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Folic acid is known to be associated with inflammatory diseases, but the relationship between folic acid and allergic diseases is unclear.
The purpose of the study was to examine the relationship between serum folate levels and markers of atopy, wheeze, and asthma.
Data were obtained from the 2005–2006 National Health and Nutrition Examination Survey (NHANES) in which serum folate and total IgE levels were measured in 8,083 subjects 2 years of age and older. A high total IgE level was defined as >100kU/L. Allergen-specific IgE levels were measured for a panel of 5 common aeroallergens. Atopy was defined as at least 1 positive allergen-specific IgE level. Doctor-diagnosed asthma and wheeze in the previous 12 months were assessed by questionnaire.
Serum folate levels were inversely associated with total IgE levels (p<.001). The odds of a high total IgE, atopy, and wheeze decreased across quintiles of serum folate, indicating a dose-response relationship between serum folate levels and these outcomes. Each of these associations remained statistically significant after adjusting for age, sex, race/ethnicity and poverty index ratio. Adjusted odds ratios[95% confidence intervals] associated with the fifth quintile (Q5) of folate relative to the first quintile (Q1) were as follows: High IgE: 0.70[0.53–0.92]; atopy: 0.69[0.57–0.85]; and wheeze: 0.60[0.44–0.82]. Higher folate levels were also associated with a lower risk of doctor-diagnosed asthma, but this finding was not statistically significant (OR[95% CI] for Q5 vs. Q1: 0.84 [0.70–1.02]).
Serum folate levels are inversely associated with high total IgE, atopy, and wheeze.
Folic acid status may influence the development and/or progression of atopy and wheeze.
The prevalence of asthma and allergy has risen dramatically over the past 20–30 years in developed countries and the reasons behind this striking trend remain unclear. This increase in prevalence cannot be attributed to changes in the genetic makeup of the population because this trend has occurred over a relatively short period of time. As such, there has been active investigation in changes in environmental exposures as a potential cause of the recent rise in allergic diseases.
Allergen exposure(1–7), pollutant exposure(8–12), endotoxin exposure(13–15), immunizations(16–19), diet(20–23), and exposure to parasitic(24–26) and viral(27–29) infections have all been implicated in the epidemic. These exposures could act directly on the immune system or end-organs to elicit or attenuate sensitization and disease, but they may also result in epigenetic changes that could tilt the immunophenotypic balance in favor of allergic disease. One particular dietary component that could directly influence the propensity for epigenetic modifications is folic acid which serves as a source for methyl donors for DNA methylation. In fact, a methyl donor-enriched diet that included folic acid enhanced allergic humoral responses and lung inflammation in a mouse model(30), suggesting that epigenetic changes can indeed promote the development of allergic disease.
Although the findings from this mouse model are intriguing, it is unclear how they might translate to humans. On the one hand, the concomitant rise in allergic diseases(31;32) and serum folate levels in the United States(33) suggests that the relatively recent enrichment of the US diet with folic acid may be a risk factor for allergic disease, an observation that would be consistent with the findings in mice. On the other hand, lower folic acid levels have been implicated in a variety of inflammation-mediated diseases such as cardiovascular disease(34–37) and rheumatoid arthritis(38;39), so it is possible that folate may mitigate against, rather than promote, allergic diseases, which are also inflammation-mediated.
In order to gain insight into the role of folate in the development of allergic disease, we examined relationships between serum folate levels and measures of atopy and airways disease in the 2005–6 NHANES population.
The 2005–6 NHANES dataset(40) was used to examine relationships between serum folate levels and measures of atopy, wheeze, and asthma. The NHANES is a nationally representative survey of the non-institutionalized United States civilian population. NHANES uses a complex multistage probability design to select a representative population and oversamples certain underserved groups to increase the precision of estimates generated during analysis. The NHANES was approved by the IRB of the National Center for Health Statistics, Centers for Disease Control and Prevention, and informed consent was obtained from all participants.
