Inhibition of MTOR signaling impairs rat embryo organogenesis by affecting folate availability.

Mechanistic target of rapamycin (MTOR) is essential for embryo development by acting as a nutrient sensor to regulate cell growth, proliferation and metabolism. Folate is required for normal embryonic development and it was recently reported that MTOR functions as a folate sensor. In this work, we tested the hypothesis that MTOR functions as a folate sensor in the embryo and its inhibition result in embryonic developmental delay affecting neural tube closure and that these effects can be rescued by folate supplementation. Administration of rapamycin (0.5 mg/kg) to rats during early organogenesis inhibited embryonic ribosomal protein S6, a downstream target of MTOR Complex1, markedly reduced embryonic folate incorporation (-84%, P < 0.01) and induced embryo developmental impairments, as shown by an increased resorption rate, reduced embryo somite number and delayed neural tube closure. These alterations were prevented by folic acid administered to the dams. Differently, although an increased rate of embryonic rotation defects was observed in the rapamycin-treated dams, this alteration was not prevented by maternal folic acid supplementation. In conclusion, MTOR inhibition during organogenesis in the rat resulted in decreased folate levels in the embryo, increased embryo resorption rate and impaired embryo development. These data suggest that MTOR signaling influences embryo folate availability, possibly by regulating the transfer of folate across the maternal-embryonic interface.

Folate deficiency prevents neural crest fate by disturbing the epigenetic Sox2 repression on the dorsal neural tube.

Folate deficiency has been known to contribute to neural tube and neural crest defects, but why these tissues are particularly affected, and which are the molecular mechanisms involved in those abnormalities are important human health questions that remain unanswered. Here we study the function of two of the main folate transporters, FolR1 and Rfc1, which are robustly expressed in these tissues. Folate is the precursor of S-adenosylmethionine, which is the main donor for DNA, protein and RNA methylation. Our results show that knockdown of FolR1 and/or Rfc1 reduced the abundance of histone H3 lysine and DNA methylation, two epigenetic modifications that play an important role during neural and neural crest development. Additionally, by knocking down folate transporter or pharmacologically inhibiting folate transport and metabolism, we observed ectopic Sox2 expression at the expense of neural crest markers in the dorsal neural tube. This is correlated with neural crest associated defects, with particular impact on orofacial formation. By using bisulfite sequencing, we show that this phenotype is consequence of reduced DNA methylation on the Sox2 locus at the dorsal neural tube, which can be rescued by the addition of folinic acid. Taken together, our in vivo results reveal the importance of folate as a source of the methyl groups necessary for the establishment of the correct epigenetic marks during neural and neural crest fate-restriction.

Folic acid and diseases - supplement it or not?

INTRODUCTION: folic acid is a water soluble vitamin, which is synthetically-produced and found in fortified foods and supplements. Folate is found naturally in plants, such as the dark green leafy vegetables. Folate is not synthesized de novo by humans, therefore the daily requirements are met from the dietary intake of folic acid supplements or food rich in this vitamin. Folate deficiency could lead to numerous common health problems. Hyperhomocysteinemia and the possibility of malignancy developments are the long term consequences of this deficit albeit contradictory findings on these claims. METHODS: the articles included in this review focused on recent updated evidence-based reports and meta-analyses on the associations of the serum folate/folic acid and the various diseases found globally. RESULTS: the benefit of folic acid supplementation in the pre-conception period for the prevention of neural tube defects (NTDs) was well established and it was suggested that counseling sessions should be given to women with previous pregnancies affected by NTDs. However, supplementation of folic acid and its medicinal effects in the treatment of other diseases were contradictory and unclear. CONCLUSION: more detailed investigations into the health benefits of folic acid are needed before it could be recommended for supplementation, treatment or prevention of some of the diseases discussed in this review.

Folic acid and diseases - supplement it or not? / Ácido fólico e doenças - suplementá-lo ou não?

