High Dose (5mg) Daily Folic Acid Supplement in Healthy Brazilian Volunteers Increases Mononuclear TNF-Î± Expression and Reduces NK Cell Number and Activity
Authors: Bertinato Juliano , Paniz Clovis , Lucena Maylla Rodrigues , Amorim Patrícia Mendonça da Silva , Gomes Guilherme Wataru , Figueiredo Maria Stella , Green Ralph , Shinohara Elvira Maria Guerra 
Published: October 2019

Abstract
Background
In Brazil, wheat and corn flour is fortified with 150 µg of folic acid (FA), the synthetic form of folate. Individuals with increased cell duplication, including pregnant women and patients with hemolytic anemia need increased amounts of folate. The effects of amounts of FA higher than the defined tolerable upper intake of 1 mg/day are poorly understood. Some Brazilian patients with hemolytic anemia, such as hereditary spherocytosis (HS), have been receiving 5mg/day supplemental FA, in addition to being exposed to mandated food fortification with FA. Our previous data has shown that patients with HS have higher serum folate levels than healthy controls, as well as higher mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes (1). However, it was not clear whether the increased mRNA expression resulted from folic acid use or underlying disease.
Objective
The aim of this study was to verify the effects of an intervention with 5mg/day FA on folate levels (serum and whole blood), serum inflammatory markers levels, mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes and cytotoxicity of NK cells in healthy Brazilian volunteers.
Material and methods
Fifteen male and fifteen female healthy subjects were given 5mg/day FA for 90 days. Blood was collected at baseline, day 45 and day 90 for blood count, including reticulocytes, C-reactive protein and lactate dehydrogenase (LDH). Folate (serum and whole blood) and vitamin B12 were determined by a microbiological method. Serum cytokines levels were measured using a Milliplex Map kit. The mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes in mononuclear cells were performed using Real Time PCR. Cytotoxicity of lymphocytes and NK cell number were measured by flow cytometry.
Results
All blood count parameters were unaffected by FA intervention, whereas there was a slight increase in concentrations of LDH (P = 0.001) after 90 days compared with baseline and 45 day measurements. The folate levels (serum and whole blood) were higher at 45 and 90 days of intervention with 5mg/day of FA (P<0.001 for both). There were no differences among the basal and the follow up for serum vitamin B12, total homocysteine, cytokines IL6, IL8, IL10, IFNγ and TNFα levels (P>0.05). The mRNA expression of IL8 was higher at 45 days of intervention (Fig 1), while mRNA expressions of TNF-α were elevated at 45 and 90 days compared with baseline (Fig 1). No difference was found in mRNA of DHFR, MTHFR and IFNγ in this study.
The data are median and interquartile intervals. The groups were compared using the Friedman test, when significant was performed for multiple comparisons Dunn`s test. Different letters show significant differences between groups
After 5 mg FA daily there was a reduction in the number and cytotoxic capacity of NK cells (Table 1).
The data are median and interquartile intervals. The groups were compared using the Friedman test, when significant was performed for multiple comparisons Dunn`s test. Different letters show significant differences between groups.
Conclusions
Intervention with 5 mg/day of FA in healthy people was associated with around 4-fold increase in serum and whole blood folate, accompanied by increased mRNA expression of proinflammatory cytokines IL8 and TNF-α and a reduction in NK cell number and cytotoxicity. High dose FA fortification may result in changes in innate immune parameters that could perturb immune surveillance pathways.
Financing: FAPESP 2012/12912-1, CNPq 4826412012-6 and CNPq 401586/2014-6
References
1. Paniz, C et al. Blood 2014;124:4005. Presented at the ASH 2014.
Figure 1. mRNA expression of IL8 (A) and TNFα (B) genes in healthy subjects before and 45 and 90 days after 5 mg FA daily Figure 1. mRNA expression of IL8 (A) and TNFα (B) genes in healthy subjects before and 45 and 90 days after 5 mg FA daily Tabel 1 Number, lytic activity and cytotoxic capacity of NK cells after intervention with 5 mg/day of folic acid Tabel 1. Number, lytic activity and cytotoxic capacity of NK cells after intervention with 5 mg/day of folic acid

Disclosures
No relevant conflicts of interest to declare.