ABSTRACT

Folic acid (FA) supplements and food fortification are used to prevent neural tube defects and to lower plasma homocysteine. Through exposure to food fortification and vitamin supplement use, large populations in the United States and elsewhere have an unprecedented high FA intake. We evaluated dietary and supplemental intakes of folate and FA in relation to an index of immune function, natural killer cell (NK) cytotoxicity, among 105 healthy, postmenopausal women. Among women with a diet low in folate (<233 μg/d), those who used FA-containing supplements had significantly greater NK cytotoxicity (P = 0.01). However, those who consumed a folate-rich diet and in addition used FA supplements > 400 μg/d had reduced NK cytotoxicity compared with those consuming a low-folate diet and no supplements (P = 0.02). Prompted by this observation, we assessed the presence of unmetabolized FA in plasma as a biochemical marker of excess FA. Unmetabolized folic acid was detected in 78% of plasma samples from fasting participants. We found an inverse relation between the presence of unmetabolized FA in plasma and NK cytotoxicity. NK cytotoxicity was ∼23% lower among women with detectable folic acid (P = 0.04). This inverse relation was stronger among women ≥ 60 y old and more pronounced with increasing unmetabolized FA concentrations (P-trend = 0.002). Because of the increased intake of FA in many countries, our findings highlight the need for further studies on the effect of long-term high FA intake on immune function and health.

Folic acid (FA)4 is the parent compound of folate coenzymes whose crucial role in one-carbon transfers for the synthesis of thymidylate, purines, and biological methylation reactions renders this vitamin essential for health and well-being throughout life. The importance of adequate folate intake to public health is underlined by evidence that neural tube defects can be prevented by periconceptional intake of supplemental FA, as well as epidemiologic data linking the prevalence of cancer, cardiovascular, and other diseases to poor folate intake and status. In light of such data, the United States government mandated the fortification of flour and cereal grain products with FA (1).

The perceived safety of regular FA intake (2,3), and the clear benefits of food FA fortification for the reduction of the incidence of neural tube defect and homocysteine-lowering (48) lend support to calls to increase food FA fortification to even higher levels (9). However, few data exist on the potential effect of long-term high FA intake with respect to potentially harmful health outcomes. Such evaluation is necessary because current levels of food FA are in excess by as much as twice the target set for fortification (1012). Fortification comes on top of consumption by as much of 35% of the U.S. population of unregulated over-the-counter vitamin pills containing folic acid (13) and the availability of many breakfast cereal products that are also enriched with FA by as much as 400 μg FA/serving.

Concerns about exposure of the population to excessive FA intake have focused on the known risk of masking the hematological symptoms of vitamin B-12 deficiency, which may also lead to neurological disease (14,15). It was mainly for this reason that the Institute of Medicine recommended an Upper Limit for FA intake of 1 mg/d for adults (16). It is more difficult to define the scope of other potentially adverse effects of excessive FA intake (17). For example, there is concern that excess folate may enhance the development and progression of already existing, undiagnosed premalignant and malignant lesions (18). However, the currently available human data are insufficient to evaluate this possibility. One way of addressing this problem is to examine the relation of folate status to various health outcomes in existing studies.

In light of such concerns, we evaluated dietary and supplemental intakes of folate and FA in relation to an index of immune function, natural killer cell (NK) cytotoxicity. NK cells are important in fighting viral infections and can also kill cancer cells (19). Prompted by preliminary findings that high supplementary FA intakes were associated with reduced NK cytotoxicity among some women, we assessed the presence of unmetabolized FA in plasma as a biochemical marker of excess FA intake (2022), and determined its relation to NK cytotoxicity.

SUBJECTS AND METHODS

Study population.

The study population was described previously (23). Briefly, participants (n = 105) were a subset (who met eligibility criteria for participation in a study of immune function) of a study population of women in the greater Seattle area recruited for an exercise intervention trial during 1998–2000 (24,25). Eligibility criteria were as follows: postmenopausal; age 50–75 y; in good health; nonsmoking; sedentary; no hormone-replacement therapy in the past 6 mo; alcohol consumption <2 drinks/d (∼26 g); BMI between 25 and 40 kg/m2 or BMI 24.0–24.9 kg/m2 if body fat > 33% by bioelectric impedance; no history of invasive cancer, diabetes, cardiovascular disease, asthma; no current serious allergies; no regular (≥2 times/wk) use of aspirin or other nonsteroidal anti-inflammatory medications; no use of corticosteroids or other medications known to affect immune function. We also excluded study participants with a reported energy intake <600 kcal/d (2.510 MJ) or > 4000 kcal/d (16.736 MJ) (n = 4), because nutrient calculations in this range are not reliable.

This study reports the relation between dietary folate intakes or unmetabolized FA and NK cytotoxicity, all measured before entering any intervention. The study procedures were approved by the Fred Hutchinson Cancer Research Center's Institutional Review Board and all study participants provided written informed consent.

Dietary and supplemental folate intakes.

Nutrient intakes from dietary sources were obtained at the time of entry into the study using a 120-item FFQ designed and validated at the Fred Hutchinson Cancer Research Center (26,27). Intake of individual nutrients was calculated using algorithms for nutrient calculation from the University of Minnesota Nutrition Coding Center nutrient database (26,28). The calculation of folate intake was based on revised values of the database reflecting the fortified levels.