SUMMARY Introduction: folic acid is a water soluble vitamin, which is synthetically-produced and found in fortified foods and supplements. Folate is found naturally in plants, such as the dark green leafy vegetables. Folate is not synthesizedde novo by humans, therefore the daily requirements are met from the dietary intake of folic acid supplements or food rich in this vitamin. Folate deficiency could lead to numerous common health problems. Hyperhomocysteinemia and the possibility of malignancy developments are the long term consequences of this deficit albeit contradictory findings on these claims. Methods: the articles included in this review focused on recent updated evidence-based reports and meta-analyses on the associations of the serum folate/folic acid and the various diseases found globally. Results: the benefit of folic acid supplementation in the pre-conception period for the prevention of neural tube defects (NTDs) was well established and it was suggested that counseling sessions should be given to women with previous pregnancies affected by NTDs. However, supplementation of folic acid and its medicinal effects in the treatment of other diseases were contradictory and unclear. Conclusion: more detailed investigations into the health benefits of folic acid are needed before it could be recommended for supplementation, treatment or prevention of some of the diseases discussed in this review.

RESUMO Introdução: ácido fólico é uma vitamina solúvel em água produzida sinteticamente e encontrada em alimentos e suplementos enriquecidos. O folato é encontrado naturalmente em plantas, como vegetais folhosos verde-escuros. O folato não é sintetizado de novo por seres humanos; portanto, as necessidades diárias são satisfeitas a partir da ingestão de suplementos de ácido fólico ou alimentos ricos nessa vitamina. A deficiência de folato pode levar a inúmeros problemas de saúde comuns. Hiper-homocisteinemia e a possibilidade de desenvolver malignidades são as consequências a longo prazo desse déficit, ainda que os resultados sejam contraditórios sobre essas afirmações. Métodos: os artigos incluídos nesta revisão tratam de relatórios recentes atualizados com base em provas e metanálises sobre a associação entre o folato/ácido fólico e várias doenças encontradas globalmente. Resultados: o benefício da suplementação de ácido fólico no período de pré-concepção para a prevenção de defeitos do tubo neural (DTN) foi bem estabelecido e foi sugerido que sessões de aconselhamento devem ser providas às mulheres com gravidezes anteriores afetadas por DTN. No entanto, os benefícios da suplementação de ácido fólico e os efeitos medicinais no tratamento de outras doenças são contraditórios e pouco claros. Conclusão: investigações mais detalhadas sobre os benefícios do ácido fólico são necessárias antes que a suplementação seja recomendada para tratamento ou prevenção de algumas das doenças discutidas nesta revisão.

Folate, vitamin B12 and Homocysteine status in the post-folic acid fortification era in different subgroups of the Brazilian population attended to at a public health care center.

BACKGROUND: Folate and vitamin B12 are essential nutrients, whose deficiencies are considerable public health problems worldwide, affecting all age groups. Low levels of these vitamins have been associated with high concentrations of homocysteine (Hcy) and can lead to health complications. Several genetic polymorphisms affect the metabolism of these vitamins. The aims of this study were to assess folate, vitamin B12 and homocysteine status in distinct Brazilian individuals after the initiation of folic acid fortification by Brazilian authorities and to investigate the effects of RFC1 A80G, GCPII C1561T and MTHFR C677T polymorphisms on folate, vitamin B12 and Hcy levels in these populations. METHODS: A total of 719 individuals including the elderly, children, as well as pregnant and lactating women were recruited from our health care center. Folate, vitamin B12 and Hcy levels were measured by conventional methods. Genotype analyses of RFC1 A80G, GCPII C1561T and MTHFR C677T polymorphisms were performed by PCR-RFLP. RESULTS: The overall prevalence of folate and vitamin B12 deficiencies were 0.3% and 4.9%, respectively. Folate deficiency was observed only in the elderly (0.4%) and pregnant women (0.3%), whereas vitamin B12 deficiency was observed mainly in pregnant women (7.9%) and the elderly (4.2%). Plasma Hcy concentrations were significantly higher in the elderly (33.6%). Pregnant women carrying the MTHFR 677TT genotype showed lower serum folate levels (p = 0.042) and higher Hcy levels (p = 0.003). RFC1 A80G and GCPII C1561T polymorphisms did not affect folate and Hcy levels in the study group. After a multivariate analysis, Hcy levels were predicted by variables such as folate, vitamin B12, gender, age and RFC1 A80G polymorphism, according to the groups studied. CONCLUSION: Our results suggest that folate deficiency is practically nonexistent in the post-folic acid fortification era in the subgroups evaluated. However, screening for vitamin B12 deficiency may be particularly relevant in our population, especially in the elderly.

