Folate plays an important role in one-carbon transfer reactions, participating in DNA, RNA, and protein methylation reactions; nucleic acid synthesis; and gene expression regulation (1, 2). Folate requirements are increased during pathological conditions, such as cancer and hemolytic anemias, but also during physiological conditions, such as certain age groups, pregnancy, and lactation (3–5).

To avoid folate deficiency, Brazilian patients with chronic hemolytic anemia are usually given folic acid (FA) supplements at 5 mg/d during virtually their entire lives. Furthermore, the Brazilian government recommends that women who want to conceive or are pregnant take 5 mg FA/d (6), which is higher than the 0.4–0.6 mg/d recommended in other countries with FA fortification programs, such as the United States. In fact, the 5-mg FA supplement is the main formulation available in Brazil. This vitamin dose is 5 times that of the Tolerable Upper Intake Levels for healthy individuals (7). The effects of high-dose FA supplementation on blood folate concentrations and inflammatory markers, and on the number and cytotoxicity of NK cells, are not known.

Different from the naturally occurring food folate, FA is a synthetic, oxidized form of folate that is found in fortified foods and supplements. FA must be reduced first to dihydrofolate and then to tetrahydrofolate (THF) by dihydrofolate reductase (DHFR) before it can be incorporated into the active cellular folate pool (8). Higher amounts of FA intake exceed the enzyme’s capacity to reduce the vitamin, resulting in the appearance of unmetabolized FA (UMFA) in plasma (9, 10). Thus, circulating UMFA implies, hypothetically, that the body’s capacity to convert FA to metabolically active folate has been exceeded (11).

Currently, there is concern about the possibility of potential adverse effects of FA when used in high amounts over extended periods of time. The 2015 report from the National Toxicology Program on safe use of high intakes of FA provides a current review and discussion, with special focus on four categories of potential adverse health effects: cancer, cognition in conjunction with vitamin B-12 deficiency, hypersensitivity-related outcomes, and thyroid and diabetes-related disorders (12). However, these findings still need to be reproduced in a more rigorous way, if they are detected in specific populations.

In a select population of obese, postmenopausal, white women, Troen et al. (13) found an association between plasma UMFA concentrations and reduced NK cell cytotoxicity, whereas an in vitro study carried out by Hirsch et al. (14) did not find such an association. NK cells comprise 5–25% of peripheral blood lymphocytes (15), and provide an important effector arm of the innate immune system, mediating spontaneous “natural” cytotoxicity toward certain tumor and virus-infected cells. Furthermore, NK cells are also a major source of some cytokines, such as IFN-γ and TNF-α (16, 17). However, at present, it is unclear whether increased or decreased NK activity has any beneficial or adverse health effects.

The effects of daily high dosages of FA on blood folate concentrations are well described (18, 19), but the effects on immune function in healthy individuals are less well known. Thus, the objective of this study was to evaluate the effect of a high-dose FA supplement (5 mg/d over 90 d) in common use in Brazil and other countries on blood concentrations of folate and inflammatory markers, number and cytotoxicity of NK cells, and mRNA expression of 5 genes in a small convenience sample of healthy Brazilian individuals.

Serum folate was determined by a microbiologic assay with the use of Lactobacillus casei (chloramphenicol-resistant strain NCIB 10463), and FA (F7876 Sigma Aldrich folic acid 98%) was used as calibrator (20). The limit of detection was 0.03 nmol/L.

Concentrations of serum folate forms [UMFA, tetrahydrofolate (THF), 5-methyl-THF, 5-formyl-THF, 5,10-methenyl-THF, and MeFox (an oxidation product of 5-methyl-THF)] were analyzed by HPLC-tandem MS (HPLC-MS/MS) (21). The limit of detection to UMFA, 5-methyl-THF, THF, 5-formyl-THF, 5,10-methenyl-THF, and MeFox were 0.14, 0.13, 0.25, 0.20, 0.20, and 0.10 nmol/L, respectively. Serum total folate was calculated as the sum of these folate forms excluding MeFox.

