| Sumario: | Background: One-carbon metabolism supports methyl-donor availability for cellular methylation reactions. (6S)-5-methyltetrahydrofolate dicholine salt (5-MTHF-DCh) is a source of bioactive folate and choline, and a potential modifier of one-carbon metabolism and DNA methylation. However, despite the potential relevance of this dual folate-choline formulation to dietary supplement science, its effects on methyl-donor metabolism and downstream epigenetic endpoints remain insufficiently characterized. Study Design: Here, we tested whether 5-MTHF-DCh modifies one-carbon metabolism endpoints and DNA methylation of selected repetitive elements (RE) in HepaRG cells. Differentiated HepaRG cells were treated with 5-MTHF-DCh (0, 1, 10, or 40 nM) for 96 h. Cellular S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and methionine were quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). DNA methylation was assessed for five RE (L1PA2, L1PA13, L1PREC1, Alu, and HERVK11I long terminal repeat, LTR). Transcript levels were measured by quantitative real time PCR (qRT-PCR) for DNA methylation machinery and one-carbon enzymes as well as for selected RE. Dose effects were evaluated by ANOVA with Holm-adjusted comparisons. Results: 5-MTHF-DCh increased cellular SAM by 71% at 40 nM (p < 0.01). SAH decreased modestly at 1 nM (-31%, p < 0.01), while methionine was unchanged across doses. Among the RE, L1PA2 methylation increased at all doses with an inverted U-shaped pattern, while HERVK11I LTR methylation increased in a dose-dependent manner, with a 14-fold increase at 40 nM (p < 0.001). These changes were accompanied by a trend toward increased BHMT expression at 40 nM (2.1-fold; p = 0.074), while the mRNA levels of the DNA methylation machinery remained unchanged. In HepaRG cells, 5-MTHF-DCh increased SAM and was associated with selective increases in DNA methylation at specific RE, without detectable changes in core DNA methylation machinery transcripts. Conclusions: These findings support 5-MTHF-DCh as a modulator of methyl-donor status with measurable epigenetic endpoints in a human hepatic cell model, and provide mechanistic evidence relevant to further evaluation of this folate-choline ingredient.
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