Use of nutritional supplements was ascertained during an in-person interview. Study participants were asked to bring all nutritional supplements currently used to the clinic visit. Labels of the supplements were photocopied, abstracted, and data entered. The number of months the supplements were used during the past 12-mo period was recorded, as was the number of pills per week or day. From these data, the current daily FA intake from nutritional supplements was calculated. Only supplements that were used at least 1 time/wk during the past 3 mo were included.

Questionnaires were used to collect information on demographic and other factors that may affect immune function.

Folate and FA assays.

Plasma concentrations of 5-methyl-tetrahydrofolate (THF), unmetabolized FA, and total plasma folate were measured in fasting subjects in 2003 by combined affinity HPLC with electrochemical detection at the Jean Mayer USDA Human Nutrition Aging Center at Tufts University, Boston, MA as described previously (22). Briefly, plasma was diluted 10-fold with extraction buffer (0.05 mol/L potassium tetraborate, 1% sodium ascorbate, pH 9.2), heat extracted (100°C for 15 min), and centrifuged for 15 min at 36,000 × g. The supernatant fraction (2 mL) was injected onto the affinity column (10 × 4.6 mm) that contained purified milk folate binding protein covalently bound to AffiPrep 10 support (Bio-Rad). After the affinity column was washed sequentially with 0.05 mol/L potassium phosphate, pH 7, and water, the folates were eluted onto the analytical column (Betasil Phenyl, 250 × 4.6 mm; Keystone Scientific) with an acid mobile phase (0.028 mol/L dipotassium phosphate and 0.06 mol/L phosphoric acid in water). Folates then were eluted from the analytical column using the same aqueous mobile phase at a flow rate of 1 mL/min for 6 min followed by a linear gradient over 50 min to the same mobile phase containing 20% acetonitrile (v:v). This elution separates folates according to both their pteridine ring structure and the number of glutamate residues. Plasma folate forms eluted in the order of 5-methyl-THF followed by FA (pteroylglutamate). Folate activity was determined using an ESA Four Channel Coularray Detector with channels 1–4 set at 0, 300, 500, and 600 mV, respectively. Quantification and identification of individual folates were done by comparison with external folate standards of known concentration.

NK Cytotoxicity.

Blood was drawn from fasting subjects between 0730–0830 at the University of Washington under observance of strict criteria [described in reference (23)] that excluded the presence of any infectious symptoms, or the possible influence of other factors known to affect immune function (exercise, sleep patterns, use of nonsteroidal or immunosuppressive medications). All blood draws occurred between May 1998 and July 2000, a time period in which FA fortification had been mandated. Blood samples were processed within 1–2 h of the blood draw; the NK cytotoxicity assay was begun at that time and completed within the same day. A 4-color flow cytometer (XL-MCL, Beckman Coulter) was used to enumerate NK in blood samples, as described previously (23). The flow-cytometric NK cytotoxicity assay used here was also described previously (23), and results correlate well with the chromium release assay. Briefly, mononuclear cells were prepared by Ficoll-Hypaque differential centrifugation of blood effector cells, diluted according to the final effector-to-target (E:T) cell ratios of 50:1, 25:1, 12.5:1, and 6.25:1, and incubated with the DiO-labeled K562 cell suspension (target cells) for 4 h at 37°C with 5% CO2. After incubation, propidium iodide (PI, 0.03 g/L final concentration) was added to each tube to identify dead cells. The percentage of dead target cells (i.e., dual positive for DiO and PI) among total DiO-identified target cells was used as the measure of NK cytotoxicity. Each assay was performed in duplicate and with appropriate controls. We repeated the NK cytotoxicity assay in 13 study participants who underwent additional blood draws between 1 wk and 9 mo after the initial blood draw, using identical blood draw criteria. Intraclass correlation coefficients between the initial and repeat blood draws were: r = 0.84 (E:T 6.25:1), r = 0.91 (E:T 12.5:1), r = 0.90 (E:T 25:1), r = 0.79 (E:T 50:1), demonstrating high reproducibility. We used the intermediate dilutions (E:T 12.5:1 and 25:1) in our analysis because they showed the highest reproducibility (r ≥ 0.90) and were in the linear range of the NK cytotoxicity curve; NK cytotoxicity frequently reached a plateau at the 50:1 ratio, and there was little target-cell cytotoxicity at the 6.25:1 ratio.

Statistical analysis.

Regression analysis was used to investigate associations between dietary intakes, plasma folate or FA, and NK cytotoxicity, adjusting for potential confounding factors (see below). NK cytotoxicity (E:T 25:1 and 12.5:1) was investigated as a paired measurement; we also fitted models including all 4 E:T ratios and confirmed similar results and identical trends. The paired NK cytotoxicity measurements were analyzed via the Generalized Estimating Equation, accounting for the potential within-person correlation of the 2 NK cytotoxicity measurements (29). Exposures of interest were dietary and supplemental intakes of folate or FA, and subsequent plasma concentrations of unmetabolized FA, 5-methyl-THF, and total plasma folate, which were entered into regression models as categorical variables (tertiles) to allow a nonlinear trend. We also investigated the association between unmetabolized FA, 5-methyl-THF, and total plasma folate both as continuous linear variables and grouped by approximate tertiles. All models were adjusted for factors that were associated previously with NK cytotoxicity in this population: age (continuous), education (3 categories), employment (3 categories), income (3 categories), race (2 categories), energy intake (continuous), and multivitamin use (yes/no). Because immune function declines with age, we also investigated the associations stratified by age group (50–59 and 60–75 y). The percentage (relative) difference was computed by comparing adjusted means of NK cytotoxicity for ET 12.5 and ET 25:1. Analyses were performed using SAS version 8.02 (SAS Institute). Values in the text are means ± SD.

