Metabolomic Effects of Folic Acid Supplementation in Adults: Evidence from the FACT Trial.

BACKGROUND: Folic acid (FA) is the oxidized form of folate found in supplements and FA-fortified foods. Most FA is reduced by dihydrofolate reductase to 5-methyltetrahydrofolate (5mTHF); the latter is the form of folate naturally found in foods. Ingestion of FA increases the plasma levels of both 5mTHF and unmetabolized FA (UMFA). Limited information is available on the downstream metabolic effects of FA supplementation, including potential effects associated with UMFA. OBJECTIVE: We aimed to assess the metabolic effects of FA-supplementation, and the associations of plasma 5mTHF and UMFA with the metabolome in FA-naïve Bangladeshi adults. METHODS: Sixty participants were selected from the Folic Acid and Creatine Trial; half received 800 µg FA/day for 12 weeks and half placebo. Plasma metabolome profiles were measured by high-resolution mass spectrometry, including 170 identified metabolites and 26,541 metabolic features. Penalized regression methods were used to assess the associations of targeted metabolites with FA-supplementation, plasma 5mTHF, and plasma UMFA. Pathway analyses were conducted using Mummichog. RESULTS: In penalized models of identified metabolites, FA-supplementation was associated with higher choline. Changes in 5mTHF concentrations were positively associated with metabolites involved in amino acid metabolism (5-hydroxyindoleacetic acid, acetylmethionine, creatinine, guanidinoacetate, hydroxyproline/n-acetylalanine) and 2 fatty acids (docosahexaenoic acid and linoleic acid). Changes in 5mTHF concentrations were negatively associated with acetylglutamate, acetyllysine, carnitine, propionyl carnitine, cinnamic acid, homogentisate, arachidonic acid, and nicotine. UMFA concentrations were associated with lower levels of arachidonic acid. Together, metabolites selected across all models were related to lipids, aromatic amino acid metabolism, and the urea cycle. Analyses of nontargeted metabolic features identified additional pathways associated with FA supplementation. CONCLUSION: In addition to the recapitulation of several expected metabolic changes associated with 5mTHF, we observed additional metabolites/pathways associated with FA-supplementation and UMFA. Further studies are needed to confirm these associations and assess their potential implications for human health. TRIAL REGISTRATION NUMBER: This trial was registered at https://clinicaltrials.gov as NCT01050556.

Uncovering the Hidden Dangers and Molecular Mechanisms of Excess Folate: A Narrative Review.

This review delves into the intricate relationship between excess folate (vitamin B9) intake, especially its synthetic form, namely, folic acid, and its implications on health and disease. While folate plays a pivotal role in the one-carbon cycle, which is essential for DNA synthesis, repair, and methylation, concerns arise about its excessive intake. The literature underscores potential deleterious effects, such as an increased risk of carcinogenesis; disruption in DNA methylation; and impacts on embryogenesis, pregnancy outcomes, neurodevelopment, and disease risk. Notably, these consequences stretch beyond the immediate effects, potentially influencing future generations through epigenetic reprogramming. The molecular mechanisms underlying these effects were examined, including altered one-carbon metabolism, the accumulation of unmetabolized folic acid, vitamin-B12-dependent mechanisms, altered methylation patterns, and interactions with critical receptors and signaling pathways. Furthermore, differences in the effects and mechanisms mediated by folic acid compared with natural folate are highlighted. Given the widespread folic acid supplementation, it is imperative to further research its optimal intake levels and the molecular pathways impacted by its excessive intake, ensuring the health and well-being of the global population.

Association of folic acid dosage with circulating unmetabolized folic acid in Chinese adults with H-type hypertension: a multicenter, double-blind, randomized controlled trial.

