Associations Between Red Blood Cell Folate Forms and Demographic, Physiologic, and Lifestyle Factors in the US Population: Cross-Sectional Data from the National Health and Nutrition Examination Survey August 2021–August 2023

Background Red blood cell (RBC) total folate (TFOL) is an indicator of long-term folate status. Data on RBC folate forms are lacking. They may inform associations with health-related indicators. Objective We aim to describe associations between RBC folate forms and different factors in the US population. Methods We used data for 5 biologically active RBC folate forms (5-methyltetrahydrofolate [5-methylTHF], unmetabolized folic acid [UMFA], tetrahydrofolate [THF], 5-formyltetrahydro-folate [5-formylTHF], and 5,10-methenyltetrahydrofolate [5,10-methenylTHF]; sum of which is TFOL) and 1 oxidation product of 5-methylTHF (MeFox) in persons ≥6 y participating in the NHANES 2021–2023 cycle. We excluded pregnant/lactating women. Using SUDAN, we assessed bivariate associations between folate biomarkers (concentrations for TFOL, 5-methylTHF, THF, and MeFox; prevalences ≥limit of detection [LOD] for UMFA, 5-formylTHF, and 5,10-methenylTHF) and demographic, physiologic, and lifestyle variables in persons ≥20 y. We estimated the impact of covariates on biomarker concentrations or odds ratios after covariate adjustment. Results In persons ≥6 y ( n =7126), geometric mean concentrations (nmol/L) were: 913 (TFOL), 835 (5-methylTHF), 26.3 (THF), and 161 (MeFox); prevalences ≥LOD (%) were: 43.6 (UMFA), 40.6 (5-formylTHF), and 73.0 (5,10-methenylTHF); and contributions to RBC TFOL were: 92.7% (5-methylTHF), 5.02% (THF), and 1.61% (5,10-methenylTHF), varying by race/Hispanic origin. In persons ≥20 y, fasting status was not associated with biomarker concentrations or the prevalence ≥LOD. Biomarkers were significantly associated before and after covariate adjustment with age (TFOL; 5-methylTHF; THF; 5,10-methenylTHF), sex (MeFox), race/Hispanic origin (TFOL; 5-methylTHF; THF; MeFox), BMI (TFOL; 5-methylTHF), tobacco use (TFOL; THF; MeFox), and folic acid supplement use (TFOL; 5-methylTHF; THF; MeFox; 5-formylTHF; 5,10-methenylTHF). Covariates explained ≤21% of the variation of 5-methylTHF, THF, or MeFox concentrations. Conclusions Folic acid supplement use was the strongest predictor of RBC folate biomarker concentrations and detection. Differences in the methyl-to-nonmethyl makeup of RBC TFOL by race/Hispanic origin suggest potential biological and/or genetic influences.