RESULTS

Characteristics of the study participants are described in Table 1. All women were postmenopausal and overweight or obese (BMI ≥25.0 mg/kg2), with 49% classified as obese (BMI ≥30.0 mg/kg2). Per study exclusion criteria, none were taking hormone-replacement therapy or were current smokers. Multivitamins were used by 54% of the study population and 3% used FA supplements. Characteristics of the study population did not differ depending on the presence of unmetabolized FA in their plasma.

TABLE 1

Characteristics of the study population

Characteristic
Age, y 60.2 ± 6.6 
BMI, kg/m2 30.3 ± 3.9 
Race/Ethnicity, n (% 
    Caucasian 93 (89) 
    Non-Caucasian 11 (11) 
Energy intake,2kcal/d 1678 ± 614 
Dietary folate intake, μg/d 304 ± 126 
Supplemental FA intake, μg/d 255 ± 285 
Multivitamin use, n (%57 (54) 
Plasma FA, nmol/L 2.31 ± 1.91 
Plasma 5-methyl-THF, nmol/L 42.7 ± 20.8 
Plasma total folate, nmol/L 45.0 ± 21.1 
NK cytotoxicity, 12.5:1 E:T ratio 20.1 ± 12.3 
NK cytotoxicity, 25:1 E:T ratio 27.2 ± 13.6 
NK, n 160 ± 98 
Characteristic
Age, y 60.2 ± 6.6 
BMI, kg/m2 30.3 ± 3.9 
Race/Ethnicity, n (% 
    Caucasian 93 (89) 
    Non-Caucasian 11 (11) 
Energy intake,2kcal/d 1678 ± 614 
Dietary folate intake, μg/d 304 ± 126 
Supplemental FA intake, μg/d 255 ± 285 
Multivitamin use, n (%57 (54) 
Plasma FA, nmol/L 2.31 ± 1.91 
Plasma 5-methyl-THF, nmol/L 42.7 ± 20.8 
Plasma total folate, nmol/L 45.0 ± 21.1 
NK cytotoxicity, 12.5:1 E:T ratio 20.1 ± 12.3 
NK cytotoxicity, 25:1 E:T ratio 27.2 ± 13.6 
NK, n 160 ± 98 
1

Values are means ± SD or n (%), n = 105.

2

To convert to kJ/d, multiply by 4.184.

TABLE 1

Characteristics of the study population

Characteristic
Age, y 60.2 ± 6.6 
BMI, kg/m2 30.3 ± 3.9 
Race/Ethnicity, n (% 
    Caucasian 93 (89) 
    Non-Caucasian 11 (11) 
Energy intake,2kcal/d 1678 ± 614 
Dietary folate intake, μg/d 304 ± 126 
Supplemental FA intake, μg/d 255 ± 285 
Multivitamin use, n (%57 (54) 
Plasma FA, nmol/L 2.31 ± 1.91 
Plasma 5-methyl-THF, nmol/L 42.7 ± 20.8 
Plasma total folate, nmol/L 45.0 ± 21.1 
NK cytotoxicity, 12.5:1 E:T ratio 20.1 ± 12.3 
NK cytotoxicity, 25:1 E:T ratio 27.2 ± 13.6 
NK, n 160 ± 98 
Characteristic
Age, y 60.2 ± 6.6 
BMI, kg/m2 30.3 ± 3.9 
Race/Ethnicity, n (% 
    Caucasian 93 (89) 
    Non-Caucasian 11 (11) 
Energy intake,2kcal/d 1678 ± 614 
Dietary folate intake, μg/d 304 ± 126 
Supplemental FA intake, μg/d 255 ± 285 
Multivitamin use, n (%57 (54) 
Plasma FA, nmol/L 2.31 ± 1.91 
Plasma 5-methyl-THF, nmol/L 42.7 ± 20.8 
Plasma total folate, nmol/L 45.0 ± 21.1 
NK cytotoxicity, 12.5:1 E:T ratio 20.1 ± 12.3 
NK cytotoxicity, 25:1 E:T ratio 27.2 ± 13.6 
NK, n 160 ± 98 
1

Values are means ± SD or n (%), n = 105.

2

To convert to kJ/d, multiply by 4.184.

Unmetabolized FA was detected in the plasma of 78% of the women in this study. Concentrations of plasma folate metabolites were 2.29 ± 1.91 nmol/L for unmetabolized FA, 42.7 ± 20.9 nmol/L for 5-methyl-THF, and 45.0 ± 21.2 nmol/L for total folate. The presence and concentration of plasma unmetabolized FA were not correlated with plasma total folate.

Our analyses of unmetabolized FA in plasma were prompted by an initial observation of an apparent inverse U-shaped relation between dietary folate or supplemental FA and NK cytotoxicity (Fig. 1). For this analysis between source and dose of folate and NK cytotoxicity, we dichotomized dietary folate at <233 μg/d (equivalent to the lowest tertile of folate intake in this population); further, we categorized supplemental FA use into 0 μg/d, 0–400 μg/d, and >400 μg/d, reflecting common amounts used in nutritional supplements. Among women with a low dietary intake (<233 μg folate/d), supplemental folate intake up to 400 μg/d was associated with higher mean NK cytotoxicity (P = 0.01); however, women who had greater dietary intakes (>233 μg/d) did not show additional benefits if they also consumed supplemental FA. Concern arose over our observation that women with higher dietary intakes of folate had significantly lower NK cytotoxicity if they also consumed supplemental FA in excess of 400 μg (P = 0.02).