Background: There is growing concern regarding elevated levels of circulating unmetabolized folic acid (UMFA) due to excessive intake of folic acid (FA). However, no randomized clinical trial has been conducted to examine the FA-UMFA dose-response relationship. Objective: This study aimed to investigate the FA-UMFA dose-response relationship in Chinese adults with hypertension and elevated homocysteine (H-type hypertension), a population with clear clinical indication for FA treatment. Methods: The data for this study were derived from a randomized, double-blind, multicenter clinical trial of 8 FA dosages on efficacy of homocysteine (Hcy) lowering. The parent trial had three 3 stages: screening period (2-10 days), run-in period (0-2 weeks, baseline visit), and double-blind treatment period (8 weeks) with follow-up visits at the end of the 2nd, 4th, 6th, and 8th weeks of treatment. Participants were randomly assigned to 8 treatment groups corresponding to FA dosages of 0, 0.4, 0.6, 0.8, 1.2, 1.6, 2.0 mg to 2.4 mg. Results: This study included 1,567 Chinese adults aged ≥45 years with H-type hypertension. There was a positive but non-linear association between FA supplementation and UMFA levels in the dosage range of 0 mg to 2.4 mg. In the regression analysis, the coefficients for the linear and quadratic terms of FA dosage were both statistically significant (P < 0.001). Notably, the slope for UMFA was greater for FA dosages >0.8 mg (ß = 11.21, 95% CI: 8.97, 13.45) compared to FA dosages ≤0.8 mg (ß = 2.94, 95% CI: 2.59, 3.29). Furthermore, FA dosages higher than 0.8 mg did not confer additional benefits in terms of increasing 5-methyl tetrahydrofolic acid (5-MTHF, active form of folate) or reducing homocysteine (Hcy). Conclusion: In Chinese adults with H-type hypertension, this study showed a positive, non-linear, dosage-response relationship between FA supplementation ranging from 0 to 2.4 mg and circulating UMFA levels. It revealed that 0.8 mg FA is an optimal dosage in terms of balancing efficacy (increasing 5-MTHF and lowering Hcy) while minimizing undesirable elevation of UMFA. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03472508?term=NCT03472508&draw=2&rank=1, identifier NCT03472508.

Associations between folic acid supplement use and folate status biomarkers in the first and third trimesters of pregnancy in the Maternal-Infant Research on Environmental Chemicals (MIREC) Pregnancy Cohort Study.

BACKGROUND: Achieving optimal folate status during early gestation reduces the risk of neural tube defects (NTDs). While inadequate folate intake remains a concern, it is becoming increasingly common for individuals to consume higher than recommended doses of folic acid (FA) with minimal additional benefit. OBJECTIVE: Here, we sought to investigate the determinants, including FA supplement dose and use, of plasma total and individual folate vitamer concentrations in the first and third trimesters of pregnancy. METHODS: Using data from the Maternal-Infant Research on Environmental Chemicals (MIREC) Study, a cohort exposed to mandatory FA fortification, we measured plasma total folate and individual folate vitamer [5-methyltetrahydrofolate (5-methylTHF), unmetabolized FA (UMFA), and non-methyl folates (sum of THF, 5-formylTHF, 5,10-methenyl-THF)] concentrations in the first and third trimesters (n = 1,893). Using linear mixed models, we estimated associations between plasma folate concentrations, total daily supplemental FA intake, plasma vitamin B-12 concentrations, and multiple demographic, maternal, and reproductive factors. RESULTS: Almost 95% of MIREC study participants met or exceeded the recommended daily supplemental FA intake from supplements (≥400 µg/d), with approximately 25% consuming more than the Tolerable Upper Intake Level (>1000 µg/d). Over 99% of MIREC participants had a plasma total folate status indicative of maximal NTD risk reduction (25.5 nmol/L) regardless of FA supplement dose. UMFA was detected in almost all participants, with higher concentrations associated with higher FA doses. Determinants of adequate FA supplement intake and folate status associated with reduced NTD risk included indicators of higher socioeconomic position, higher maternal age, nulliparity, and lower prepregnancy BMI. CONCLUSIONS: In the context of mandatory FA fortification, our data indicate that higher-than-recommended FA doses are unwarranted, with the exception of individuals at higher risk for NTDs. Ideally, prenatal supplements would contain 400 rather than 1000 µg FA, thereby enabling the consumption of optimal and safe FA doses.

MR1-dependence of unmetabolized folic acid side-effects.

The fortification of flour with folic acid for the prevention of neural tube defects (NTD) is currently mandated in over eighty countries worldwide, hence compelling its consumption by the greater part of the world's population. Notwithstanding its beneficial impact on rates of NTD, pervasive folic acid supplementation has invariably led to additive daily intakes reaching well beyond their original target, resulting in the circulation of unmetabolized folic acid. Associated idiopathic side-effects ranging from allergies to cancer have been suggested, albeit inconclusively. Herein, we hypothesize that their inconsistent detection and elusive etiology are linked to the in vivo generation of the immunosuppressive folic acid metabolite 6-formylpterin, which interferes with the still emerging and varied functions of Major Histocompatibility Complex-related molecule 1 (MR1)-restricted T cells. Accordingly, we predict that fortification-related adverse health outcomes can be eliminated by substituting folic acid with the bioequivalent folate vitamer 5-methyltetrahydrofolate, which does not break down into 6-formylpterin.

