Although simultaneous supplementation with iron and folic acid is justified, the potential interactions between these micronutrients are unknown. The aim of this study was to determine the effects of oral iron and folic acid, administered together or separately, on iron concentration in tissues in rats with a deficiency of both these micronutrients. In the first stage of the experiment (28 days), 150 8-week-old female Wistar rats were randomly assigned to a control group (C; n = 30) fed the standard diet and to a study group (n = 120) fed a diet deficit in iron and folate. The study group was then randomly divided to four groups: D group fed a deficit diet, FE group fed a deficit diet with iron gluconate, the FOL group fed a deficit diet with folate acid, and the FEFOL group fed a deficit diet with iron gluconate and folate acid. After 2, 10, and 21 days of supplementation, ten animals from each group were killed. Morphological parameters were measured in whole blood. Iron concentration was assayed in serum, liver, spleen, pancreas, heart, and kidneys. Folic acid supplementation more significantly decreased iron concentrations in the pancreas and spleen than in the D group after 10 and 21 days of supplementation. Moreover, the combination of iron with folic acid markedly decreased iron levels in the liver and spleen, in comparison with iron alone, after 10 and 21 days of the experiment. In conclusion, folic acid affects iron status in female rats deficient in these micronutrients in moderate and long-term supplementation.

Core binding factor β (Cbfb) is a cofactor of the Runx family of transcription factors. Among these transcription factors, Runx1 is a prerequisite for anterior-specific palatal fusion. It was previously unclear, however, whether Cbfb served as a modulator or as an obligatory factor in the Runx signaling process that regulates palatogenesis. Here, we report that Cbfb is essential and indispensable in mouse anterior palatogenesis. Palatal fusion in Cbfb mutants is disrupted owing to failed disintegration of the fusing epithelium specifically at the anterior portion, as observed in Runx1 mutants. In these mutants, expression of TGFB3 is disrupted in the area of failed palatal fusion, in which phosphorylation of Stat3 is also affected. TGFB3 protein has been shown to rescue palatal fusion in vitro TGFB3 also activated Stat3 phosphorylation. Strikingly, the anterior cleft palate in Cbfb mutants is further rescued by pharmaceutical application of folic acid, which activates suppressed Stat3 phosphorylation and Tgfb3 expression in vitro With these findings, we provide the first evidence that Cbfb is a prerequisite for anterior palatogenesis and acts as an obligatory cofactor in the Runx1/Cbfb-Stat3-Tgfb3 signaling axis. Furthermore, the rescue of the mutant cleft palate using folic acid might highlight potential therapeutic targets aimed at Stat3 modification for the prevention and pharmaceutical intervention of cleft palate.

Renal fibrosis is a pathologic feature of chronic kidney disease, which can lead to end-stage kidney disease. Myeloid fibroblasts play a central role in the pathogenesis of renal fibrosis. However, the molecular mechanisms pertaining to myeloid fibroblast activation remain to be elucidated. In the present study, we examine the role of signal transducer and activator of transcription 6 (STAT6) in myeloid fibroblast activation, macrophage polarization, and renal fibrosis development in a mouse model of folic acid nephropathy. STAT6 is activated in the kidney with folic acid nephropathy. Compared with folic-acid-treated wild-type mice, STAT6 knockout mice had markedly reduced myeloid fibroblasts and myofibroblasts in the kidney with folic acid nephropathy. Furthermore, STAT6 knockout mice exhibited significantly less CD206 and PDGFR-β dual-positive fibroblast accumulation and M2 macrophage polarization in the kidney with folic acid nephropathy. Consistent with these findings, STAT6 knockout mice produced less extracellular matrix protein, exhibited less severe interstitial fibrosis, and preserved kidney function in folic acid nephropathy. Taken together, these results have shown that STAT6 plays a critical role in myeloid fibroblasts activation, M2 macrophage polarization, extracellular matrix protein production, and renal fibrosis development in folic acid nephropathy. Therefore, targeting STAT6 may provide a novel therapeutic strategy for fibrotic kidney disease.