All participants who had serum folate and total IgE levels measured were included in the analyses, which resulted in a final sample size of 8,083. Ages ranged from 2 years to greater than 85 years. Sociodemographic, serum folate, total and specific IgE levels, the respiratory disease questionnaire and medical conditions questionnaire data were included in the dataset. A participant was considered to have had wheeze in the preceding 12 months if he/she responded affirmatively to the following question: “In the past 12 months, have you had wheezing or whistling in your chest?” A participant was considered to have doctor-diagnosed asthma if he/she responded affirmatively to the following question: “Has a doctor or other health professional ever told you that you have asthma?”
Serum total IgE levels were measured using the ImmunoCAP system (Phadia, Uppsala, Sweden). Cat, dog (e5), Dermatophagoides farinae, Dermatophagoides pteronyssinus, Alternaria, and cockroach-specific IgE levels were also measured using the ImmunoCAP system. A level ≥ 0.35 kU/L was considered positive and atopy was defined as at least one positive allergen-specific IgE. Serum folate levels were measured by radioassay with the Quantaphase II Folate kit (Bio-Rad Laboratories).
Statistical analyses were performed with StataSE 8.0 (StataCorp, College Station, TX). The primary sampling units and strata were taken into account using the variables provided in the NHANES dataset to account for the complex survey design. Sampling weights provided by NHANES were used to generate estimates that are representative of the United States non-institutionalized civilian population.
Relationships between variables of interest were examined using logistic or linear regression methods that accounted for the sampling design and the weighting of the observations in the NHANES dataset. Bivariate analyses of folate and the outcome variables of interest were used to examine trends in the outcomes across a continuous measure of serum folate as well as quintiles of folate. A test for trend was performed by using either the continuous folate variable or the variable for quintiles of folate, without inclusion of dummy variables. Multivariate models were adjusted for age, sex, race/ethnicity, and poverty income ratio which is the ratio of family income to the poverty threshold. Analysis of variance (ANOVA) was used to compare serum folate levels among race/ethnicity subgroups. A two-tailed p < .05 was considered statistically significant.
The study population reflected the national representation of the survey design and consisted of approximately equal proportions of males and females, with a mean age of 38 years. (Table I.) The population was predominantly Non-hispanic white, with 13% self-identified as Mexican American or another Hispanic ethnicity, and 12% Non-hispanic Black. Approximately 14% of the population had doctor-diagnosed asthma, with 15.7% reporting wheeze in the previous 12 months. Twenty-seven percent had a total IgE level above 100kU/L and 32% were atopic, having at least one positive allergen-specific IgE level.
Serum folate levels ranged from 0.7–171.0 ng/ml, and were stratified by quintile. The quintile cutpoints were as follows: Q1: 0.7–8.1; Q2: 8.2–10.9; Q3: 11.0–13.8; Q4: 13.9–17.9; and Q5: 18.0–171.0 ng/ml. Non-hispanic Blacks and Hispanics had lower serum folate levels than Non-hispanic whites (mean serum folate levels: 12.0, 12.5, and 15.0 ng/ml, respectively; p < .001). Adjustment for income did not alter these racial/ethnic associations with serum folate levels, but higher income tended to be associated with higher serum folate levels. Specifically, serum folate levels were estimated to be 2.8 ng/ml lower among Non-hispanic Blacks than Non-hispanic Whites and 2.1 ng/ml lower among Hispanics than Non-hispanic Whites. (Table II.)
Total IgE levels decreased across quintiles of serum folate levels (p < .001 for trend) (Figure 1). After adjusting for age, sex, race/ethnicity and poverty index ratio, higher serum folate levels remained statistically significantly associated with lower total IgE levels (data not shown). Relationships between serum folate levels and high total IgE (>100 kU/L) and atopy (≥1 positive allergen-specific IgE level) were also examined. The odds of both a high total IgE level and atopy decreased across quintiles of serum folate in unadjusted analyses (p < .001 for trend) as well as multivariate analyses that adjusted for age, sex, race/ethnicity, and poverty index ratio. (Table III.)