Women Taking a Folic Acid Supplement in Countries with Mandatory Food Fortification Programs May Be Exceeding the Upper Tolerable Limit of Folic Acid: A Systematic Review.

BACKGROUND: In preconception and pregnancy, women are encouraged to take folic acid-based supplements over and above food intake. The upper tolerable limit of folic acid is 1000 mcg per day; however, this level was determined to avoid masking a vitamin B12 deficiency and not based on folic acid bioavailability and metabolism. This review's aim is to assess the total all-source intake of folate in women of childbearing age and in pregnancy in high-income countries with folate food fortification programs. METHODS: A systematic search was conducted in five databases to find studies published since 1998 that reported folate and folic acid intake in countries with a mandatory fortification policy. RESULTS: Women of childbearing age do not receive sufficient folate intake from food sources alone even when consuming fortified food products; however, almost all women taking a folic acid-based supplement exceed the upper tolerable limit of folic acid intake. CONCLUSIONS: Folic acid supplement recommendations and the upper tolerable limit of 1000 mcg set by policy makers warrant careful review in light of potential adverse effects of exceeding the upper tolerable limit on folic acid absorption and metabolism, and subsequent impacts on women's health during their childbearing years.

Detectable Unmetabolized Folic Acid and Elevated Folate Concentrations in Folic Acid-Supplemented Canadian Children With Sickle Cell Disease.

Sickle cell disease (SCD) is an inherited hemoglobinopathy caused by a variant (rs344) in the HBB gene encoding the ß-globin subunit of hemoglobin. Chronic hemolytic anemia and increased erythropoiesis and RBC turnover in individuals with SCD can result in increased needs for folate and other B-vitamins. We assessed B-vitamin status, and the distribution of folate forms, including unmetabolized folic acid (UMFA), in Canadian children with SCD supplemented with 1 mg/d folic acid (current routine practice). Non-fasted serum and plasma samples were analyzed for concentrations of folate, and vitamins B-2, B-6, and B-12. Eleven individuals (45% male; SCD type: HbSS n = 8, HbSC n = 2, HbSß0-Thal n = 1), with a median (IQR) age of 14 (7, 18) years, were included. Total folate concentrations were 3-27 times above the deficiency cut-off (10 nmol/L), and 64% of children had elevated folate levels (>45.3 nmol/L). UMFA (>0.23 nmol/L) was detected in all children, and 36% of participants had elevated levels of UMFA (>5.4 nmol/L). All children were vitamin B-12 sufficient (>150 pmol/L), and the majority (55%) had sufficient B-6 status (>30 nmol/L). Among this sample of Canadian children with SCD, there was limited evidence of B-vitamin deficiencies, but UMFA was detectable in all children.

Unmetabolized folic acid is associated with TNF-α, IL-1ß and IL-12 concentrations in a population exposed to mandatory food fortification with folic acid: a cross-sectional population-based study in Sao Paulo, Brazil.

PURPOSE: The study assessed associations between inflammatory markers, as cytokines, adhesion molecules and unmetabolized folic acid (UMFA) among a population exposed to mandatory fortification. METHODS: Data were collected from a cross-sectional population-based survey (n = 302) conducted in São Paulo City, Brazil. UMFA was assayed by a modified affinity-HPLC method with electrochemical detection to measure the different forms of the folate in plasma. We used a commercial test kit to analyze cytokines and adhesion molecules. Multiple logistic regressions were applied to investigate the association between inflammatory markers and UMFA. Multiple models were adjusted for sex, age, self-reported skin color, BMI and smoking status. RESULTS: The prevalence of detectable UMFA in this population was high (81.2%), with median concentration of 1.67 nmol/L. The odds ratios (95% CIs) for having higher immunological markers levels among individuals in the highest tertile of UMFA were 0.44 (0.24; 0.81) for TNF-α, 0.92 (0.49; 1.75) for CRP, 1.32 (0.70; 2.48) for ICAM, 0.99 (0.54; 1.81) for VCAM, 0.45 (0.25; 0.83) for IL-1ß, 0.74 (0.40; 1.38) for IL-6, 1.34 (0.73; 2.44) for IL-8, 0.65 (0.36; 1.18) for IL-10 and 0.49 (0.27; 0.89) for IL-12. CONCLUSION: UMFA concentrations were inversely associated with elevated proinflammatory markers (TNF-α, IL-1ß and IL-12). These results signalize a link between folate metabolism and the inflammatory status of adults in an apparently folate-replete population.

