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. 2025 Aug;122(2):413-423.
doi: 10.1016/j.ajcnut.2025.05.029. Epub 2025 May 31.

One-carbon metabolism-related compounds are associated with epigenetic aging biomarkers: results from the cross-sectional National Health and Nutrition Examination Survey 1999-2002

Anne K Bozack  1 , Dennis Khodasevich  2 , Jamaji C Nwanaji-Enwerem  3 , Nicole Gladish  2 , Hanyang Shen  2 , Saher Daredia  4 , Mary Gamble  5 , Belinda L Needham  6 , David H Rehkopf  7 , Andres Cardenas  8
Affiliations

One-carbon metabolism-related compounds are associated with epigenetic aging biomarkers: results from the cross-sectional National Health and Nutrition Examination Survey 1999-2002

Anne K Bozack et al. Am J Clin Nutr. 2025 Aug.

Abstract

Background: One-carbon metabolism (OCM), a biochemical pathway dependent on micronutrients including B vitamins, plays an essential role in aging-related physiological processes. DNA methylation-based aging biomarkers may be influenced by OCM.

Objectives: This study investigated associations of OCM-related biomarkers with epigenetic aging biomarkers in the cross-sectional National Health and Nutrition Examination Survey (1999-2002).

Methods: Blood DNA methylation was measured in adults aged ≥50 y. The following epigenetic aging biomarkers were included: Horvath1, Horvath2, Hannum, PhenoAge, GrimAge2, Dunedin Pace-of-Aging (DunedinPoAm), and DNA methylation telomere length (DNAmTL). We tested for associations of serum folate, red blood cell folate, vitamin B12, homocysteine (Hcy), and methylmalonic acid (MMA) concentrations with epigenetic age deviation (EAD) among 2346 participants with epigenetic and nutritional status biomarkers using adjusted survey-weighted general linear regression models.

Results: A doubling of serum folate concentration was associated with -0.82 y (95% confidence interval: -1.40, -0.23) lower GrimAge2 EAD, -0.13 SDs (-0.22, -0.03) lower DunedinPoAm, and 0.02 kb (0.00, 0.04) greater DNAmTL EAD. Conversely, a doubling in Hcy concentration was associated with 1.05 y (0.06, 2.04) greater PhenoAge EAD, 1.93 y (1.16, 2.71) greater GrimAge2 EAD, and 0.26 SDs (0.10, 0.41) greater DunedinPoAm. Associations of serum folate with EAD were attenuated after adjusting for smoking status, alcohol intake, and estimated glomerular filtration rate. Furthermore, smoking modified the associations of Hcy with GrimAge2 EAD. Chronic kidney disease modified associations of B12 and MMA with Horvath1 and GrimAge2 EAD, respectively.

Conclusions: In a nationally representative sample of United States adults, higher concentration of folate, a carbon donor, was associated with lower EAD, and higher concentration of Hcys, an indicator of OCM deficiencies, was associated with greater EAD; however, some associations were influenced by smoking and renal function. Future research should focus on high-risk populations. Long-term randomized controlled trials are also needed to establish causality and investigate the clinical relevance of changes in EAD.

Keywords: DNA methylation; National Health and Nutrition Examination Survey (NHANES); epigenetic aging; folate; homocysteine; one-carbon metabolism; vitamin B12.

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Conflict of interest statement

Conflict of interest The authors report no conflicts of interest.

Figures

Figure 1
Figure 1
One-carbon metabolism. Compounds included in this study are shown in orange. DHF, dihydrofolate; MMA, methylmalonic acid; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF, tetrahydrofolate. Adapted from Bozack et al. [5].
FIGURE 2
FIGURE 2
Associations of promoters of one-carbon metabolism (OCM) with epigenetic aging biomarkers. Effect estimates (95% confidence intervals [CIs]) and P values are shown for a doubling in concentration of each compound. Results are from weighted generalized linear regression models adjusted for age, age2, sex, race and ethnicity, BMI, education level, occupation, and poverty-to-income ratio. Effect estimates for Horvath1, Horvath2, Hannum, PhenoAge, and GrimAge2 EAD are in years; effect estimates for DunedinPoAm are in standard deviations; and effect estimates for DNAmTL EAD are in kilobases. EAD, epigenetic age deviation; eGFR, estimated glomerular filtration rate; RBC, red blood cell.
FIGURE 3
FIGURE 3
Associations of markers of one-carbon metabolism (OCM) deficiencies with epigenetic aging biomarkers. Effect estimates (95% confidence intervals [CIs]) and P values are shown for a doubling in concentration of each OCM-related compound. Results are from weighted generalized linear regression models adjusted for age, age2, sex, race and ethnicity, BMI, education level, occupation, and poverty-to-income ratio. Effect estimates for Horvath1, Horvath2, Hannum, PhenoAge, and GrimAge2 EAD are in years; effect estimates for DunedinPoAm are in standard deviations; and effect estimates for DNAmTL EAD are in kilobases. EAD, epigenetic age deviation; eGFR, estimated glomerular filtration rate; MMA, methylmalonic acid.
FIGURE 4
FIGURE 4
Scatter plots and associations of homocysteine concentration with GrimAge2 epigenetic age deviation (EAD). The transparency of points corresponds to their survey weights. Estimates are from weighted generalized linear regression models and shown for average age, BMI, alcohol intake, estimated glomerular filtration rate, and poverty-to-income ratio, and for the reference level of race and ethnicity, education level, and occupation. Interaction P value: never smokers compared with former smokers = 0.028; never smokers compared with current smokers = 0.23. ∗ P < 0.05; ∗∗ P < 0.01.
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