Craniofacial development requires extremely fine-tuned developmental coordination of multiple specialized tissues. It has been evidenced that a folate deficiency (vitamin B9), or its synthetic form, folic acid (FA), in maternal diet could trigger multiple craniofacial malformations as oral clefts, tongue, or mandible abnormalities. In this study, a folic acid-deficient (FAD) diet was administered to eight-week-old C57/BL/6J female mouse for 2-16 weeks. The head symmetry, palate and nasal region were studied in 24 control and 260 experimental fetuses. Our results showed a significant reduction in the mean number of fetuses per litter according to maternal weeks on FAD diet (p < 0.01). Fetuses were affected by cleft palate (3.8%) as well as other severe congenital abnormalities, for the first time related to maternal FAD diet, as head asymmetries (4.6%), high arched palate (3.5%), nasal septum malformed (7.3%), nasopharynx duct shape (15%), and cilia and epithelium abnormalities (11.2% and 5.8%). Dysmorphologies of the nasal region were the most frequent, appearing at just four weeks following a maternal FAD diet. This is the first time that nasal region development is experimentally related to this vitamin deficiency. In conclusion, our report offers novel discoveries about the importance of maternal folate intake on midface craniofacial development of the embryos. Moreover, the longer the deficit lasts, the more serious the consequent effects appear to be.

Recent efforts have revealed the microRNA (miRNA) pathways in the pathogenesis of Alzheimer's disease (AD). Epidemiological studies have revealed an association between folic acid deficiency and AD risk. However, the effects of folic acid deficiency on miRNA expression in AD animals have not been observed. We aimed to find if folic acid deficiency may enhance amyloid-β (Aβ) peptide deposition and regulate amyloid-associated miRNAs and their target genes expression in APP/PS1 mice. APP/PS1 mice and N2a cells were treated with folic acid-deficient diet or medium. Cognitive function of mice was assessed using the Morris water maze. miRNA profile was tested by polymerase chain reaction (PCR) array. Different expressional miRNAs were validated by real-time PCR. The deposition of Aβ plaques was evaluated by immunohistochemistry and enzyme-linked immunosorbent assay. APP and BACE1 proteins in mice brain and N2a cells were determined by Western blot. Folic acid deficiency aggravated amyloid pathology in AD mice. The AD+FD group showed shorter time spent in the target zone during the probe test. Analysis of miRNAs predicted to target these genes revealed several miRNA candidates that were differentially modulated by folic acid deficiency. In APP/PS1 mice brains and N2a cells with folic acid-deficient treatment, miR-106a-5p, miR-200b-3p and miR-339-5p were down-regulated, and their target genes APP and BACE1 were up-regulated. In conclusion, folic acid deficiency can enhance Aβ accumulation in APP/PS1 mice brain and decrease amyloid-associated miRNAs expression.

Increased consumption of folic acid is prevalent, leading to concerns about negative consequences. The effects of folic acid on the liver, the primary organ for folate metabolism, are largely unknown. Methylenetetrahydrofolate reductase (MTHFR) provides methyl donors for S-adenosylmethionine (SAM) synthesis and methylation reactions.

Supplementation with iron and folic acid is widely recommended in women of childbearing age and during pregnancy; however, the effect of such supplementation on mineral status is not well-known. The aim of this study was to determine the effects of oral iron and folic acid, administered together and separately, on copper, zinc, calcium, and magnesium concentrations in the tissues of rats with a deficiency of both these micronutrients. The experiment was performed on 8-week-old female Wistar rats. In the first stage of the experiment, the animals were randomly assigned to a control group of rats fed the standard diet (AIN-93 M), and to a study group of rats fed a diet deficient in iron and folate. The study group was then randomly divided to four groups: group D was fed a deficit diet, group FE was fed a deficit diet with iron gluconate, the FOL group was fed a deficit diet with folate acid, and the FEFOL group was fed a deficit diet with iron gluconate and folate acid. After 2, 10, and 21 days of the intervention, ten animals from each group were killed. Mineral concentrations were assayed in the liver, spleen, pancreas, heart, and kidneys using atomic absorption spectrometry. Statistical analysis was performed using Statistica 12.0 with the ANOVA test (p < 0.05). It was found that separate supplementation with iron and folic acid significantly decreased copper concentrations in tissues. The deficit in iron and folic acid decreased, and their simultaneous supplementation increased calcium content in the organs. Separate and simultaneous supplementation decreased magnesium status in deficient rats. In conclusion, iron and folic acid, supplemented separately or simultaneously, affect the copper, calcium, and magnesium level in tissues.