FIGURE 1

Association between FA intake from supplements and NK cytotoxicity among postmenopausal women, stratified by folate intake from diet. NK cytotoxicity is displayed at an E:T cell ratio of 25:1; bars represent adjusted means ± SEM, n = 105. In the presence of low dietary folate, modest supplementary FA appears to increase NK cytotoxicity; in the presence of higher dietary folate, high supplementary FA may suppress NK cytotoxicity.

In light of this observation, we subsequently investigated the relation between the presence of unmetabolized FA and NK cytotoxicity. NK cytotoxicity was significantly lower in women for whom unmetabolized FA was detected in plasma (Fig. 2A). In the multivariable-adjusted regression models among all study participants, NK cytotoxicity (%) was 6.2% lower in women with unmetabolized plasma FA compared with those without plasma FA present (P = 0.04). This corresponds to a relative difference of ∼23% (e.g., the ratio of the mean NK cytotoxicity between women with and without detectable FA was 0.77) (Table 2).

FIGURE 2

NK cytotoxicity among postmenopausal women stratified by the presence of unmetabolized FA in plasma. NK cytotoxicity is displayed at the E:T cell ratios of 25:1 (black bars) and 12.5:1 (white bars). Bars represent adjusted means ± SEM, n = 105. P-values are derived from a combined analysis of both E:T ratios by multivariable generalized estimating equation. Panels represent: (A) all women, any plasma FA vs. none; (B) women 50–59 y old, approximate tertiles of plasma FA; (C) women 60–75 y old, approximate tertiles of plasma FA. Overall, NK cytotoxicity was lower in women with detectable unmetabolized FA in plasma, an association explained largely by the pattern seen for older women in which there was a significant inverse trend.

TABLE 2

NK cytotoxicity across tertiles of folate or FA among postmenopausal women stratified by age12

All womenAge 50–59 yAge 60–75 y
nDifference (β)P-valuenDifference (β)P-valuenDifference (β)P-value
FA, nmol/L          
    0–1.7 34 Ref. Ref. 17 Ref. Ref. 17 Ref. Ref. 
    1.8–3.0 38 −2.30 0.43 23 −3.10 0.40 15 −9.13 0.07 
    3.0+ 33 −5.12 0.09 20 −2.96 0.36 13 −12.83 0.001 
    (P-trend)   (0.09)   (0.38)   (0.002) 
5-Methyl-THF, nmol/L          
    0–31.9 35 Ref. Ref. 19 Ref. Ref. 16 Ref. Ref. 
    32.0–51.9 37 −2.90 0.30 25 −6.00 0.04 12 −3.63 0.57 
    52.0+ 33 4.48 0.17 16 2.55 0.48 17 −0.19 0.98 
    (P-trend)   (0.25)   (0.50)   (0.79) 
Total plasma folate, nmol/L          
    0–34.9 31 Ref. Ref. 16 Ref. Ref. 15 Ref. Ref. 
    35.0–53.9 40 −4.86 0.09 27 −8.09 0.02 13 −5.05 0.43 
    54.0+ 34 2.90 0.39 17 0.63 0.86 17 −0.52 0.93 
 (P-trend)   (0.47)   (0.64)   (0.66) 
All womenAge 50–59 yAge 60–75 y
nDifference (β)P-valuenDifference (β)P-valuenDifference (β)P-value
FA, nmol/L          
    0–1.7 34 Ref. Ref. 17 Ref. Ref. 17 Ref. Ref. 
    1.8–3.0 38 −2.30 0.43 23 −3.10 0.40 15 −9.13 0.07 
    3.0+ 33 −5.12 0.09 20 −2.96 0.36 13 −12.83 0.001 
    (P-trend)   (0.09)   (0.38)   (0.002) 
5-Methyl-THF, nmol/L          
    0–31.9 35 Ref. Ref. 19 Ref. Ref. 16 Ref. Ref. 
    32.0–51.9 37 −2.90 0.30 25 −6.00 0.04 12 −3.63 0.57 
    52.0+ 33 4.48 0.17 16 2.55 0.48 17 −0.19 0.98 
    (P-trend)   (0.25)   (0.50)   (0.79) 
Total plasma folate, nmol/L          
    0–34.9 31 Ref. Ref. 16 Ref. Ref. 15 Ref. Ref. 
    35.0–53.9 40 −4.86 0.09 27 −8.09 0.02 13 −5.05 0.43 
    54.0+ 34 2.90 0.39 17 0.63 0.86 17 −0.52 0.93 
 (P-trend)   (0.47)   (0.64)   (0.66) 
1

Combined analysis of E:T ratios 25:1 and 12.5:1 by generalized estimating equation, P-values in comparison to referent (Ref.) group.

2

Models adjusted for age (continuous), education (3 categories), employment (3 categories), race (2 categories), energy intake (continuous), and multivitamin use (yes/no).

