1. Study Design This is an interventional study designed to evaluate the efficacy of oral butyrate supplementation in patients diagnosed with metabolic dysfunction-associated steatotic liver disease (MASLD).

2. Introduction MASLD is becoming an increasingly significant global public health issue, though the underlying mechanisms of this disorder are not fully understood. Emerging research highlights the crucial role of the gut microbiota, particularly through the production of short-chain fatty acids such as butyrate, in regulating metabolic processes related to MASLD. Animal studies have shown that butyrate has beneficial effects on liver function, but large-scale human studies exploring this potential are lacking. Butyrates have long been used in the treatment of irritable bowel syndrome without significant adverse effects.

3. Study Objectives

   Primary Objective:

   To assess the impact of oral butyrate supplementation on liver steatosis in patients with MASLD, measured by changes in the attenuation coefficient (ATT) using point shear wave elastography (pSWE).

   Secondary Objectives:

   • Evaluate changes in alanine aminotransferase (ALT) levels.
   • Assess the impact on inflammatory markers (high-sensitivity C-reactive protein [hsCRP], tumor necrosis factor-alpha [TNF-α], interleukin-1, interleukin-6, and cytokeratin-18).
   • Measure changes in lipid profile and stool short-chain fatty acids (SCFA).
   • Evaluate microRNA expression (miR-192, miR-885, miR-122).

4. Methods/Study Design 4.1 Study Population and Inclusion Criteria

   Inclusion Criteria:

   • Patients aged 18-70 years with confirmed MASLD based on:

     • ATT > 0.63 dB/cm/MHz measured by pSWE.
     • ALT level > 40 IU/L.

   • Patients capable of providing informed consent.

   Exclusion Criteria:

   • History of other liver diseases (e.g., viral hepatitis, alcoholic liver disease).
   • Current use of medications known to affect liver function (e.g., statins, corticosteroids).
   • Pregnant and breastfeeding women. 4.2 Sample Size and Power Calculation The study will include 200 patients with MASLD, randomly assigned in a 1:1 ratio to receive either calcium butyrate or sodium butyrate. A sample size of 200 patients was determined to ensure sufficient power to detect significant differences in primary and secondary outcomes with an alpha value of 0.05 and 80% power.

   4.3 Randomization and Blinding Participants will be randomly assigned to two groups using computer-generated randomization. The study will be single-blind, meaning that patients will not know which type of butyrate they receive.

   4.4 Interventions

   • Group 1 (Calcium Butyrate): 1000 mg/day calcium butyrate.
   • Group 2 (Sodium Butyrate): 1000 mg/day sodium butyrate.
   • Both groups will follow a Mediterranean diet (20-30 kcal/kg body weight; 35-40% fats, 20% protein, 40-45% carbohydrates).
   • Treatment will last for 12 weeks. 4.5 Outcome Measures

   Primary Outcome:

   • Change in liver steatosis measured by ATT after 12 weeks compared to baseline.

   Secondary Outcomes:

   • Changes in ALT, inflammatory markers (hsCRP, TNF-α, IL-1, IL-6, cytokeratin-18), lipid profile, and stool SCFA levels.
   • Changes in microRNA expression (miR-192, miR-885, miR-122).

5. Data Collection and Analysis 5.1 Data Collection At baseline and after 12 weeks, the following assessments will be conducted:

   • Liver steatosis measured by pSWE.
   • Blood samples for ALT, inflammatory markers, and lipid profile.
   • Stool samples for SCFA analysis.
   • MicroRNA panel analysis (miR-192, miR-885, miR-122). 5.2 Statistical Analysis Data will be analyzed using SPSS software (v. XX). Baseline and 12-week data will be compared using paired t-tests or Wilcoxon tests. ANOVA will be used to assess differences between groups. Statistical significance will be set at p < 0.05.

6. Ethics and Dissemination 6.1 Ethical Considerations The study protocol has been submitted for review by the Ethics Committee of the Clinical Center of Serbia, Belgrade. Written consent will be obtained from all participants.

6.2 Data Management All patient data will be anonymized, securely stored, and managed following local data protection regulations.

6.3 Dissemination The study results will be published in peer-reviewed scientific journals and presented at international conferences. No commercial application of the results is planned.

Inclusion Criteria:

• Patients aged over 18 years with confirmed MASLD based on:

ATT > 0.63 dB/cm/MHz measured by pSWE.

ALT level > 40 IU/L.

• Patients capable of providing informed consent.

Exclusion Criteria:

• History of other liver diseases (e.g., viral hepatitis, alcoholic liver disease).
• Current use of medications known to affect liver function (e.g., statins, corticosteroids).
• Pregnant and breastfeeding women.

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