Supplementation with engineered Lactococcus lactis improves the folate status in deficient rats.

OBJECTIVE: The aim of this study was to establish the bioavailability of different folates produced by engineered Lactococcus lactis strains using a rodent depletion-repletion bioassay. METHODS: Rats were fed a folate-deficient diet, which produces a reversible subclinical folate deficiency, supplemented with different L. lactis cultures that were added as the only source of folate. Three bacterial strains that overexpressed the folC, folKE, or folC +KE genes were used. These strains produce folates with different poly glutamyl tail lengths. The growth response of the rats and the concentration of folates in different organs and blood samples were monitored. RESULTS: The folate produced by the engineered strains was able to compensate the folate depletion in the diet and showed similar bioavailability compared with commercial folic acid that is normally used for food fortification. Folate concentrations in organ and blood samples increased significantly in animals that received the folate-producing strains compared with those that did not receive bacterial supplementation. Hematologic studies also showed that administration of the L. lactis strains was able to revert a partial megaloblastic anemia caused by folate deficiency. No significant differences were observed in the bioavailability of folates containing different glutamyl tail lengths. CONCLUSION: To our knowledge, this is the first study that demonstrated that folates produced by engineered lactic acid bacteria represent a bioavailable source of this essential vitamin.

Lack of effect of diet-induced hypomethylation on endothelium-dependent relaxation in rats.

BACKGROUND: Endothelial dysfunction is a key process in atherosclerosis. Hypomethylation is one of the postulated mechanisms involved in atherogenesis and is mainly secondary to a decrease in essential factors such as, folate and vitamin B12 for the biosynthesis of S-adenosylmethionine (SAM), the main methyl-group donor for methylation reactions. AIM: To investigate in an animal model, whether hypomethylation, secondary to folate or vitamin B12 deficiency, affects endothelium-dependent relaxation (EDR) induced by acetylcholine (ACh). METHODS: Adult male Wistar rats were divided into 4 groups of 12 rats each: folate and B12 deficiency (FB12D 0mg folate/kg, 0 microg/kg B12), folate deficiency (FD 0mg folate/kg and 50 microg/kg B12), B12 deficiency (B12D: 8 mg/kg folate and 0 microg/kg B12 and control diet (CD)). After eight weeks the animals were killed and thoracic aorta and liver removed. Serum concentration of homocysteine, folate and vitamin B12 were determined. Hepatic levels of SAM and S-adenosylhomocysteine (SAH) were measured, as indicator of hypomethylation. ACh-induced EDR and sodium nitroprusside (SNP)-induced endothelium-independent relaxation (EIR), in isolated aorta rings were evaluated. RESULTS: Hcy concentrations were significantly increased in the folate and B12 deficient groups. SAM and the SAM/SAH ratio were lower in the FD and FB12D than in the control and B12D group. Folate, B12 deficiency, serum Hcy levels and hepatic SAM/SAH ratio did not affect EDR neither EIR. CONCLUSIONS: In adult Wistar rats, chronic folate or folate plus vitamin B12 deficiency generates hypomethylation which is not related to an alteration of endothelial function.