TABLE 2

NK cytotoxicity across tertiles of folate or FA among postmenopausal women stratified by age12

All womenAge 50–59 yAge 60–75 y
nDifference (β)P-valuenDifference (β)P-valuenDifference (β)P-value
FA, nmol/L          
    0–1.7 34 Ref. Ref. 17 Ref. Ref. 17 Ref. Ref. 
    1.8–3.0 38 −2.30 0.43 23 −3.10 0.40 15 −9.13 0.07 
    3.0+ 33 −5.12 0.09 20 −2.96 0.36 13 −12.83 0.001 
    (P-trend)   (0.09)   (0.38)   (0.002) 
5-Methyl-THF, nmol/L          
    0–31.9 35 Ref. Ref. 19 Ref. Ref. 16 Ref. Ref. 
    32.0–51.9 37 −2.90 0.30 25 −6.00 0.04 12 −3.63 0.57 
    52.0+ 33 4.48 0.17 16 2.55 0.48 17 −0.19 0.98 
    (P-trend)   (0.25)   (0.50)   (0.79) 
Total plasma folate, nmol/L          
    0–34.9 31 Ref. Ref. 16 Ref. Ref. 15 Ref. Ref. 
    35.0–53.9 40 −4.86 0.09 27 −8.09 0.02 13 −5.05 0.43 
    54.0+ 34 2.90 0.39 17 0.63 0.86 17 −0.52 0.93 
 (P-trend)   (0.47)   (0.64)   (0.66) 
All womenAge 50–59 yAge 60–75 y
nDifference (β)P-valuenDifference (β)P-valuenDifference (β)P-value
FA, nmol/L          
    0–1.7 34 Ref. Ref. 17 Ref. Ref. 17 Ref. Ref. 
    1.8–3.0 38 −2.30 0.43 23 −3.10 0.40 15 −9.13 0.07 
    3.0+ 33 −5.12 0.09 20 −2.96 0.36 13 −12.83 0.001 
    (P-trend)   (0.09)   (0.38)   (0.002) 
5-Methyl-THF, nmol/L          
    0–31.9 35 Ref. Ref. 19 Ref. Ref. 16 Ref. Ref. 
    32.0–51.9 37 −2.90 0.30 25 −6.00 0.04 12 −3.63 0.57 
    52.0+ 33 4.48 0.17 16 2.55 0.48 17 −0.19 0.98 
    (P-trend)   (0.25)   (0.50)   (0.79) 
Total plasma folate, nmol/L          
    0–34.9 31 Ref. Ref. 16 Ref. Ref. 15 Ref. Ref. 
    35.0–53.9 40 −4.86 0.09 27 −8.09 0.02 13 −5.05 0.43 
    54.0+ 34 2.90 0.39 17 0.63 0.86 17 −0.52 0.93 
 (P-trend)   (0.47)   (0.64)   (0.66) 
1

Combined analysis of E:T ratios 25:1 and 12.5:1 by generalized estimating equation, P-values in comparison to referent (Ref.) group.

2

Models adjusted for age (continuous), education (3 categories), employment (3 categories), race (2 categories), energy intake (continuous), and multivitamin use (yes/no).

When stratified by age (age 50–59 vs. 60–75 y), a strong inverse association between plasma FA and NK cytotoxicity was apparent among women 60–75 y old (Fig. 2C). Mean NK cytotoxicity decreased significantly and linearly with higher concentrations of unmetabolized FA (P-trend = 0.002), resulting in a relative difference of ∼25% lower NK cytotoxicity among women with plasma concentrations >3.1 nmol/L compared with those without detectable unmetabolized FA. The associations between unmetabolized FA and NK cytotoxicity remained significant when 5-methyl-THF or plasma folate were simultaneously included in the model, thus demonstrating that the association was independent of these other plasma concentrations (Table 2).

Among women 50–59 y old, the presence of FA was also associated with lower NK cytotoxicity but this was not statistically significant (Fig. 2B). Although NK cytotoxicity was reduced within the intermediate category of 5-methyl or total plasma folate, there were no clear trends for 5-methyl-THF in either age group, suggesting that this was a chance association.

No statistically significant associations were observed in corresponding analyses when the absolute number of NK cells (CD3CD15+CD56+CD45+) rather than NK cytotoxicity was used as an outcome.

DISCUSSION

In the present study, unmetabolized FA was present in the circulation of 78% of women in the study population. Natural dietary folates occur in reduced and substituted forms of the vitamin, whereas FA, the synthetic form of the vitamin, is fully oxidized and unsubstituted.

Ingested FA can be converted to its physiological forms. This process is initiated by dihydrofolate reductase (DHFR) in a two-step reaction; the first step, conversion to dihydrofolate (DHF), is a slow and rate-limiting step (30). In the second, more rapid, step DHF is further reduced to tetrahydrofolate (THF). THF can then be converted into additional physiological folates including 5-methyl-THF, the form that is normally found in the circulation (31). The human intestine contains DHFR. However, the capacity of this enzyme is limited and when supplemental FA is in excess, a large proportion of ingested FA appears in its unmetabolized form in blood (20,21). Eventually, however, the unmetabolized FA is converted to the reduced forms of folate by peripheral tissues.

In the present study, which used a highly sensitive methodology, FA was detectable in plasma collected from individuals after an overnight fast. In most cases, the amount of FA represented only a small fraction of total folate, which was otherwise comprised exclusively of 5-methyl-THF. In other studies that measured plasma FA, the concentrations were much higher than those seen in the present study (20,21). This difference is likely due to the use of plasma samples from nonfasting subjects after the ingestion of excess FA. Our study population of well-educated postmenopausal women consumed more multivitamins and other supplements than the average U.S. population (13), which may help explain the high prevalence of circulating plasma FA in this population.

