A Daily Dose of 5 mg Folic Acid for 90 Days Is Associated with Increased Serum Unmetabolized Folic Acid and Reduced Natural Killer Cell Cytotoxicity in Healthy Brazilian Adults

J Nutr. 2017 Sep;147(9):1677-1685. doi: 10.3945/jn.117.247445. Epub 2017 Jul 19.


Background: The effects of high-dose folic acid (FA) supplementation in healthy individuals on blood folate concentrations and immune response are unknown.Objective: The aim of the study was to evaluate the effects of daily consumption of a tablet containing 5 mg FA on serum folate; number and cytotoxicity of natural killer (NK) cells; mRNA expression of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR), interferon γ (IFNG), tumor necrosis factor α (TNFA), and interleukin 8 (IL8) genes; and concentrations of serum inflammatory markers.Methods: This prospective clinical trial was conducted in 30 healthy Brazilian adults (15 women), aged 27.7 y (95% CI: 26.4, 29.1 y), with a body mass index (in kg/m2) of 23.1 (95% CI: 22.0, 24.3). Blood was collected at baseline and after 45 and 90 d of the intervention. Serum folate concentrations were measured by microbiological assay and HPLC-tandem mass spectrometry [folate forms, including unmetabolized folic acid (UMFA)]. We used real-time polymerase chain reaction to assess mononuclear leukocyte mRNA expression and flow cytometry to measure the number and cytotoxicity of NK cells.Results: Serum folate concentrations increased by ∼5-fold after the intervention (P < 0.001), and UMFA concentrations increased by 11.9- and 5.9-fold at 45 and 90 d, respectively, when compared with baseline (P < 0.001). UMFA concentrations increased (>1.12 nmol/L) in 29 (96.6%) participants at day 45 and in 26 (86.7%) participants at day 90. We observed significant reductions in the number (P < 0.001) and cytotoxicity (P = 0.003) of NK cells after 45 and 90 d. Compared with baseline, DHFR mRNA expression was higher at 90 d (P = 0.006) and IL8 and TNFA mRNA expressions were higher at 45 and 90 d (P = 0.001 for both).Conclusion: This noncontrolled intervention showed that healthy adults responded to a high-dose FA supplement with increased UMFA concentrations, changes in cytokine mRNA expression, and reduced number and cytotoxicity of NK cells. This trial was registered at www.ensaiosclinicos.gov.br as RBR-2pr7zp.

Keywords: 5-methyl-THF; NK cell cytotoxicity; folic acid supplementation; mRNA expression; serum folate; unmetabolized folic acid.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Brazil
  • Dietary Supplements / adverse effects*
  • Female
  • Folic Acid / administration & dosage
  • Folic Acid / adverse effects*
  • Folic Acid / blood
  • Humans
  • Immunity / drug effects
  • Inflammation Mediators / blood*
  • Interleukin-8 / blood*
  • Killer Cells, Natural*
  • Male
  • Methylenetetrahydrofolate Reductase (NADPH2) / genetics
  • Methylenetetrahydrofolate Reductase (NADPH2) / metabolism
  • Nutritional Status
  • Prospective Studies
  • RNA, Messenger / metabolism
  • Real-Time Polymerase Chain Reaction
  • Reference Values
  • Tetrahydrofolate Dehydrogenase / genetics
  • Tetrahydrofolate Dehydrogenase / metabolism
  • Tumor Necrosis Factor-alpha / blood*
  • Tumor Necrosis Factor-alpha / genetics
  • Vitamin B Complex / administration & dosage
  • Vitamin B Complex / adverse effects
  • Vitamin B Complex / blood


  • Inflammation Mediators
  • Interleukin-8
  • RNA, Messenger
  • Tumor Necrosis Factor-alpha
  • Vitamin B Complex
  • Folic Acid
  • Methylenetetrahydrofolate Reductase (NADPH2)
  • Tetrahydrofolate Dehydrogenase