Knowledge gaps in understanding the metabolic and clinical effects of excess folates/folic acid: a summary, and perspectives, from an NIH workshop.

Folate, an essential nutrient found naturally in foods in a reduced form, is present in dietary supplements and fortified foods in an oxidized synthetic form (folic acid). There is widespread agreement that maintaining adequate folate status is critical to prevent diseases due to folate inadequacy (e.g., anemia, birth defects, and cancer). However, there are concerns of potential adverse effects of excess folic acid intake and/or elevated folate status, with the original concern focused on exacerbation of clinical effects of vitamin B-12 deficiency and its role in neurocognitive health. More recently, animal and observational studies have suggested potential adverse effects on cancer risk, birth outcomes, and other diseases. Observations indicating adverse effects from excess folic acid intake, elevated folate status, and unmetabolized folic acid (UMFA) remain inconclusive; the data do not provide the evidence needed to affect public health recommendations. Moreover, strong biological and mechanistic premises connecting elevated folic acid intake, UMFA, and/or high folate status to adverse health outcomes are lacking. However, the body of evidence on potential adverse health outcomes indicates the need for comprehensive research to clarify these issues and bridge knowledge gaps. Three key research questions encompass the additional research needed to establish whether high folic acid or total folate intake contributes to disease risk. 1) Does UMFA affect biological pathways leading to adverse health effects? 2) Does elevated folate status resulting from any form of folate intake affect vitamin B-12 function and its roles in sustaining health? 3) Does elevated folate intake, regardless of form, affect biological pathways leading to adverse health effects other than those linked to vitamin B-12 function? This article summarizes the proceedings of an August 2019 NIH expert workshop focused on addressing these research areas.

A prospective birth cohort study on cord blood folate subtypes and risk of autism spectrum disorder.

BACKGROUND: We previously reported that extremely high concentrations of maternal plasma folate were associated with increased risk of autism spectrum disorder (ASD) in children. This study explored whether specific types of folate in cord blood have differential association with ASD. OBJECTIVES: In the Boston Birth Cohort (BBC), we assessed the association between cord blood unmetabolized folic acid (UMFA), 5-methyl tetrahydrofolate (THF), and total folate and a child's ASD risk. In a subset, we explored whether the association between UMFA and ASD risk can be affected by the dihydrofolate reductase (DHFR) genotype and cord plasma creatinine. We also examined prenatal correlates of cord UMFA concentrations. METHODS: This report included 567 BBC children (92 ASD, 475 neurotypical), who were recruited at birth and prospectively followed at the Boston Medical Center. ASD was defined from International Classification of Diseases (ICD)-9 and ICD-10 codes documented in electronic medical records. RESULTS: Children with cord UMFA in the highest, versus lowest quartile, had a greater ASD risk (adjusted OR, aORquartile4: 2.26; 95% CI: 1.08, 4.75). When stratified by race/ethnicity, the association was limited to 311 (45 ASD) Black children (aORquartile4: 9.85; 95% CI: 2.53, 38.31); a test of interaction between race/ethnicity and cord UMFA concentrations was significant (P = 0.007). The UMFA-ASD association in Black children slightly attenuated after adjusting for cord plasma creatinine (P = 0.05). There was no significant association between cord 5-methyl THF, total folate, DHFR genotype, and ASD risk. Cord total folate and maternal supplement intake during second trimester were associated with higher cord UMFA. CONCLUSIONS: Higher concentrations of cord UMFA, but not 5-methyl THF or total folate, were associated with a greater risk of ASD in Black children. This study in a preterm-birth-enriched cohort raises more questions than it could answer and underscores the need for additional investigations on the sources and role of cord UMFA in children's neurodevelopmental outcomes and underlying mechanisms.

Serum folate and cytokines in heterozygous ß-thalassemia.