Our data showing that unmetabolized FA in plasma is associated with decreased NK cytotoxicity are a cause for concern. This association with FA was independent of circulating 5-methyl-THF and total folate. The association was strong and significant among women 60–75 y old. This is consistent with findings showing that immune function may be more easily modulated among the elderly (32).

Nutritional factors such as vitamin E, zinc, and multivitamin supplements are widely recognized as important determinants of immune function (32). However, few studies have examined the relation of folate to immune function in general and to NK cytotoxicity in particular. Kim et al. (33) showed that folate deficiency can diminish NK cytotoxicity in rats, findings that are consistent with our findings of greater NK cytotoxicity with FA supplement use among women with low dietary folate intakes. A study in an Italian population of healthy 90- to 106-y olds found no correlation between plasma total folate concentration and NK cytotoxicity (34). However, as mentioned above, the decrease that we observed in NK cytotoxicity in association with unmetabolized plasma FA was independent of total folate and 5-methyl-THF concentrations. Subjects in the Italian study did not use nutritional supplements, and thus plasma folic acid was not measured.

NK cells are part of the nonspecific immune response and can kill a variety of normal and virus-infected cells without prior sensitization. Experimental and clinical evidence supports a role of NK cells in tumor cell destruction; thus, this component of the immune system may be considered a first line of host defense against carcinogenesis (19). Although the relation between in vitro NK cytotoxicity and in vivo cytotoxicity is incompletely understood, decreased NK cytotoxicity may increase the risk or severity of infections and was associated with increased future cancer incidence in a Japanese cohort study (35). If excess folate does in fact suppress NK cytotoxicity in vivo, then this would suggest another way in which excess folate might promote existing premalignant and malignant lesions.

At present, we lack a clear mechanistic explanation for our observation. Preliminary data from mathematical modeling indicate that very high folate intakes may create biochemical conditions similar to those of folate deficiency (36). Our study raises concern about possible direct toxicity of FA, but it does not exclude the possibility of an underlying biologic defect among some women, which may result in the appearance of unmetabolized FA in plasma and concurrently have adverse effects on NK cytotoxicity. Although our study was prompted by initial observations of lower NK cytotoxicity among women with high folate supplementation, we did not observe a correlation between dietary/supplementary FA intake based on the questionnaire data and measured plasma FA concentrations. It is possible that a low capacity to metabolize large amounts of FA might be a metabolic correlate of immune function. To explore this possibility, the metabolic and functional effects of polymorphisms in the DHFR gene (37,38), and their relation to immune function merit further investigation.

Much of our knowledge of the relation between folate and the immune system derives from studies of nutritional deficiencies or the use of antifolate drugs. Few studies were conducted on the condition of high folate intake. The possibility of an adverse effect of excess total folate or FA intake in humans, in the range of current population levels of dietary intake, was raised recently in relation to a possible adverse effect on cognitive function (39). Further, several studies in rats showed adverse effects of excess FA. Although dietary FA enrichment of 8 mg FA/kg diet (3 times the recommended levels for rodents) did not diminish NK cytotoxicity in rats, diets containing 40 mg FA/kg diet (20 times the recommended dietary folate concentration for rats) accelerated cancer progression in rodent models of cancer (18,40,41). More recent studies found that dietary intake of 40 mg FA/kg diet reduced birth weight and size in the offspring of pregnant rats consuming high FA compared with rats consuming a control diet (2 mg/kg). High dietary FA was also associated with elevations of the methyl donor S-adenosyl-methionine and decreases in the efficiency of nitrogen metabolism (4244). Finding low NK cytotoxicity in elderly people with unmetabolized FA in the circulation is a concern that has possible public health implications.

This study highlights the need for a better understanding of the relation of folate metabolism, immune function, and health. Considering the increased intake of FA in the population (10,11), our finding of an adverse relation between circulating FA and NK cytotoxicity must be corroborated in larger studies. Until additional studies are conducted, calls for further increases in food FA fortification and intake should be viewed with caution.

We thank Maggie Mayes and Judy Schwartz for performing the immune assays, and Kristin LaCroix and the FHCRC study staff for management of the IMEX study.

Literature Cited

1.

U.S. Food and Drug Administration
FDA food standards: amendments of the standards identity for enriched grain products to require addition of folic acid.
United States Department of Health and Human Services, Food and Drug Administration
;
1996
. p.
8781
807
.

2.

Brattstrom
LE
,
Israelsson
B
,
Jeppsson
JO
,
Hultberg
BL
.
Folic acid—an innocuous means to reduce plasma homocysteine
.
Scand J Clin Lab Invest.
1988
;
48
:
215
21
.

3.

Butterworth
C
Jr,
Tamura
T
.
Folic acid safety and toxicity: a brief review
.
Am J Clin Nutr.
1989
;
50
:
353
8
.

4.

Centers for Disease Control and Prevention
Spina bifida and anencephaly before and after folic acid mandate—United States, 1995–1996 and 1999–2000
.
MMWR Morb Mortal Wkly Rep.
2004
;
53
:
362
5
.

5.

Mathews
TJ
,
Honein
MA
,
Erickson
JD
.
Spina bifida and anencephaly prevalence—United States, 1991–2001
.
MMWR Recomm Rep.
2002
;
51
:
9
11
.