To determine if increasing folate levels were associated with a decreasing number of positive specific IgE tests, an ordinal logistic regression model that accounted for the survey design of NHANES was constructed. The outcome variable was a categorical variable that indicated the number of positive specific IgE tests as follows: 0, 1, 2–3, and 4 or more positives allergen-specific IgE tests. Sixty-eight percent had no positive specific IgE tests, 9% had one, 16% had two or three, and 7% had four or more positive specific IgE tests. For every one unit increase in log10(folate), there was approximately a 50% decrease in the odds of having a greater number of positive specific IgE tests (OR[95%CI]: 0.51[0.37–0.69]). This marked protective effect of log10(folate) on number of positive specific IgE tests was also seen between log10(folate) and atopy (OR[95%CI]: 0.27[0.15–0.47]), while the effects of log10(folate) on asthma and wheeze were less strong. (Table E1, online supplement)
Higher folate levels were also associated with a lower odds of doctor-diagnosed asthma and wheeze in the previous 12 months in unadjusted analyses (p = .02 and p < .001 for trend, respectively). Higher serum folate levels remained significantly associated with a lower odds of wheeze in the previous 12 months after adjusting for age, sex, race/ethnicity, and poverty index ratio, but the association with doctor-diagnosed asthma was not statistically significant. (Table III.)
To determine if the effects of serum folate levels on atopy and high total IgE were independent of the effect of folate on wheeze, wheeze was included as a covariate in the final multivariate models. (Table IV.) In models with high total IgE or atopy as the outcomes, the addition of wheeze as a covariate did not alter the relationship between serum folate levels and these outcomes as there continued to be a decreasing risk of these outcomes across quintiles of serum folate. Similarly, in the models with wheeze as the outcome, the addition of either high total IgE or atopy as covariates did not alter the relationship between serum folate levels and wheeze. These findings suggest that the relationships between serum folate level and measures of atopic status (high total IgE, atopy) are independent of wheeze, and vice versa.
It is also possible that the effect of serum folate levels on either total IgE levels or atopic status could vary depending on wheezing status or that the effect of serum folate on wheezing could vary depending on atopic status. To address these questions, final multivariate models were stratified by wheezing status, atopic status, and total IgE status, depending on the outcome that was being evaluated. Overall, the risk of the outcomes of interest was lowest at the highest serum folate levels, regardless of atopic status, wheeze status, or total IgE status, suggesting that neither atopic status nor wheeze modified the relationships between serum folate levels and these outcomes (data not shown).
The effects of age on the relationships between serum folate levels and atopy, high IgE, and wheeze were also examined. Across all age groups, higher serum folate levels were associated with a decreased odds of high IgE levels and wheeze, with a trend towards a decreased odds of asthma. Across all age groups, except for those 60 years and older, higher serum folate levels were associated with a decreased odds of atopy. For those 60 years and older, there was no relationship between serum folate levels and atopy. (Tables E1 and E2, online repository)
To determine if income or race/ethnicity modified the relationship between serum folate levels and our outcomes of interest, we conducted analyses stratified by tertiles of poverty income ratio and by race/ethnicity (Table V.). The inverse relationship between serum folate levels and odds of high total IgE level, atopy, and wheeze persisted across all tertiles of poverty income ratio, although some of the point estimates lost statistical significance with stratification.
When analyses were stratified by race/ethnicity, the inverse relationships between serum folate and high total IgE, atopy, and wheeze were present among Non-hispanic Whites and Blacks (Table V.). The relationships were somewhat stronger among Non-hispanic Whites than Blacks, but these differences were not statistically significant. Among Hispanics, there was no association between serum folate levels and atopy or wheeze, and some suggestion that this group may have a higher risk of wheeze with higher serum folate levels.