INTRODUCTION: Folate deficiency is commonly reported in ß-thalassemia. Individuals heterozygous for ß-thalassemia may have higher folate requirements than normal individuals. OBJECTIVES: To document the concentration of serum total folate and its forms in ß-thalassemia heterozygote users (ß-TmU) and nonusers (ß-TmN) of 5 mg folic acid/d; to determine whether folic acid (FA) consumption from fortified foods allows beta-Tm patients, who do not take FA supplements, to meet their dietary folate requirements; and to investigate the association between higher serum unmetabolized folic acid (UMFA) and inflammatory cytokine concentrations. METHODS: Serum total folate and forms were measured in 42 ß-Tm (13 ß-TmU and 29 ß-TmN) and 84 healthy controls. The mononuclear leucocyte mRNA expression of relevant genes and their products and hematological profiles were determined. RESULTS: ß-TmU had higher serum total folate, 5-methyltetrahydrofolate, UMFA, and tetrahydrofolate (THF) compared with ß-TmN. The ß-TmN had lower serum total folate and THF than controls. Plasma total homocysteine (tHcy) was lower in ß-TmU compared with both ß-TmN and controls, while ß-TmN had higher tHcy than controls. ß-TmU had higher IL-8 than their controls while ß-TmN had higher IL-6 and IL-8 than their controls. ß-TmU have higher levels of serum total folate, 5- methyltetrahydrofolate, UMFA, and THF than controls. There was no association between UMFA concentrations and cytokine levels. CONCLUSIONS: Mandatory flour fortification with FA in Brazil may be insufficient for ß-TmN, since they have higher tHcy and lower serum total folate than controls. Furthermore, ß-TmN have higher IL-6 levels than ß-TmU. UMFA was not associated with inflammatory cytokine levels.

Demographic, Physiologic, and Lifestyle Characteristics Observed with Serum Total Folate Differ Among Folate Forms: Cross-Sectional Data from Fasting Samples in the NHANES 2011-2016.

BACKGROUND: Serum folate forms were measured in the US population during recent NHANES to assess folate status. OBJECTIVE: We describe post-folic acid-fortification concentrations of serum folate forms in the fasting US population ≥1 y from the NHANES 2011-2016. METHODS: We measured 5 biologically active folates and 1 oxidation product (MeFox) of 5-methyltetrahydrofolate (5-methyl-THF). We calculated geometric means of 5-methyl-THF, unmetabolized folic acid (UMFA), nonmethyl folate (sum of tetrahydrofolate, 5-formyltetrahydrofolate, and 5,10-methenyltetrahydrofolate), total folate (sum of above biomarkers), and MeFox by demographic, physiologic, and lifestyle variables; estimated the magnitude of variables on biomarker concentrations after covariate adjustment; and determined the prevalence of UMFA >2 nmol/L. RESULTS: After demographic adjustment, age, sex, and race-Hispanic origin were significantly associated with most folate forms. MeFox increased with age, while 5-methyl-THF, UMFA, and nonmethyl folate displayed U-shaped age patterns. Compared with non-Hispanic whites, non-Hispanic blacks had 23% lower predicted 5-methyl-THF but comparable UMFA; non-Hispanic Asians had comparable 5-methyl-THF but 28% lower UMFA; Hispanics, non-Hispanic Asians, and non-Hispanic blacks had ∼20% lower MeFox. After additional physiologic and lifestyle adjustment, predicted UMFA and MeFox concentrations were 43% and 112% higher, respectively, in adults with chronic kidney disease and 17% and 15% lower, respectively, in adults consuming daily 1-<2 alcoholic beverages; 5-methyl-THF concentrations were 20% lower in adult smokers. The prevalence of UMFA >2 nmol/L was highest in persons aged ≥70 y (9.01%) and lowest in those aged 12-19 y (1.14%). During 2011-2014, the prevalence was 10.6% in users and 2.22% in nonusers of folic acid-containing supplements. CONCLUSIONS: In fasting persons ≥1 y, the demographic, physiologic, and lifestyle characteristics observed with serum total folate differed among folate forms, suggesting biological and/or genetic influences on folate metabolism. High UMFA was mostly observed in supplement users and older persons.

The MTHFR 677C>T polymorphism is associated with unmetabolized folic acid in breast milk in a cohort of Canadian women.