Vitamins B9 (folate) and B12 act as methyl donors in the one-carbon metabolism which influences epigenetic mechanisms. We previously showed that an embryofetal deficiency of vitamins B9 and B12 in the rat increased brain expression of let-7a and miR-34a microRNAs involved in the developmental control of gene expression. This was reversed by the maternal supply with folic acid (3 mg/kg/day) during the last third of gestation, resulting in a significant reduction of associated birth defects. Since the postnatal brain is subject to intensive developmental processes, we tested whether further folate supplementation during lactation could bring additional benefits. Vitamin deficiency resulted in weaned pups (21 days) in growth retardation, delayed ossification, brain atrophy and cognitive deficits, along with unchanged brain level of let-7a and decreased expression of miR-34a and miR-23a. Whereas maternal folic acid supplementation helped restore the levels of affected microRNAs, it led to a reduction of structural and functional defects taking place during the perinatal/postnatal periods, such as learning/memory capacities. Our data suggest that a gestational B-vitamin deficiency could affect the temporal control of the microRNA regulation required for normal development. Moreover, they also point out that the continuation of folate supplementation after birth may help to ameliorate neurological symptoms commonly associated with developmental deficiencies in folate and B12.

Embryonic development is a critical period wherein brain neurons are generated and organized. Maternal dietary folate, a cofactor in one-carbon metabolism, modulates neurogenesis and apoptosis in foetal brain neurons. We hypothesized that aberrant neuronal apoptosis may affect the development of the central nervous system during maternal folic acid deficiency, with evident effects because maternal folic acid deficiency modulates the microRNA-34a associated with Bcl-2 pathway during embryonic development. Four-week-old female Sprague-Dawley rats were divided randomly into two groups (10 rats per group): a folate-deficient diet group and a folate-normal diet group. The diets were administered to the rats 60 d before mating, which was continued for the pregnant dams until parturition. Maternal folic acid deficiency increased neuronal apoptosis in the hippocampus and the cortex in the offspring. Furthermore, maternal folic acid deficiency increased the ratio of cleaved caspase-3/caspase-3, followed by an increase in caspase-3 activity. Moreover, maternal folic acid deficiency downregulated Bcl-2 and upregulated Bax, and this effect associate with maternal folic acid deficient increases expression of microRNA-34a. Together, the present results indicate that maternal folic acid deficiency stimulates neuronal apoptosis via microRNA-34a associated with Bcl-2 signalling in rat offspring.

Disturbed epigenetic modifications have been linked to the pathogenesis of Neural Tube Defects (NTDs) in those with folate deficiency during pregnancy. However, evidence is lacking to delineate the critical region in epigenome regulated by parental folic acid and mechanisms by which folate deficiency affects normal embryogenesis. Our data from clinical samples revealed the presence of aberrant DNA methylation in GNAS imprinting cluster in NTD samples with low folate concentrations. Results from mouse models indicated that the establishment of GNAS imprinting was influenced by both maternal and paternal folate-deficient diets. Such aberrant GNAS imprinting was present prior to the gametogenesis period. Imprinting in Exon1A/GNAS gDMR was abolished in both spermatozoa and oocytes upon treating with a parental folate-deficient diet (3.6% in spermatozoa, 9.8% in oocytes). Interestingly, loss of imprinting in the GNAS gene cluster altered chromatin structure to an overwhelmingly open structure (58.48% in the folate-free medium group vs. 39.51% in the folate-normal medium group; P < 0.05), and led to a disturbed expression of genes in this region. Furthermore, an elevated cyclic AMP levels was observed in folate acid deficiency group. Our results imply that GNAS imprinting plays major roles in folic acid metabolism regulation during embryogenesis. Aberrant GNAS imprinting is an attribute to NTDs, providing a new perspective for explaining the molecular mechanisms by which folate supplementation in human pregnancy provides protection from NTDs.