6.

Neuhouser
ML
,
Beresford
SA
.
Folic acid: are current fortification levels adequate?
Nutrition.
2001
;
17
:
868
72
.

7.

Williams
LJ
,
Mai
CT
,
Edmonds
LD
,
Shaw
GM
,
Kirby
RS
,
Hobbs
CA
,
Sever
LE
,
Miller
LA
,
Meaney
FJ
,
Levitt
M
.
Prevalence of spina bifida and anencephaly during the transition to mandatory folic acid fortification in the United States
.
Teratology.
2002
;
66
:
33
9
.

8.

Selhub
J
,
Jacques
PF
,
Bostom
AG
,
Wilson
PW
,
Rosenberg
IH
.
Relationship between plasma homocysteine and vitamin status in the Framingham study population. Impact of folic acid fortification
.
Public Health Rev.
2000
;
28
:
117
45
.

9.

Oakley
GP
Jr,
Adams
MJ
,
Dickinson
CM
.
More folic acid for everyone, now
.
J Nutr.
1996
;
126
:
751S
5
.

10.

Quinlivan
EP
,
Gregory
JF
3rd
.
Effect of food fortification on folic acid intake in the United States
.
Am J Clin Nutr.
2003
;
77
:
221
5
.

11.

Choumenkovitch
SF
,
Selhub
J
,
Wilson
PW
,
Rader
JI
,
Rosenberg
IH
,
Jacques
PF
.
Folic acid intake from fortification in United States exceeds predictions
.
J Nutr.
2002
;
132
:
2792
8
.

12.

Shane
B
.
Folate fortification: enough already?
Am J Clin Nutr.
2003
;
77
:
8
9
.

13.

Radimer
K
,
Bindewald
B
,
Hughes
J
,
Ervin
B
,
Swanson
C
,
Picciano
MF
.
Dietary supplement use by US adults: data from the National Health and Nutrition Examination Survey, 1999–2000
.
Am J Epidemiol.
2004
;
160
:
339
49
.

14.

Herbert
V
,
Bigaouette
J
.
Call for endorsement of a petition to the Food and Drug Administration to always add vitamin B-12 to any folate fortification or supplement
.
Am J Clin Nutr.
1997
;
65
:
572
3
.

15.

Reynolds
EH
.
Benefits and risks of folic acid to the nervous system
.
J Neurol Neurosurg Psychiatry.
2002
;
72
:
567
71
.

16.

Institute of Medicine
Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline.
Washington (DC)
:
National Academy Press
;
1998
.

17.

Yetley
EA
,
Rader
JI
.
Modeling the level of fortification and post-fortification assessments: U.S. experience
.
Nutr Rev.
2004
;
62
:
S50
9
.

18.

Kim
YI
.
Will mandatory folic acid fortification prevent or promote cancer?
Am J Clin Nutr.
2004
;
80
:
1123
8
.

19.

Janeway
CA
,
Travers
P
,
Walport
MJ
,
Capra
JD
.
Immunobiology: the immune system in health and disease.
4th ed.
New York
:
Elsevier Science Ltd/Garland Publishing
;
1999
.

20.

Kelly
P
,
McPartlin
J
,
Goggins
M
,
Weir
DG
,
Scott
JM
.
Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements
.
Am J Clin Nutr.
1997
;
65
:
1790
5
.

21.

Ghandour
H
,
Bagley
PJ
,
Shemin
D
,
Hsu
N
,
Jacques
PF
,
Dworkin
L
,
Bostom
AG
,
Selhub
J
.
Distribution of plasma folate forms in hemodialysis patients receiving high daily doses of L-folinic or folic acid
.
Kidney Int.
2002
;
62
:
2246
9
.

22.

Bagley
PJ
,
Selhub
J
.
Analysis of folate form distribution by affinity followed by reversed-phase chromatography with electrical detection
.
Clin Chem.
2000
;
46
:
404
11
.

23.

Shade
ED
,
McTiernan
A
,
Wener
MH
,
Wood
B
,
Yasui
Y
,
LaCroix
K
,
Potter
J
,
Ulrich
CM
.
Frequent intentional weight loss is associated with lower natural killer cell cytotoxicity constituting possible long-term effects on immune function
.
J Am Diet Assoc.
2004
;
104
:
903
12
.

24.

Irwin
ML
,
Yasui
Y
,
Ulrich
CM
,
Bowen
D
,
Rudolph
RE
,
Schwartz
RS
,
Yukawa
M
,
Aiello
E
,
Potter
JD
,
McTiernan
A
.
Effect of exercise on total and intra-abdominal body fat in postmenopausal women: a randomized controlled trial
.
JAMA.
2003
;
289
:
323
30
.

25.

McTiernan
A
,
Ulrich
CM
,
Yancey
D
,
Slate
S
,
Nakamura
H
,
Oestreicher
N
,
Bowen
D
,
Yasui
Y
,
Potter
J
,
Schwartz
R
.
The Physical Activity for Total Health (PATH) Study: rationale and design
.
Med Sci Sports Exerc.
1999
;
31
:
1307
12
.

26.

Kristal
AR
,
Vizenor
NC
,
Patterson
RE
,
Neuhouser
ML
,
Shattuck
AL
,
McLerran
D
.
Precision and bias of food frequency-based measures of fruit and vegetable intakes
.
Cancer Epidemiol Biomarkers Prev.
2000
;
9
:
939
44
.