Serum vitamin B-12 was determined by microbiologic assay with the use of colistin sulfate-resistant strain of Lactobacillus leishmannii (22,23). The limit of detection was 36.9 pmol/L. Plasma total homocysteine (tHcy) was measured by using a chemiluminescent immunoassay on the IMMULITE 2000 analyzer (Siemens Healthcare). The limit of detection was 2 μmol/L.

A complete blood count and reticulocyte counts were determined in EDTA whole-blood samples on an electronic analyzer Pentra 120 (Horiba).

High-sensitivity C-reactive protein (hs-CRP) was determined by an immunoturbidimetric assay using the Roche-CRPL kit on the Cobas 8000 analyzer (Roche Diagnostics). Lactate dehydrogenase (LDH) activity was determined by an enzymatic assay using the Vitros 250 analyzer (Ortho Clinical Diagnostics).

The cytokines IL-6, IL-8, IL-10, IFN-γ and TNF-α were determined by a multiplex immunoassay, the high-sensitivity panel T Cell Magnetic Bead Milliplex Map (EMD Millipore Corporation) on the Bio-PLex 200 analyzer (Bio-Rad Laboratories, Inc.), following the manufacturer’s protocols.

Sample preparation (effector and target cells) and NK cell identification, as well as determining the number of NK cells, were performed on the day of blood collection as previous described (24). Briefly, target cells (human chronic myelogenous leukemia cell line K562 in the log phase) were previously labelled by adding Dioctadecyloxacarbocyanine perchlorate (DiO) and adjusted to a concentration of 10⁶ cells/mL in phosphate buffer. Effector (mononuclear cells) and target cells were added in tubes to create four different effector-to-target ratios of 25:1, 12.5:1, 6.25:1 and 3.125:1 (each proportion in duplicate) and incubated for 2h at 37° C in a 5% CO2 incubator, with addition of 0.15 mM propidium iodide (PI). The NK cytotoxicity assay was than performed by using FACS Canto II (BD Biosciences) equipment. A total of 20,000 events were acquired. Cells marked with both DiO and PI were considered dead target cells, whereas those positive only for DiO represented surviving target cells. Controls comprised target cells only plus PI and effector cells only plus PI and were used to determine spontaneous lysis and nonviable effector cells, respectively.

In this assay, the lytic unit was defined as the number of effector cells (NK) required to lyse 15% of target cells. Cytotoxic capacity of NK cells was defined as the ratio of the “number of NK cells in 1 mL of whole blood” per lytic unit.

DNA was obtained from total blood with the use of QIAMP Blood DNA Mini Kit (Qiagen). Genotyping for DHFR 19-bp deletion (rs70991108) was performed by PCR according to the previously described methods (25).

Peripheral blood mononuclear cell total RNA was extracted by using Trizol^® reagent (Invitrogen), following the manufacturer’s instructions. The concentration of total RNA extracted was determined by spectrophotometry at 260 nm with the use of Nanodrop ND-1000 (Thermo Scientific), and integrity was evaluated by 1% agarose gel electrophoresis stained with GelRed. The cDNA was synthesized from 500 ng of RNA by using the High-Capacity RNA-to-cDNA kit (Invitrogen Life Technologies) in a final volume of 20 μL according to the manufacturer’s instructions.

mRNA expression of DHFR, methylenetetrahydrofolate reductase (MTHFR), interferon-γ (IFNG), tumor necrosis factor-α (TNFA) and IL8 genes were performed by real-time PCR with the use of TaqMan assays (Hs00758822_s1, Hs00195560_m1, Hs00989291_m1, Hs01113624_g1, Hs01553824_g1; Applied Biosystems). Six genes [β2-microglobulin (B2M), GAPDH, hydroxymethylbilane sinthase (HMBS), hypoxantine phosphoribolsyltransferase 1 (HPRT1), actin β (ACTB), and ubiquitin C (UBC) were tested by using geNorm software (26) in order to evaluate the most stable genes under experimental conditions, and ACTB and HPRT1 was chosen as the reference genes (TaqMan assays Hs01060665_g1 and Hs02800695_m1, respectively). The mRNA expression results were normalized by ACTB and HPRT1 housekeeping mRNA expression mean and calculated by 2^−ΔCT as described by Livak and Schmittgen (27). All reactions were executed in duplicate, and each reaction plate was analyzed in the presence of a negative control to assess possible reagent contamination.