The link between folate deficiency and congenital spina bifida defects was first suggested in the 1960s. Although the prevention of these defects by preconception folic acid supplementation was confirmed in a large multi-centre controlled trial in 1991, its subsequent implementation as health education advice has made very little difference. North America's policy of folic acid fortification of flour and bread has had a beneficial impact. No European country has implemented fortification due to concern over possible adverse effects on older subjects, but a recent review shows these to be largely hypothetical and far outweighed by beneficial effects. Recent research by Menezo et al. has, however, shown that folic acid is ineffective for some women with severe fertility problems including recurrent miscarriage and failed in vitro fertilisation. There is a genetically determined bottleneck (677TT) in their folate metabolism that can be successfully overridden by going straight to the next step in the metabolic pathway and taking 5-methylytetrahydrofolate, as a preconception supplement. Menezo suggests that all women with fertility problems should be tested for 677TT. If fortification of flour and bread is to be implemented in the UK, there should be monitoring for possible adverse effects including the incidence of colorectal cancers and cognitive decline. In conclusion, whilst there are concerns that fortification could have a detrimental effect on these conditions, there is sound evidence that it would have much greater beneficial effects.

Human Amylin, or islet amyloid polypeptide (hIAPP), is a small hormone secreted by pancreatic β-cells that forms aggregates under insulin deficiency metabolic conditions, and it constitutes a pathological hallmark of type II diabetes mellitus. In type II diabetes patients, amylin is abnormally increased, self-assembled into amyloid aggregates, and ultimately contributes to the apoptotic death of β-cells by mechanisms that are not completely understood. We have screened a library of approved drugs in order to identify inhibitors of amylin aggregation that could be used as tools to investigate the role of amylin aggregation in type II diabetes or as therapeutics in order to reduce β-cell damage. Interestingly, three of the compounds analyzed-benzbromarone, quercetin, and folic acid-are able to slow down amylin fiber formation according to Thioflavin T binding, turbidimetry, and Transmission Electron Microscopy assays. In addition to the in vitro assays, we have tested the effect of these compounds in an amyloid toxicity cell culture model and we have found that one of them, quercetin, has the ability to partly protect cultured pancreatic insulinoma cells from the cytotoxic effect of amylin. Our data suggests that quercetin can contribute to reduce oxidative damage in pancreatic insulinoma β cells by modulating the aggregation propensity of amylin.

The most important function of vitamin B12 is to accomplish DNA synthesis, which is necessary for cell division and proliferation. Deficiency of vitamin B12 causes megaloblastic anemia, retardation of growth, and delay in neuromotor maturation. Newborns whose mothers have vitamin B12 deficiency are born with low vitamin B12 storages, and are at risk in terms of vitamin B12 deficiency symptoms during infancy. The aim of our study was to investigate the frequency of anemia and deficiency of vitamin B12, folic acid, and iron in pregnant women living in our region, in their newborn babies, and during the infancy period of these babies. Another aim of our study was to investigate the correlation between the levels of these vitamins in newborns and in their mothers.

To demonstrate the role of IL-6 and pSTAT3 in the inflammatory response to cerebral ischemia/reperfusion following folic acid deficiency (FD).

Moderate folic acid (FA) intake is an effective strategy that slows colorectal cancer (CRC) progression. However, high consumption of FA may trigger the transition of precancerous tissue towards malignancy. MicroRNAs (miRNAs) are considered to be potential biomarkers of CRC. Thus, identification of miRNAs of dysregulated genes in CRC cells by detailed analysis of mRNA and miRNA expression profile in the context of FA deficiency could substantially increase our understanding of its oncogenesis. mRNA-seq and miRNA-seq analyses were utilized to investigate the expression of miRNAs in FA-deficient CRC cell line-HCT116 through massive parallel sequencing technology. A total of 38 mRNAs and 168 miRNAs were identified to be differentially expressed between CRC groups with or without FA deficiency. We constructed an miRNA-mRNA network for the vital regulatory miRNAs altered in FA-deficient CRC cells. The mRNAs and miRNAs validated by Western blotting and RT-qPCR were consistent with the sequencing results. Results showed that FA deficiency upregulated some miRNAs thereby inhibiting the expression of critical genes in the endoplasmic reticulum (ER) stress pathway. Dysregulated miRNAs in our miRNA-mRNA network could contribute to CRC cell in response to deficient FA. This work reveals novel molecular targets that are likely to provide therapeutic interventions for CRC.