BACKGROUND: Maternal nutrition and genetics are determinants of breast-milk nutrient composition and, as such, are determinants of the nutritional exposure of breastfed infants. OBJECTIVES: The aim of this study was to determine whether common maternal single nucleotide polymorphisms (SNPs) in folate-dependent enzymes are associated with breast-milk folate content in a cohort of mothers enrolled in the Maternal-Infant Research on Environmental Chemicals (MIREC) study. METHODS: The MIREC study is a Canadian prospective pregnancy cohort study that recruited 2001 participants between 2008 and 2011. Five folate-related SNPs-MTHFR 677C>T (rs1801133), MTHFR 1298A>C (rs1801131), MTHFR 1793G>A (rs2274976), MTR 2756A>G (rs1805087), and MTRR 66A>G (rs1801394)-were genotyped. Breast milk was sampled ∼1 mo postpartum, and tetrahydrofolate (THF), 5-methyl-THF, 5-formyl-THF, 5,10-methenyl-THF, and unmetabolized folic acid (UMFA) were measured using liquid chromatography-tandem mass spectrometry in a subset of participants (n = 551). Associations were assessed using Wald's test. Associations were considered significant if P ≤ 0.01 (Bonferroni correction for multiple testing). RESULTS: None of the SNPs were associated with total breast-milk folate. However, the MTHFR 677C>T SNP was associated with breast-milk UMFA (R2 = 0.01; unadjusted P = 0.004), explaining a small portion of total variance; this association remained significant when adjusted for other covariates, including supplemental folic acid consumption. The MTHFR 1793G>A and MTRR 66A>G SNPs tended to be associated with 5-methyl-THF (R2 = 0.008, P = 0.04) and reduced folates (THF + 5-methyl-THF + 5-formyl-THF + 5,10-methenyl-THF; R2 = 0.01, P = 0.02), respectively. CONCLUSIONS: We found that total breast-milk folate content was not associated with any of the folate-related SNPs examined. The association between the MTHFR 677C>T SNP and breast-milk UMFA, albeit modest, highlights the need to better understand the determinants of breast-milk folate and the impact they might have on milk folate bioavailability.

Is High Folic Acid Intake a Risk Factor for Autism?-A Review.

Folate is required for metabolic processes and neural development. Insuring its adequate levels for pregnant women through supplementation of grain-based foods with synthetic folic acid (FA) in order to prevent neural tube defects has been an ongoing public health initiative. However, because women are advised to take multivitamins containing FA before and throughout pregnancy, the supplementation together with natural dietary folates has led to a demographic with high and rising serum levels of unmetabolized FA. This raises concerns about the detrimental effects of high serum synthetic FA, including a rise in risk for autism spectrum disorder (ASD). Some recent studies have reported a protective effect of FA fortification against ASD, but others have concluded there is an increased risk for ASD and other negative neurocognitive development outcomes. These issues are accompanied by further health questions concerning high, unmetabolized FA levels in serum. In this review, we outline the reasons excess FA supplementation is a concern and review the history and effects of supplementation. We then examine the effects of FA on neuronal development from tissue culture experiments, review recent advances in understanding of metabolic functional blocks in causing ASD and treatment for these with alternative forms such as folinic acid, and finally summarize the conflicting epidemiological findings regarding ASD. Based on the evidence evaluated, we conclude that caution regarding over supplementing is warranted.

Association between Serum Unmetabolized Folic Acid Concentrations and Folic Acid from Fortified Foods.

OBJECTIVE: To investigate the association between serum unmetabolized folic acid (UMFA) concentrations and folic acid from fortified foods and nutrients known as dietary methyl-group donors (folate, methionine, choline, betaine and vitamins B2, B6 and B12) in participants exposed to mandatory fortification of wheat and maize flours with folic acid. METHODS: Cross-sectional study carried out with 144 healthy Brazilian participants, both sexes, supplement nonusers. Serum folate, UMFA, vitamin B12 and total plasma homocysteine (tHcy) were biochemically measured. Dietary intake was assessed by 2 non-consecutive 24-hour dietary recalls (24-HRs) and deattenuated energy-adjusted nutrient data were used for statistical analysis. RESULTS: Ninety eight (68.1%) participants were women. Median (interquartile range) age was 35.5 (28.0-52.0) years. Elevated serum folate concentrations (>45 nmol/L) were found in 17 (11.8%), while folate deficiency (<7 nmol/L) in 10 (6.9%) participants. No one had vitamin B12 deficiency (<148 pmol/L). An elevated serum UMFA concentration was defined as > 1 nmol/L (90th percentile). UMFA concentrations were positively correlated with folic acid intake and negatively correlated to choline, methionine and vitamin B6 intakes. Participants in the lowest quartile of UMFA concentrations had lower dietary intake of total folate (DFEs) and folic acid, and higher dietary intake of methionine, choline and vitamin B6 than participants in the highest quartile of UMFA. Folic acid intake (OR [95% CI] = 1.02 [1.01-1.04)] and being a male (OR [95% CI] = 0.40 [0.19-0.87) were associated with increased and reduced odds for UMFA concentrations > 0.55 nmol/L (median values), respectively. CONCLUSION: UMFA concentrations were directly influenced by folic acid intake from fortified foods in a healthy convenience sample of adult Brazilians exposed to mandatory flour fortification with folic acid.