Estratégias de combate às microdeficiências no grupo materno-infantil / Strategies to combat the microdeficiencies in the maternal-infantile group / Estrategias del combate a las microdeficiencias en el grupo materno-infantil

As microdeficiências nutricionais, representadas especialmente pelas carências de vitamina A, ferro e ácido fólico, são amplamente reconhecidas como um importante problema de saúde e nutrição entre mulheres e crianças, refletindo de forma negativa na saúde reprodutiva e no desenvolvimento infantil. O presente trabalho objetivou fazer uma revisão da literatura sobre fortificação e suplementação de alimentos como estratégias para o combate às microdeficiências no grupo materno-infantil, com ênfase nos referidos nutrientes, no período de 1986 a 2006. Neste cenário, a suplementação nutricional, a fortificação de alimentos e o estímulo à diversificação alimentar representam estratégias promissoras no combate às carências de micronutrientes. Por outro lado, a suplementação deve ser criteriosa, baseada no limite tolerável para cada faixa etária e em estados fisiológicos, como a gestação. Por fim, o estímulo ao aleitamento materno e à prática da alimentação saudável são importantes aliados na tentativa de combater as deficiências nutricionais de vitaminas e minerais no grupo materno-infantil, em especial, reconhecidamente as de maior magnitude.

Micronutrient deficiencies, especially represented by vitamin A, iron and folic acid deficiencies, are widely recognized as an important health and nutritional problem among women and children reflecting, in a negative way, upon reproductive health and child development. The aim of the present study was to conduct a literature review about food fortification and supplementation as strategies of fighting against micronutrient deficiencies in the mother-child group, emphasizing the referred nutrients, in the period between 1986and 2006. According to the analyzed bibliography, the nutritional supplementation, food fortification and incentive to food diversification represent promising strategies towards fighting against micronutrient deficiencies. On the other hand, the supplementation must be established according to the tolerable limit for each age level and physiological state, like pregnancy. Finally, incentive to breast feeding and healthy eating practices are important allies in the attempt to fight against micronutrient deficiencies of vitamins and minerals in the mother-child group, especially those with greater magnitude.

Las microdeficiencias nutricionales, que son representadas especialmente, por la carencia de Vitamina A, hierro y ácido fólico, son ampliamente reconocidas como un importante problema de salud y nutrición en mujeres y niños, por repercutir negativamente en la salud reproductiva y en el desenvolvimiento infantil. El presente trabajo tuvo como objetivo la revisión de literatura científica que enfocara la fortificación y la suplementación de alimentos como estrategias de lucha contra microdeficiencias, en el grupo maternoinfantil, principalmente en los referidos nutrientes, en el periodo de 1986 a 2006. Eneste contexto, la suplementación nutricional, la fortificación de los alimentos y el estimulo a una alimentación variada, representan estrategias prometedoras de lucha contra las carencias de micronutrientes. Sin embargo, la suplementación debe ser hecha con criterio, basandose en los límites tolerables para cada grupo de edad y los estados fisiológicos, como la gestación. Además, el estimulo al amamantamiento materno y la practica de una alimentación saludable son importantes aliados en la tentativa de combatir, en el grupo materno infantil, las deficiencias nutricionales de vitaminas y de minerales, especialmente de aquellos de reconocida importancia.

Folate intake at RDA levels is inadequate for Mexican American men with the methylenetetrahydrofolate reductase 677TT genotype.

