The Effects of 5-methyltetrahydrofolate Supplementation in Patients With Metabolic Dysfunction-Associated Steatotic Liver Disease

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly termed non-alcoholic fatty liver disease (NAFLD), is diagnosed via liver biopsy or imaging when steatosis is present in the absence of alcohol intake or other hepatic disorders. As the liver manifestation of metabolic syndrome, it commonly coexists with obesity, diabetes, dyslipidemia, hypertension, and related conditions. Global prevalence of MASLD continues to rise.

Evidence from an earlier systematic review and meta-analysis indicated that MASLD patients had significantly lower serum folate and higher homocysteine concentrations. Folate is an essential water-soluble B vitamin that occurs in multiple chemically related forms. Food folates are mainly reduced and polyglutamated, with 5-MTHF predominating in both the diet and systemic circulation. 5-MTHF does not require reduction by DHFR and can enter the bloodstream directly for use. Reduced folates act as methyl donors in one carbon metabolism, supporting cellular proliferation, homocysteine re-methylation to methionine, nucleic acid synthesis and methylation of DNA, RNA, proteins and phospholipids.

Experimental studies have demonstrated that diet-induced hyperhomocysteinemia promotes hepatic steatosis and liver injury and folate as a key regulator of homocysteine concentration, may exert hepatoprotective effects. Evidence suggests that folate may improve hepatic lipid metabolism by activating peroxisome proliferator-activated receptor alpha (PPARα) signaling and modulate the immune response and reduce inflammatory mediators. Nevertheless, no evidence on the effects of folate on PPARα and TNFα gene expression in MASLD patients exist. Moreover, PPARα gene expression is dysregulated in MASLD and related metabolic conditions; PPARα is highly expressed in the liver, skeletal muscle and brown adipose tissue, stimulates β-oxidation and suppresses fatty-acid synthesis. Although the effect of 5-MTHF supplementation on gene expression of PPARα and TNFα in MASLD patients has not been examined, evidence showed that folate can modulate PPARα and TNFα. As folate has been shown to affect lipid metabolism and inflammation, we hypothesized that 5-MTHF supplementation might regulate PPARα and TNFα expression in MASLD patients. This randomized, double-blind, placebo-controlled clinical trial will therefore be undertaken to determine the effects of 5-MTHF supplementation on serum levels of folate and homocysteine, and gene expression of PPARα and TNFα in MASLD patients.

Inclusion criteria

• Adult men or women (18-50 years)
• Diagnosis of MASLD (grade 1 or 2 of steatosis confirmed by ultrasound)
• Body mass index (BMI) = 25-34.9 kg/m²
• Providing written informed consent

Exclusion criteria

• Pregnancy, lactation, or plans to get pregnant during the next three months.
• Liver disease (viral hepatitis, autoimmune liver disease, cirrhosis, drug-induced hepatotoxicity, or alcoholic fatty liver disease), heart or renal failure, kidney stones, any neoplasia, inflammatory disease, hypothyroidism, hypercortisolism, or hypertension
• Taking drugs affecting glucose or lipid metabolism, folate supplements, anti-obesity medications, weight-loss diets, or dietary supplements
• Lifestyle factors known to impact folate status (current smoking, alcohol intake, recreational drug use)
• Pre-existing conditions affecting folate status (malabsorptive or inflammatory bowel diseases, active celiac disease, gastric bypass surgery, atrophic gastritis, epilepsy, advanced liver disease, kidney dialysis, type 1 or 2 diabetes mellitus, or sickle cell trait/anemia)
• Medications that interfere with B-vitamin metabolism (chloramphenicol, methotrexate, metformin, sulfasalazine, phenobarbital, phenytoin, primidone, triamterene, barbiturates)