Body weight was measured with each participant wearing only light clothing and no shoes, to the nearest 0.1 kg by using a digital weight scale (Kratos). Height was measured to the nearest 0.1 cm by using a portable stadiometer (Alturexata). BMI was calculated by dividing weight (kg) divided by height- squared (in meters). Participants were classified as underweight [BMI (kg/m²) <18.5], normal weight (BMI: 18.5–24.9), overweight (BMI 25.0-29.9), or obese (BMI ≥30.0) (28).

Serum folate concentrations <7.0 and >45 nmol/L were considered to represent folate deficiency and high concentration, respectively (29). High serum UMFA was defined as >1.12 nmol/L, according to the 95th percentile in the distribution of values obtained from 1730 individuals (aged 20–39 y) by Pfeiffer et al. (30) in an NHANES study.

Plasma tHcy concentrations >13.9 μmol/L were considered to be high (31). Serum vitamin B-12 concentrations <148 pmol/L were considered to represent vitamin B1-2 deficiency (32). Anemia was defined as a hemoglobin level <120 g/L in women and <130 g/L in men (33).

Two 24-h dietary recalls were obtained in each period of the study, the first one on the day of blood collection and the second one a few days after blood collection. A randomization schedule for 24-h dietary recall collections was created to allow dietary intake representativeness for every day of the week, including weekend days. Data were entered into the Nutrition Data System for Research software version 2014, developed by the Nutrition Coordinating Center, University of Minnesota. This software uses the USDA food-composition table as its main database, from which 3 estimates are calculated: the folate naturally present in foods, the dietary FA added to fortified foods, and the total dietary folate expressed as dietary folate equivalents (DFEs). The synthetic FA, and consequently DFE, values were corrected to account for differences in the mandatory fortification amounts between Brazil (150 μg FA/100 g flour) and the United States (140 μg FA/100 g flour). The Multiple Source Method (version 1.0.1, v2008–2011; Department of Epidemiology of the German Institute of Human Nutrition Potsdam-Rehbrücke) was used to correct the effect of within-individual variability on data in order to estimate usual dietary intake distributions (deattenuated data) (34).

Statistical analyses were carried out by using SPSS version 22.0 (IBM), GraphPad Prism version 5.04 (GraphPad Software, Inc.) and Minitab version 17 software. The chi-square test, the likelihood ratio, or Fisher’s exact tests were used to analyze the frequencies of categorical variables. One-factor repeated-measure ANOVA was used to compare numeric variables at baseline and after the daily intervention with 5 mg FA/d. When we found a significant difference by using ANOVA, Tukey test was performed. When data were not distributed, Box-cox transformation was performed for normalization [all forms of folate, vitamin B-12, tHcy, hemoglobin, white blood cells, reticulocytes, LDH, hs-CRP, serum cytokines, expression of mRNA of 4 genes (DHFR, IFNG, IL8 and TNFA), and number and cytotoxicity capacity of NK cells], before using 1-factor repeated-measure ANOVA. Furthermore, transformed variables were used for the Pearson correlation. Unpaired t test was used to compare FA forms and vitamin B-12 and tHcy concentrations according to sexes at each blood collection time. Means and 95% confidence interval of back-transformed values are presented in Tables 1–4 and Figures 1 and 2.

Twenty-two univariate linear regression models were conducted for each dependent variable: log numbers of NK cells and log cytotoxic capacity of NK cells. One multiple linear regression model was performed for each dependent variable cited above. Only variables that had significant P values in univariate linear regression were included as independent variable in multiple linear regression mode. There were strong Pearson correlations between intervention with 5 mg FA/d, serum folate, serum total folate, THF, UMFA and 5-methyl-THF. Because of this, only 5-methyl-THF was included together with other variables in multiple linear regression models. The Bonferroni correction is shown in the legends for Tables 3 and 4. The level of significance was set at P <0.05.