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

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

Total folate and unmetabolized folic acid in the breast milk of a cross-section of Canadian women.

Background: Folate requirements increase during pregnancy and lactation. It is recommended that women who could become pregnant, are pregnant, or are lactating consume a folic acid (FA)-containing supplement.Objectives: We sought to determine breast-milk total folate and unmetabolized folic acid (UMFA) contents and their relation with FA-supplement use and doses in a cohort of Canadian mothers who were enrolled in the MIREC (Maternal-Infant Research on Environmental Chemicals) study.Design: Breast-milk tetrahydrofolate (THF), 5-methyl-THF, 5-formyl-THF, 5,10-methenyl-THF, and UMFA were measured with the use of liquid chromatography-tandem mass spectrometry (n = 561). Total daily supplemental FA intake was based on self-reported FA-supplement use.Results: UMFA was detectable in the milk of 96.1% of the women. Total daily FA intake from supplements was associated with breast folate concentration and species. Breast-milk total folate was 18% higher (P < 0.001) in supplement users (n = 401) than in nonusers (n = 160), a difference driven by women consuming >400 µg FA/d (P ≤ 0.004). 5-Methyl-THF was 19% lower (P < 0.001) and UMFA was 126% higher (P < 0.001) in supplement users than in nonusers. Women who consumed >400 µg FA/d had proportionally lower 5-methyl-THF and higher UMFA than did women who consumed ≤400 µg FA/d.Conclusions: FA-supplement use was associated with modestly higher breast-milk total folate. Detectable breast-milk UMFA was nearly ubiquitous, including in women who did not consume an FA supplement. Breast-milk UMFA was proportionally higher than 5-methyl-THF in women who consumed >400 µg FA/d, thereby suggesting that higher doses exceed the physiologic capacity to metabolize FA and result in the preferential uptake of FA in breast milk. Therefore, FA-supplement doses >400 µg may not be warranted, especially in populations for whom FA fortification is mandatory.

Synthetic folic acid intakes and status in children living in Ireland exposed to voluntary fortification.

BACKGROUND: In the context of mandatory and voluntary folic acid fortification, the exposure of children to folic acid has been a focus of concern, particularly regarding the possibility of whether any potentially adverse effects will emerge in the future. OBJECTIVE: We explored concentrations of fasting unmetabolized folic acid (UFA) in the circulation of children living in Ireland who were exposed to the voluntary folic acid-fortification regimen in place in Ireland. DESIGN: Healthy children who were attending Our Lady's Children's Hospital, Crumlin, for routine minor surgery were recruited to provide a fasting 3-mL blood sample that was taken while a general anesthetic was administered. The samples were analyzed for plasma folate, red blood cell folate, and UFA concentrations. A short dietary questionnaire that captured recent and habitual intakes of folic acid, both as supplements and as fortified foods, was completed face to face with parents. RESULTS: We collected fasting samples (n = 68) and completed questionnaires that captured recent and habitual daily folic acid intakes of children grouped as follows: 0-5 y of age: 6 girls and 21 boys (27 children total); 6-10 y of age: 10 girls and 10 boys (20 children total); and 11-16 y of age: 10 girls and 11 boys (21 children total). UFA was detected in 10.3% of the samples tested (range: 0.5-1.3 nmol/L). Mean plasma folate and red blood cell folate concentrations were 35.1 nmol/L (range: 21-47 nmol/L) and 956 nmol/L (range: 305-2319 nmol/L), respectively. Mean daily intake of folic acid from fortified foods and supplements was 109 µg (range: 0-767 µg). CONCLUSIONS: We showed that there was UFA in the plasma of just >10% of the children sampled after an overnight fast. These findings should be considered by policy makers who are responsible for folic acid fortification. This trial was registered at www.isrctn.com as ISRCTN90038765.