27.

Patterson
RE
,
Kristal
AR
,
Levy
L
,
McLerran
D
,
White
E
.
Validity of methods used to assess vitamin and mineral supplement use
.
Am J Epidemiol.
1998
;
148
:
643
9
.

28.

Schakel
SF
,
Sievert
YA
,
Buzzard
IM
.
Sources of data for developing and maintaining a nutrient database
.
J Am Diet Assoc.
1988
;
88
:
1268
71
.

29.

Zeger
SL
,
Liang
KY
.
Longitudinal data analysis for discrete and continuous outcomes
.
Biometrics.
1986
;
42
:
121
30
.

30.

Wagner
C
.
Biochemical role of folate in cellular metabolism.
New York
:
Marcel Dekker
;
1995
.

31.

Selhub
J
,
Rosenberg
IH
.
Folic acid
. In:
Ziegler
EE
,
Filer
LJJ
editors.
Present knowledge in nutrition.
7th ed.
Washington (DC)
:
ILSI Press
;
1996
. p.
207
19
.

32.

Mitchell
BL
,
Ulrich
CM
,
McTiernan
A
.
Supplementation with vitamins or minerals and immune function: can the elderly benefit?
Nutr Res.
2003
;
23
:
1117
39
.

33.

Kim
YI
,
Hayek
M
,
Mason
JB
,
Meydani
SN
.
Severe folate deficiency impairs natural killer cell-mediated cytotoxicity in rats
.
J Nutr.
2002
;
132
:
1361
7
.

34.

Ravaglia
G
,
Forti
P
,
Maioli
F
,
Bastagli
L
,
Facchini
A
,
Mariani
E
,
Savarino
L
,
Sassi
S
,
Cucinotta
D
,
Lenaz
G
.
Effect of micronutrient status on natural killer cell immune function in healthy free-living subjects aged ≥90 y
.
Am J Clin Nutr.
2000
;
71
:
590
8
.

35.

Imai
K
,
Matsuyama
S
,
Miyake
S
,
Suga
K
,
Nakachi
K
.
Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population
.
Lancet.
2000
;
356
:
1795
9
.

36.

Nijhout
HF
,
Reed
MC
,
Budu
P
,
Ulrich
CM
.
A mathematical model of the folate cycle: new insights into folate homeostasis
.
J Biol Chem.
2004
;
279
:
55008
16
.

37.

Johnson
WG
,
Stenroos
ES
,
Spychala
JR
,
Chatkupt
S
,
Ming
SX
,
Buyske
S
.
New 19 bp deletion polymorphism in intron-1 of dihydrofolate reductase (DHFR): a risk factor for spina bifida acting in mothers during pregnancy?
Am J Med Genet A.
2004
;
124
:
339
45
.

38.

Goto
Y
,
Yue
L
,
Yokoi
A
,
Nishimura
R
,
Uehara
T
,
Koizumi
S
,
Saikawa
Y
.
A novel single-nucleotide polymorphism in the 3′-untranslated region of the human dihydrofolate reductase gene with enhanced expression
.
Clin Cancer Res.
2001
;
7
:
1952
6
.

39.

Morris
MC
,
Evans
DA
,
Bienias
JL
,
Tangney
CC
,
Hebert
LE
,
Scherr
PA
,
Schneider
JA
.
Dietary folate and vitamin B12 intake and cognitive decline among community-dwelling older persons
.
Arch Neurol.
2005
;
62
:
641
5
.

40.

Kim
YI
,
Salomon
RN
,
Graeme-Cook
F
,
Choi
SW
,
Smith
DE
,
Dallal
GE
,
Mason
JB
.
Dietary folate protects against the development of macroscopic colonic neoplasia in a dose responsive manner in rats
.
Gut.
1996
;
39
:
732
40
.

41.

Song
J
,
Medline
A
,
Mason
JB
,
Gallinger
S
,
Kim
YI
.
Effects of dietary folate on intestinal tumorigenesis in the apcMin mouse
.
Cancer Res.
2000
;
60
:
5434
40
.

42.

Achon
M
,
Alonso-Aperte
E
,
Reyes
L
,
Ubeda
N
,
Varela-Moreiras
G
.
High-dose folic acid supplementation in rats: effects on gestation and the methionine cycle
.
Br J Nutr.
2000
;
83
:
177
83
.

43.

Achon
M
,
Alonso-Aperts
E
,
Varela-Moreiras
G
.
High dietary folate supplementation: effects on diet utilization and methionine metabolism in aged rats
.
J Nutr Health Aging.
2002
;
6
:
51
4
.

44.

Achon
M
,
Reyes
L
,
Alonso-Aperte
E
,
Ubeda
N
,
Varela-Moreiras
G
.
High dietary folate supplementation affects gestational development and dietary protein utilization in rats
.
J Nutr.
1999
;
129
:
1204
8
.

Abbreviations

     
  • DHF

    dihydrofolate

  •  
  • DHFR

    dihydrofolate reductase

  •  
  • E:T

    effector-to-target cell ratio

  •  
  • FA

    folic acid

  •  
  • NK

    natural killer cell

  •  
  • PI

    propidium iodide

  •  
  • THF

    tetrahydrofolate

Footnotes

1

Supported by grants from the National Institutes of Health (R01 CA69334; T32 CA80416) and the U.S. Department of Agriculture cooperative research agreement 58-1950-4-401.

Author notes

2

These authors contributed equally.