The geometric mean age for the study participants was 27.7 y (95% CI: 26.4, 29.1 y), and all of participants reported being nonsmokers. The geometric mean BMI was 23.1 kg/m² (95% CI: 22.0, 24.3). Only 1 woman and 6 men (23%) were overweight, whereas 1 man (3.3%) was classified as obese. No participant was underweight.

Natural folate intake was higher at baseline than at T45 and T90 (P <0.001), and FA intake from fortified foods was similar when comparing baseline with T45 and T90 (P >0.05). However, total food folate intake, expressed as DFEs, did not differ significantly between the study periods (P =0.103).

Serum total folate concentrations of samples measured by microbiologic assay correlated significantly with concentrations measured by HPLC-MS/MS (Pearson correlation: r= 0.987, P <0.001, n=90). On average, serum folate concentrations obtained by HPLC-MS/MS were higher than those obtained by microbiologic assay (32% at baseline and 19% at T45 and T90), as showed in Table 1.

Serum folate (both assays), UMFA, 5-methyl-THF, THF, and MeFox concentrations increased significantly after 45 and 90 d of the FA intervention compared with baseline (Table 1). At baseline, UMFA contributed 2.6% to the concentration of serum total folate (HPLC-MS/MS), however, after the intervention, the UMFA contribution increased to 7.3% and 3.5% at T45 and T90, respectively. The contribution of 5-methyl-THF to serum total folate (HPLC-MS/MS) decreased from 95.1% at baseline to 76.6% and 82.7% at T45 and T90, respectively (Table 1). The 2 minor folate forms, serum 5-formyl-THF and 5,10-methenyl-THF, were below the limit of detection at baseline and during post-intervention in most participants.

Serum folate (both microbiologic and HPLC-MS/MS methods) and 5-methyl-THF concentrations were lower in men than in women at baseline, T45 and T90 (P < 0.050). No difference was found between men and women for UMFA, MeFox and vitamin B-12 concentrations (P > 0.050). tHcy concentrations were higher in men at all blood collection times (P < 0.050).

Concentrations of serum folate >45 nmol/L were observed in 28 (93.3%) and 30 (100%) participants during the intervention period (both at T45 and T90) with the microbiologic and HPLC-MS/MS assay, respectively. UMFA concentrations increased (>1.12 nmol/L) in 29 (96.6%) participants at T45 and in 26 (86.7%) participants at T90 (Table 1). Moreover, at the end of the intervention study, 15 (50.0%) participants had UMFA concentrations between 1.12 and 1.83 nmol/L, 6 (20%) had concentrations between 2.10 and 8.68 nmol/L, and 5 (16.6%) had concentrations between 49.5 and 278 nmol/L.

Folate deficiency (serum folate <7 nmolL) was found in 4 (13.3%) participants at baseline, but not after the intervention period, on the bases of data from the microbiologic assay. On the bases of HPLC-MS/MS, none of the participants presented with folate deficiency at either time point.

We found no differences across time points for vitamin B-12 or tHcy concentrations (Table 1). No serum vitamin B-12 deficiency was found. Although only one participant (3.3%) had values above the tHcy cutoff at baseline (15.4 μmol/L); after 45 and 90 d of intervention tHcy concentrations were reduced to 14.0 and 12.2 μmol/L, respectively.

We observed no significant differences between baseline and the intervention for white blood cells and reticulocytes or concentrations of hemoglobin and serum cytokines (Table 2). LDH activity was higher at T90, whereas hs-CRP concentrations showed reduced values at T45 when compared with baseline values (Table 2).

Intervention with FA for 45 and 90 d significantly reduced the absolute NK cell count and the cytotoxicity capacity of these cells (Figure 1). The statistical powers of these tests were 0.859 and 0.961, respectively.

We observed higher DHFR mRNA expression at T90 than at baseline and T45. We also observed higher TNFA and IL8 mRNA expression at T45 and T90 than at baseline. On the other hand, IFNG mRNA expressions was lower at T45 than at baseline, whereas no significant differences were observed for MTHFR mRNA expressions with FA intervention (Figure 2).