Evidence from a Randomized Trial That Exposure to Supplemental Folic Acid at Recommended Levels during Pregnancy Does Not Lead to Increased Unmetabolized Folic Acid Concentrations in Maternal or Cord Blood.

BACKGROUND: Exposure to higher intakes of folic acid (FA) from fortified foods and supplements, although largely considered beneficial, is associated with unmetabolized FA in the circulation, which has raised some health concerns. OBJECTIVE: The effect of supplemental FA at a dose of 400 µg/d during pregnancy on unmetabolized FA concentrations in maternal plasma and newborn cord blood plasma was investigated. METHODS: A new analysis was performed of blood samples from participants in a randomized trial in pregnancy. Women aged 18-35 y, who had taken 400 µg FA/d as recommended in the first trimester, were recruited at the start of trimester 2 and randomly allocated to receive either 400 µg FA/d (n = 59) or a placebo (n = 67) throughout the second and third trimesters until delivery. Unmetabolized FA concentrations in maternal and cord blood samples were measured by LC-tandem MS analysis. RESULTS: In response to the intervention from gestational week 14 through delivery, a higher proportion of women in the FA compared with the placebo group had detectable FA (≥0.27 nmol/L) in plasma, but the difference in concentrations was not statistically significant (mean ± SD: 0.44 ± 0.80 compared with 0.13 ± 0.49 nmol/L, P = 0.38). FA treatment throughout pregnancy resulted in higher cord blood plasma total folate (50.6 ± 20.1 compared with 34.5 ± 14.4 nmol/L; P = 0.004) and 5-methyltetrahydrofolate (50.4 ± 20.3 compared with 34.5 ± 14.4 nmol/L; P = 0.005) concentrations, but FA was detected only in 8 of 53 available cord blood samples, and the proportion of samples with detectable FA concentrations was similar in FA-treated and placebo groups. CONCLUSIONS: Plasma concentrations of unmetabolized FA arising from supplemental FA at a dose of 400 µg/d, in addition to FA from fortified foods, were low or undetectable in mothers and newborns. The benefits for mothers and offspring of continuing FA supplementation beyond the first trimester of pregnancy can be achieved without posing any risk of increasing unmetabolized circulating FA, even in those already exposed to FA from fortified foods.

Folic acid causes higher prevalence of detectable unmetabolized folic acid in serum than B-complex: a randomized trial.

PURPOSE: Unmetabolized folic acid (UMFA) is common in serum of elderly individuals receiving folic acid (FA)-fortified foods or supplements. We studied the effect of supplementing FA or B-complex on serum concentrations of (6S)-5-methyltetrahydropteroylglutamate [(6S)-5-CH3-H4Pte] and UMFA in elderly people and explored factors associated with detectable UMFA post-supplementation. METHODS: This is a randomized single-blind non-controlled trial on 58 elderly people using daily 400 µg FA (n = 31) or 400 µg FA, 10 µg cyanocob(III)alamin and 8 mg pyridoxine (n = 27) for a median of 23 days. Main outcome includes changes in concentrations of serum (6S)-5-CH3-H4Pte and UMFA. RESULTS: Total homocysteine declined by a median of 1.6 (p = 0.074) in the FA and 1.3 µmol/L (p = 0.009) in the B-complex arms (p = 0.66 between the arms). Serum (6S)-5-CH3-H4Pte significantly (p < 0.001 vs. baseline) increased by a median of 9.2 and 6.5 nmol/L in the FA and B-complex groups, respectively (p = 0.152 between the groups). Compared to FA, B-complex reduced cystathionine and caused lower post-intervention serum UMFA, percentage of UMFA to (6S)-5-CH3-H4Pte and prevalence of UMFA ≥ 0.21 nmol/L. Higher serum cystathionine and whole-blood folate predicted higher post-intervention serum UMFA. CONCLUSIONS: FA caused higher UMFA as compared to B-complex. Pyridoxine appears to improve folate recycling. Data on serum UMFA should be interpreted in relation to other vitamins involved in folate metabolism. Serum UMFA is suggested to play a sensory role through which the cell recognizes FA available for metabolism via dihydrofolate reductase.