different histopathology featured by a more severe disease course [64], our PBC cohort was stratified according to relative serological characteristics. Interestingly, no differences emerged comparing PBC-AMA-positive with PBC-AMA-negative individuals in both SOS and body composition.

To the best of our knowledge, this is the first study investigating the relationship between SOS and sarcopenia, as well as SOS and pruritus severity in the human PBC context. Globally, these emerging findings must be intended as pioneering results opening the doors to various future perspectives, including the clarification of pathogenetic mechanisms linking SOS with sarcopenia and pruritus, as well as the physiopathological role of vitamin D in impacting these two worsening QoL determinants in PBC. Finally, also considering the relative relevance in cholestasis modulation, as well as the largely supported potential antioxidant implications [65,66], the investigation of the UDCA regimen in ameliorating clinical outcomes by targeting the vitamin D–oxidative stress–muscle metabolism–itch axis appears as a conceivable research field to explore in the near future.

5. Conclusions

In PBC, SOS correlates with muscle quantity impairment, and SOS imbalance is more represented in patients presenting sarcopenia. Despite sarcopenia progressively increasing along with liver disease progression, it is significantly represented even in the initial mild fibrosis stage in PBC patients, showing a higher prevalence in comparison to the other CLDs at the same DPS. In PBC, SOS also correlates with pruritus severity, configuring, simultaneously with sarcopenia, a scenario seriously impacting the QoL. In this picture, the observed correlation between cholestasis-promoted low vitamin D serum levels and both SOS and muscle quantity opens the doors, in the era of Precision Medicine, to further conduct research exploring the potential role of a targetable molecule, such as vitamin D, in influencing PBC course and complications.