With regard to the 19-bp deletion on DHFR polymorphism, 6 participants were homozygoous for the 19-bp deletion, 16 participants were heterozygous, and 6 had the wild-type genotype. DHFR mRNA expression was similar among the 3 genotypes for the DHFR 19-bp deletion.

After Bonferroni correction in multivariate linear regression models, only serum 5-methyl-THF and TNF-α were inversely associated, whereas serum IFN-γ was positively associated with the log of NK cell number (Table 3). Furthermore, serum 5-methyl-THF and TNF-α were inversely associated, whereas serum IL-6 was positively associated with the log of cytotoxicity capacity of NK cells (Table 4).

This noncontrolled intervention study showed how a small group of young, healthy adults responded to a 3-mo high-dose FA supplement regimen, namely with increased concentrations of serum UMFA and reduced number and cytotoxicity of NK cells. Our study adds to the small number of studies previously reported on this topic (13, 14).

We used 2 methods to measure serum folate, and the results of the 2 methods showed strong correlations. However, folate concentrations by HPLC/MS-MS were 30% higher at baseline and 19% higher at 45 and 90 d than were folate concentrations by microbiological assay. This difference is larger than the 610% difference between the 2 assays reported previously by Fazili et al. (35) who used Lactobacillus rhamnosus as the micro-organism for their microbiological assay. We used the L. casei strain as published by O’Broin and Kelleher (20) (chloramphenicol-resistant strain NCIB 10463). Both micro-organisms should grow in the presence of multiple forms of folate, detecting all biologically active folate species equally, while excluding those without vitamin activity (36). However, the microbiological assay has not yet been standardized, and different results have been reported from different laboratories (37).

As expected, the intervention with 5 mg FA/d, even for just a short time (45 d), produced a several fold increase in serum folate concentrations. The 5-methyl-THF form was the main contributor to serum folate concentrations, increasing at the same rate as serum folate after the intervention. Detectable concentrations of serum UMFA were present in all of the participants at baseline (geometric mean: 0.59 nmol/L; 95% CI: 0.52, 0.68 nmol/L). In a previous cross-sectional study, carried out in 144 healthy Brazilian non–supplement users exposed to mandatory wheat and maize flour fortification with FA, it was observed that dietary FA was associated with UMFA concentrations (geometric mean: 0.55 nmol/L; 95% CI: 0.50, 0.61 nmol/L) (38).

The UMFA concentrations in this study were similar to those found in the cross-sectional 2007–2008 NHANES (geometric mean: 0.64 nmol/L; 95% CI: 0.51, 0.87 nmol/L) performed in the US population ~ 10 y after the introduction of mandatory food fortification (39). Not surprisingly, after the intervention with 5 mg FA/d, the majority of participants in our study showed increased UMFA concentrations, with some >50 nmol/L. Notably, total food folate and dietary FA intakes in our study were similar at baseline, T45, and T90, which confirms that the increase in UMFA at T45 and T90 was the result of the daily use of a supplement tablet with 5 mg FA.

Men showed lower serum folate and 5-methyl-THF concentrations and higher tHcy concentrations than did women at baseline and after the intervention. By using data from the NHANES, Pfeiffer et al. (39) also reported lower serum folate concentrations in men than in women [geometric mean (95% CI): 41.5 nmol/L (39.8, 43.2 nmol/L) and 45.4 nmol/L (43.7, 47.2 nmol/L), respectively], but the observed concentrations were lower in our small study [12.2 nmol/L (9.2, 16.0 nmol/L) and 24.1 nmol/L (18.7, 31.1 nmol/L) for men and women, respectively].

Whether circulating UMFA at low or high concentrations has any health effects in general, or particularly in patients who use high doses of FA for therapeutic purposes over extended periods of time, is unknown. Animal models have shown that a high-FA diet can result in reduced NK cell cytotoxicity in aged mice (40). Likewise, mice that were supplemented with high amounts of FA before being infected with malaria (Plasmodium berghei) exhibited lower numbers of specific T and NK cell subpopulations than did mice that were infected but that were fed a normal diet (41). We observed a reduced number of NK cells and decreased cytotoxicity in healthy young participants who consumed 5 mg FA/d for 45 and 90 d. Our data corroborate previous findings by Troen et al. (13) that a high FA intake from supplementation was associated with reduced cytotoxicity of NK cells in vivo in obese postmenopausal women who consumed the greatest amount of folate from their diet. However, in the present study, BMI was not associated with cytotoxicity of NK cells. The assessment of NK cell activity could be an important measure of innate immune function of these cells in some diseases (24), especially in cancer, where several studies have shown an inverse relation between NK activity and the risk of cancer (42–45).