Since the establishment of the 1998 folate recommended dietary allowance (RDA), the methylenetetrahydrofolate reductase (MTHFR) 677C-->T variant has emerged as a strong modifier of folate status. This controlled feeding study investigated the adequacy of the RDA, 400 microg/d as dietary folate equivalents (DFE), for Mexican American men with the MTHFR 677CC or TT genotype. Because of the interdependency between folate and choline, the influence of choline intake on folate status was also assessed. Mexican American men (n = 60; 18-55 y) with the MTHFR 677CC (n = 31) or TT (n = 29) genotype consumed 438 microg DFE/d and total choline intakes of 300, 550 (choline adequate intake), 1100, or 2200 mg/d for 12 wk. Folate status response was assessed via serum folate (SF), RBC folate, plasma total homocysteine (tHcy), and urinary folate. SF decreased (P < 0.001) 66% to 7.9 +/- 0.7 nmol/L (means +/- SEM) in men with the 677TT genotype and 62% to 11.3 +/- 0.9 nmol/L in the 677CC genotype. Plasma tHcy increased (P < 0.0001) 170% to 31 +/- 3 micromol/L in men with the 677TT genotype and 18% to 11.6 +/- 0.3 micromol/L in the 677CC genotype. At the end of the study, 34% (677TT) and 16% (677CC) had SF concentrations <6.8 nmol/L and 79% (677TT) and 7% (677CC) had tHcy concentrations >14 micromol/L. Choline intake did not influence the response of the measured variables. These data showed that the folate RDA is not adequate for men of Mexican descent, particularly for those with the MTHFR 677TT genotype, and demonstrated a lack of influence of choline intake on the folate status variables measured in this study.

Methylenetetrahydrofolate reductase (MTHFR) c677t gene variant modulates the homocysteine folate correlation in a mild folate-deficient population.

BACKGROUND: A large body of evidence links plasma concentrations of homocysteine and cardiovascular disease. Several genetic and environmental variables may modulate such relationship. We investigated the influence of methylenetetrahydrofolate reductase (MTHFR) gene variants C677T, A1298C, and T1317C on homocysteine, folate, and cobalamin concentrations in a sample of individuals from a mild folate deficiency population to better clarify the complex interactions existing among these variables. METHODS: In the present study, 209 individuals belonging to an admixed urban population characterized by mild folate deficiency were investigated. MTHFR gene variants C677T, A1298C, and T1317C were genotyped and homocysteine-, folate-, and cobalamin-determined for each individual. RESULTS: Univariate analyses showed a significant association between the C677T variant with homocysteine (P<0.001) and cobalamin (P=0.005) as well as a significant relationship between the T allele and serum folate concentrations (P<0.05). The TT genotype of the C677T polymorphism remained significantly associated with log-transformed homocysteine even after adjustment for age, sex, smoking status, ethnicity, folate, and cobalamin concentrations (P<0.01). Both univariate and multivariate analysis have failed to show any effect of the A1298C and T1317C genetic variants in homocysteine concentrations in this population. Finally, a significant interaction between folate and C677T polymorphism in the determination of homocysteine was also disclosed (P<0.005). CONCLUSIONS: Taken together, these results demonstrate a significant interaction between serum folate and MTHFR genotype in predicting homocysteine concentrations. One may consider that a differential response of homocysteine to folic acid supplementation may depend on MTHFR genotype which may have important implications when attempting to lower homocysteine concentrations in populations with mild folate deficiency.

Alterações no metabolismo da homocisteína induzidas por aguardente de cana-de-açúcar em alcoólatras / Homocystein metabolism alterations induced by sugar-cane liquor in alcoholics

O alcoolismo está relacionado a má nutriçäo e baixos níveis de várias vitaminas que fazem parte do metabolismo da homocisteína (Hci). O objetivo deste estudo foi analisar a prevalência de hiper-homocisteinemia em pacientes com alta ingestäo diária de aguardente de cana-de-açúcar. Foram incluídos neste estudo 31 homens hospitalizados para tratamento de alcoolismo. Hci, folato (Fol), vitamina B12 séricos e enzimas hepáticas foram determinados e repetidos após 21 dias de abstinência alcoólica. Os valores de Hci em æmol/l antes e depois do tratamento foram, respectivamente, de 24,88 ± 2,09 e 12,48 ± 0,69. A abstinência alcoólica diminuiu significativamente os valores de aspartato aminotransferase, alanina aminotransferase e gamaglutamiltranspeptidase. Näo houve alteraçäo dos níveis de hemácias, proteínas totais e concentraçäo de hemoglobina corpuscular média. Os níveis de Hci antes do tratamento se correlacionaram com os de folato (r² = 0,333). Estes resultados sugerem que o alcoolismo crônico está acompanhado por perturbaçäo do metabolismo de aminoácidos sulfurados e que a hiper-homocisteinemia etanol-induzida através de aguardente de cana-de-açúcar pode ser acompanhada de níveis séricos baixos de folato, agravando o estado nutricional destes pacientes