In a representative US population, a higher serum folate level was associated with lower total IgE levels and a lower risk of atopy and wheeze. Although higher serum folate levels have been linked to lower risk of other inflammatory conditions such as cardiovascular disease(35–37) and rheumatoid arthritis(38), this is the first report, to our knowledge, that links higher serum folate levels to a lower risk of atopy and wheeze. These findings suggest that dietary folic acid and factors affecting its metabolism may play an important role in the development and perpetuation of allergy and asthma.
Two previous epidemiologic studies examined polymorphisms in a folate metabolism gene, methylenetetrahydrofolate reductase (MTHFR), and atopy and/or asthma. In a cross-sectional population-based Danish study(41), the TT allele of the MTHFR gene which results in impaired folate metabolism and reduces the intracellular methyl donor pool was associated with a higher prevalence of atopy.(42) Using a dietary questionnaire, that study also found that lower intake of dietary factors known to influence methyl donor metabolism (folic acid, methionine, and B vitamins) was associated with a higher risk of atopy in study participants with the TT allele of the MTHFR gene. Findings from this study are consistent conceptually with our study’s findings, as both studies suggest that a lower methyl donor pool – as evidenced by either lower serum folate levels, impaired folate metabolism, or lower dietary intake of cofactors of methyl donor metabolism – are associated with a higher risk of atopy. On the other hand, in a United Kingdom population-based cohort of mothers and children(43), there was no association between MTHFR polymorphisms and atopy or asthma. The reasons for the inconsistent findings of these two studies of MTHFR polymorphisms are unclear, but neither study examined relationships between directly measured serum folate levels, which may be a more accurate measure of the methyl donor pool, and asthma or atopy.
Although we examined serum folate levels and not dietary intake of folic acid, greater oral intake of folic acid is associated with higher serum folate levels(33;44). However, it is important to note that different dietary sources of folic acid impact serum folate levels to varying degrees. For example, supplements appear to have the greatest impact on serum folate levels followed by enriched ready-to-eat cereals and enriched cereal grain products(44). The lower limit for a “normal” serum folate level is generally considered to be in the range of 3–4.5 ng/ml(33;45), and approximately 1% of the 2005–6 NHANES population meets this criterion for a low serum folate level. There is greater debate about the definition of a high serum folate level, although a recent paper suggested that a level above 26.5 ng/ml should be considered to be high(46). In this NHANES population, approximately 5% had a serum folate level above this threshold. Thus, the findings from our study are relevant for a range of serum folate levels that would be considered to be “normal.” Whether future studies provide sufficient data to warrant a revision of the current definition of a “normal” serum folate level remains to be seen.
We found a lower prevalence of atopy in this NHANES population than reported in NHANES III(47). Skin testing was performed in NHANES III, but specific IgE testing was performed in NHANES 2005–6. In addition, in the NHANES III study, the population ranged in age from 20–59y, and in our study the age ranges from 2–85+ years. A combination of a younger study population and utilization of specific IgE testing on a fewer number of allergens is the most likely explanation for the differences in atopy prevalence between the two study populations.
In direct contrast to the two epidemiologic studies, a recently published study using a mouse model found that a diet enriched for methyl donors, including folic acid, was a risk factor, rather than a protective factor, for allergy and asthma(30). This positive association between higher dietary folate intake and enhanced allergic immune and inflammatory responses was thought to be mediated by epigenetic modification that was inherited transgenerationally since animals exposed in utero to increased methyl donors as well as their progeny who were provided normal feed exhibited increased severity of allergic airway disease. There are several possible explanations for the differences between this mouse study and the epidemiologic studies – aside from the obvious differences between mice and humans. In this study, mice were fed a cocktail of methyl donors and cofactors, including vitamin B12, choline, L-methionine, zinc, and betaine in addition to folic acid. It is also not clear how this methyl donor-enriched diet impacted serum folate levels, so it is possible that the diet resulted in supra-physiologic levels that would rarely, if ever, be observed in human populations. While our study’s findings suggest that low levels of folate may be a risk factor for allergic disease, the mouse study may indicate that very high levels of methyl donors, such as folate, could also be a risk factor for allergic disease and that optimal levels of folate (or methyl donors) fall somewhere in between.