The mechanism by which high concentrations of FA may impair the activity of NK cells remains unclear, and this may only occur in vivo, because in vitro NK cell activity was not modified by concentrations of FA and 5-methyl-THF in cell culture medium (14). We hypothesize that folate receptor 4 (FR4), present in regulatory T (Treg) cells, may be involved in this mechanism. FR4, a subtype of folate receptor, is highly expressed on the surface of Treg cells as a specific marker, distinguishing them from other native or activated T cells (46–48). In turn, Treg cells were shown to be able to profoundly inhibit NK cell functions in animal models, suggesting that Treg cells may play a major role in NK cell regulation (49, 50). However, more studies are needed to confirm this regulation and to deepen our understanding on how this mechanism works.

NK cells are main producers of inflammatory cytokines, such as IFN-γ and TNF-α, in many physiologic and pathologic conditions. They also produce a variety of immunosuppressive cytokines (e.g., IL-10), as well as growth factors (51, 52). An increase in TNF- α was observed in mice infected with malaria when they were fed a diet rich in FA but not when they were fed a normal diet (41). We did not find significant differences in serum concentrations of IL-6, IL-8, IL-10, IFN-γ, and TNF- α comparing pre- and postintervention periods, possibly because of the short time that participants were exposed to the high-dose FA supplement. However, in multiple linear regression, serum 5-methyl-THF and serum TNF- α were inversely associated, whereas serum IFN-γ was positively associated, with NK cell number. Interestingly, serum 5-methyl-THF and serum TNF-α were also inversely associated, whereas serum IL-6 was positively associated, with the cytotoxic capacity of NK cells. Furthermore, the significant increases we observed in TNFA and IL8 mRNA expression after FA supplementation further indicate a connection between higher FA concentrations and innate immunity. A longer intervention period may possibly lead to alterations in cytokine concentrations because of the increased mRNA expression we observed.

The considerable increase observed in UMFA concentrations seems to stimulate, at least at the transcription level, the production of DHFR, as observed in increased DHFR mRNA expression after 90 d of 5-mg FA/d supplementation. However, after the reduction in FA to an active form of folate, MTHFR mRNA expression was not affected. In contrast to Xu et al. (53), we did not observe any association between the presence of genotypes for 19-bp deletion in DHFR and DHFR mRNA expression.

With regard to the absence of modifications observed in vitamin B-12 and tHcy concentrations after the intervention period, it is important to highlight that both vitamin B-12 and tHcy concentrations were normal in most of our participants. Thus, it is to be expected that especially tHcy concentrations would not be further lowered by additional folate intake. One participant who showed increased tHcy concentrations at baseline showed normal concentrations after 90 d of intervention with FA. Neither vitamin B-12 nor tHcy showed any association with NK cell variables or with serum concentrations of cytokines. The slight increase in LDH activity after 90 d (P = 0.014) and a slight reduction in hs-CRP concentrations after 45 d of FA use (P = 0.006) indicate reduced inflammation and tissue damage; however, the health relevance of these modest changes is not known.

Limitations of our study include the relatively small number of individuals, the high interindividual variability in the cytotoxicity test, and the limited intervention period. Although this study represents a small convenience sample, it is important because the participants are healthy young adults. Because of the rather short intervention period, our study should be considered an exploratory laboratory study that shows how humans respond to high dosages of FA, rather than equating our findings with health outcomes. Findings could be different for patients with severe hereditary anemia, who take high FA doses for prolonged periods, sometimes during their entire life, to compensate for the higher erythropoiesis rate that results in an increased folate requirement (54, 55). Other studies with a larger number of participants are needed to confirm our findings, especially in patients who use high-FA doses for a prolonged period.