Folate levels and N(5),N(10)-methylenetetrahydrofolate reductase genotype (MTHFR) in mothers of offspring with neural tube defects: a case-control study.

BACKGROUND: Neural tube defects (NTDs) have been associated with biochemical factors involved in the conversion of homocysteine to methionine as folate deficiency and the mutation 677T in the N(5),N(10)-methylenetetrahydrofolate reductase gene (MTHFR). METHODS: A case-control study was performed to detect this mutation in 38 unrelated women with NTD deceased products and 31 mothers without antecedents of NTD offspring. All products were born in Nuevo León (northeastern Mexico) during 1997. Erythrocyte and plasmatic folate levels and the genotype of the 677 polymorphism at the MTHFR locus were analyzed in both groups. RESULTS: Although no significant differences were found in mean blood folate levels, the percentage of women in the case group with erythrocyte folate levels <160 ng/mL was significantly higher than in the control group (75 vs. 51.2%, p <0.05). The proportion of women with plasma folate levels <3.5 ng/mL was higher in the case group (16.2 vs. 0%, p <0.01). Genotype analysis demonstrated a significantly higher proportion of 677T homozygous mothers with NTD products (39.6 vs. 9.1%, p <0.05). Allele frequencies for the 677T mutation were 0.55 and 0.36 for cases and controls, respectively. The odds ratio (OR) for having a NTD product was 6.1 (95%, CI 1.56-23.6) for homozygous 677T mothers vs. homozygous 677C and heterozygous mothers. Significantly low levels of erythrocyte folate were found in the 677C homozygous case group and in plasma folate in the 677C/677T heterozygous case mothers. CONCLUSIONS: Our study suggests that folate deficiency and MTHFR unfavorable genotype in mothers are important risk factors for severe NTD phenotype in our population.

Papel del ácido fólico en la etiología de las anemias megaloblásticas / Role of folic acid in the etiology of megaloblastic anemias

Se hace una revisión sobre el metabolismo de los folatos, su estructura química, fuentes dietéticas y requerimientos diarios en lso diferentes grupos de edades y estados patológicos, así como su absorción y distribución en el organismo. Se expone además la función matabólica del ácido fólico y su papel etiológico en el desarrollo de las anemias megaloblásticas, las causas de la deficiencia de este metabolilto y su tratamiento (AU)

Papel del ácido fólico en la etiología de las anemias megaloblásticas / Role of folic acid in the etiology of megaloblastic anemias

Se hace una revisión sobre el metabolismo de los folatos, su estructura química, fuentes dietéticas y requerimientos diarios en lso diferentes grupos de edades y estados patológicos, así como su absorción y distribución en el organismo. Se expone además la función matabólica del ácido fólico y su papel etiológico en el desarrollo de las anemias megaloblásticas, las causas de la deficiencia de este metabolilto y su tratamiento

Sister-chromatid exchanges in beta-thalassaemic patients under conditions of in vivo and in vitro depletion of folic acid.

In order to investigate the effect of folate depletion, lymphocyte sister-chromatid exchange (SCE) rates were compared among homozygous beta-thalassaemic patients with low folic acid levels, heterozygous beta-thalassaemic patients with normal folate levels and healthy persons with normal haemoglobin, in cultures with both normal and depleted folate conditions. Significantly higher SCE rates were found in homozygous patients in all assays, but the in vitro folate depletion did not induce an increase in SCE frequency in any group.