Folate has received international attention regarding its role in the risk-reduction of birth defects, specifically neural tube defects (NTDs). In 1998, health officials in Canada, like the United States, mandated the addition of folic acid to white flour and select grain products to increase the folate intake of reproductive-aged women. Subsequent to this initiative there has been an increase in blood folate concentrations in Canada and a 50% reduction in NTDs. Many countries, including Korea, have not mandated folic acid fortification of their food supply. Reasons vary but often include concern over the masking of vitamin B(12) deficiency, a belief that folate intakes among womenare adequate, low priority relative to other domestic issues, and the philosophy that individuals have the right not to consume supplemental folic acid if they so choose. Prior to folic acid fortification of the food supply in Canada, the folate intakes of women were low, and their blood folate concentrations while not sufficiently low to produce overt signs of folate deficiency (eg. anemia) were inconsistent with a level known to reduce the risk of an NTD-affected pregnancy. The purpose of this article is to describe the role of folate during the periconceptional period, pregnancy, and during lactation. The rationale for, and history of recommending folic acid-containing supplements during the periconceptional period and pregnancy is described as is folic acid fortification of the food supply. The impact of folic acid fortification in Canada is discussed, and unresolved issues associated with this policy described. While the incidence of NTDs in Canada pre-folic acid fortification were seemingly higherthan that of Korea today, blood folate levels of Korean women are strikingly similar. We will briefly explore these parallels in an attempt to understand whether folic acid fortification of the food supply in Korea might be worth consideration.

The cholesterol-oxidized metabolite 27-hydroxycholesterol (27-OHC) is synthesized by CYP27A1, which is a key factor in vitamin D and oxysterol metabolism. Both vitamin D and 27-OHC are considered to play important roles in Alzheimer’s disease (AD). The study aims to research the effects of co-supplementation of vitamin D, folic acid, and vitamin B12 on learning and memory ability in vitamin D-deficient mice, and to explore the underlying mechanism. In this study, C57BL/6J mice were fed a vitamin D-deficient diet for 13 weeks to establish a vitamin D-deficient mice model. The vitamin D-deficient mice were then orally gavaged with vitamin D (VD), folic acid (FA), and vitamin B12 (VB12) alone or together for eight weeks. Following the gavage, the learning and memory ability of the mice were evaluated by Morris Water Maze and Novel object recognition test. The CYP27A1-related gene and protein expressions in the liver and brain were determined by qRT-PCR. The serum level of 27-OHC was detected by HPLC-MS. Serum levels of 25(OH)D, homocysteine (Hcy), and S-Adenosylmethionine (SAM) were measured by ELISA. After feeding with the vitamin D-deficient diet, the mice performed longer latency to a platform (p < 0.001), lower average speed (p = 0.026) in the Morris Water Maze, a lower time discrimination index (p = 0.009) in Novel object recognition, and performances were reversed after vitamin D, folic acid and vitamin B12 supplementation alone or together (p < 0.05). The gene expressions of CYP27A1 in the liver and brain were upregulated in the vitamin D-deficiency (VDD) group compared with the control (CON) group (p = 0.015), while it was downregulated in VDD + VD and VDD + VD-FA/VB12 groups compared with the VDD group (p < 0.05), with a similar trend in the protein expression of CYP27A1. The serum levels of 27-OHC were higher in the VDD group, compared with CON, VDD + VD, and VDD + VD-FA/VB12 group (p < 0.05), and a similar trend was found in the brain. The serum 25(OH)D levels were significantly decreased in the vitamin D-deficiency group (p = 0.008), and increased in the vitamin D-supplemented group (p < 0.001). The serum levels of SAM were higher in the B vitamins-supplemented group, compared with CON and VDD groups (p < 0.05). This study suggests that CYP27A1 expression may be involved in the mechanism of learning and memory impairment induced by vitamin D deficiency. Co-supplementation with vitamin D, folic acid, and vitamin B12 significantly reverses this effect by affecting the expression of CYP27A1, which in turn regulates the metabolism of 27-OHC, 25(OH)D, and SAM.