In contrast to gestational exposure, the mice that were provided high methyl donor diets during lactation or early adulthood did not demonstrate a similar increase in disease severity, suggesting that the timing of methyl donor supplementation may play a pivotal role in determining its impact on the development of allergic disease. As the NHANES is a cross-sectional survey, our study does not address this question of the impact of in utero or early post-natal serum folate levels on the development of allergic sensitization and disease. Rather, the serum folate levels in our study reflect “current” serum folate levels, which are likely the end result of a combination of current or recent dietary folic acid intake and underlying polymorphisms in genes that are involved in folate metabolism.
Although our study does not address the effects of in utero folate exposure on asthma and atopy, one recent prospective birth cohort study examined the impact of in utero exposure to folic acid supplementation on risk of respiratory outcomes in the first 18 months of life(48). The investigators found a very small increase in the risk of lower respiratory tract infection and wheeze associated with folic acid supplementation in the first trimester only and atopic outcomes were not evaluated. More prospective studies are clearly needed and current birth cohort studies are well-positioned to evaluate the impact of in utero or very early life serum folate levels on subsequent risk of allergic sensitization and disease.
A number of epidemiologic studies have described an inverse relationship between folate levels and inflammatory states(36;37), in keeping with our study’s findings. Although evidence exists that folic acid supplementation may change serum cytokine levels via non-epigenetic mechanisms(49), changes in DNA methylation have been associated with immune modulation, including the aforementioned mouse study. The precise role of epigenetics in asthma and atopy are poorly understood, but recent studies suggest potential effects on both immunity(50–53) and inflammation(54;55). In fact, we also found that the relationship between serum folate levels and measures of atopy were independent of wheeze, and vice versa. These findings suggest that serum folate levels may indeed have dual and independent functions, potentially reducing the risk of atopy through immunomodulation and also potentially reducing the risk of wheeze through modulation of inflammation.
This study also confirmed previous observations of an association between Non-hispanic Black or Hispanic race/ethnicity and lower serum folate levels(33). We also found that the relationships between serum folate levels and risk of high total IgE, atopy, and wheeze were similar across all income levels and among Non-hispanic Whites and Blacks, suggesting that low serum folate levels may contribute to risk of high total IgE, atopy, and wheeze in Black and Non-hispanic White populations of all income levels. However, relationships between folate and these outcomes among the Hispanic population are less clear, with folate having little impact on risk of atopy and wheeze, perhaps even increasing risk of wheeze in this racial/ethnic group. The reasons behind these differences across racial/ethnic groups are unclear and could be related to dietary, socio-cultural, or genetic factors.
Although the inverse association between serum folate levels and atopy and wheeze in this NHANES population is intriguing, the cross-sectional study design limits our ability to conclude that there is a causal relationship between serum folate levels and atopy and wheeze. Because the temporal relationships between serum folate levels and the development of atopy or wheeze are unknown, it is not clear whether high serum folate levels might protect against the development of allergic sensitization or elevated total IgE and/or protect against wheeze in a previously sensitized individual. Additional prospective studies will be required to lend insight into the potential role of folic acid supplementation in the primary prevention and/or treatment of allergic diseases. Ultimately, clinical trials will be needed to draw any firm conclusions about the preventive or therapeutic potential for folic acid in allergic diseases as folic acid supplementation in patients with cardiovascular disease may be related to worse outcomes in some cases(56).
In summary, higher serum folate levels are associated with lower total IgE levels, and a lower risk of allergic sensitization and wheeze. Future studies are needed to define the temporal relationships among serum folate levels and allergy and asthma and to determine whether these associations, if causal, are mediated by epigenetic changes or by other mechanisms.
Supported by grants from the NIAID (R01AI070630) and the NIEHS (DISCOVER Award: 5P50ES015903).
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