Low folate intake is associated with vascular disease. Causality has been attributed to hyperhomocysteinemia. However, human intervention trials have failed to show the benefit of homocysteine-lowering therapies. Alternatively, low folate may promote vascular disease by deregulating DNA methylation. We investigated whether folate could alter DNA methylation and atherosclerosis in ApoE null mice. Mice were fed one of six diets (n = 20 per group) for 16 weeks. Basal diets were either control (C; 4% lard) or high fat (HF; 21% lard and cholesterol, 0.15%) with different B-vitamin compositions: (1) folic acid and B-vitamin replete, (2) folic acid deficient (-F), (3) folic acid, B6 and B12 deficient (-F-B). -F diets decreased plasma (up to 85%; P < 0.05), whole blood (up to 70%; P < 0.05), and liver folate (up to 65%; P < 0.05) and hepatic SAM/SAH (up to 80%; P < 0.05). -F-B diets reduced plasma (up to 76%; P < 0.05), whole blood (up to 72%; P < 0.05), and liver B12 (up to 39%; P < 0.05) and hepatic SAM/SAH (up to 90%; P < 0.05). -F increased homocysteine 2-fold, while -F-B increased homocysteine 3.6- and 6.8-fold in the C and HF groups (P < 0.05). Plaque formation was increased 2-fold (P < 0.0001) in mice fed a HF diet. Feeding a HF-F diet increased lesion formation by 17% (P < 0.05). There was no change in 5-methyldeoxycytidine in liver or vascular tissue (aorta, periadventitial tissue and heart). These data suggest that atherogenesis is not associated with genome-wide epigenetic changes in this animal model.

Astrocytes are the most widely distributed cells in the brain, and astrocyte apoptosis may play an important role in the pathogenesis of neurodegenerative diseases. Folate is required for the normal development of the nervous system, but its effect on astrocyte apoptosis is unclear. In this study, we hypothesized that folic acid (the therapeutic form of folate) decreases astrocyte apoptosis by preventing oxidative stress-induced telomere attrition. Primary cultures of astrocytes were incubated for 12 days with various concentrations of folic acid (0-40 μmol/L), then cell proliferation, apoptosis, intracellular folate concentration, intracellular homocysteine (Hcy) concentration, intracellular reactive oxygen species (ROS) levels, telomeric DNA oxidative damage, and telomere length were determined. The results showed that folic acid deficiency decreased intracellular folate, cell proliferation, and telomere length, whereas it increased Hcy concentration, ROS levels, telomeric DNA oxidative damage, and apoptosis. In contrast, folic acid dose-dependently increased intracellular folate, cell proliferation, and telomere length but it decreased Hcy concentration, ROS levels, telomeric DNA oxidative damage, and apoptosis. In conclusion, folic acid inhibited apoptosis in astrocytes. The underlying mechanism for this protective effect may be that folic acid decreased oxidative stress and thereby prevented telomeric DNA oxidative damage and telomere attrition.

The gut-liver axis (GLA) plays an important role in the development of alcohol-induced liver injury. Alcohol consumption is typically associated with folic acid deficiency. However, no clear evidence has confirmed the effect of folic acid supplementation on alcohol-induced liver injury via GLA homeostasis. In this study, male C57BL/6J mice were given 56% (v/v) ethanol and 5.0 mg/kg folic acid daily by gavage for 10 weeks to investigate potential protective mechanisms of folic acid in alcohol-induced liver injury via GLA homeostasis. Histopathological and biochemical analyses showed that folic acid improved lipid deposition and inflammation in the liver caused by alcohol consumption and decreased the level of ALT, AST, TG, and LPS in serum. Folic acid inhibited the expression of the TLR4 signaling pathway and its downstream inflammatory mediators in the liver and upregulated the expression of ZO-1, claudin 1, and occludin in the intestine. But compared with the CON group, folic acid did not completely eliminate alcohol-induced intestine and liver injury. Furthermore, folic acid regulated alcohol-induced alterations in gut microbiota. In alcohol-exposed mice, the relative abundance of Bacteroidota was significantly increased, and the relative abundance of unclassified_Lachnospiraceae was significantly decreased. Folic acid supplementation significantly increased the relative abundance of Verrucomicrobia, Lachnospiraceae_NK4A136_group and Akkermansia, and decreased the relative abundance of Proteobacteria. The results of Spearman's correlation analysis showed that serum parameters and hepatic inflammatory cytokines were significantly correlated with several bacteria, mainly including Bacteroidota, Firmicutes, and unclassified_Lachnospiraceae. In conclusion, folic acid could ameliorate alcohol-induced liver injury in mice via GLA homeostasis to some extent, providing a new idea and method for prevention of alcohol-induced liver injury.