Folate Abstracts 12

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Mild folate deficiency induces genetic and epigenetic instability and phenotype changes in prostate cancer cells.
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BACKGROUND:  Folate (vitamin B9) is essential for cellular proliferation as it is involved in the biosynthesis of deoxythymidine monophosphate (dTMP) and s-adenosylmethionine (AdoMet). The link between folate depletion and the genesis and progression of cancers of epithelial origin is of high clinical relevance, but still unclear. We recently demonstrated that sensitivity to low folate availability is affected by the rate of polyamine biosynthesis, which is prominent in prostate cells. We, therefore, hypothesized that prostate cells might be highly susceptible to genetic, epigenetic and phenotypic changes consequent to folate restriction. RESULTS:  We studied the consequences of long-term, mild folate depletion in a model comprised of three syngenic cell lines derived from the transgenic adenoma of the mouse prostate (TRAMP) model, recapitulating different stages of prostate cancer; benign, transformed and metastatic. High-performance liquid chromatography analysis demonstrated that mild folate depletion (100 nM) sufficed to induce imbalance in both the nucleotide and AdoMet pools in all prostate cell lines. Random oligonucleotide-primed synthesis (ROPS) revealed a significant increase in uracil misincorporation and DNA single strand breaks, while spectral karyotype analysis (SKY) identified five novel chromosomal rearrangements in cells grown with mild folate depletion. Using global approaches, we identified an increase in CpG island and histone methylation upon folate depletion despite unchanged levels of total 5-methylcytosine, indicating a broad effect of folate depletion on epigenetic regulation. These genomic changes coincided with phenotype changes in the prostate cells including increased anchorage-independent growth and reduced sensitivity to folate depletion. CONCLUSIONS:  This study demonstrates that prostate cells are highly susceptible to genetic and epigenetic changes consequent to mild folate depletion as compared to cells grown with supraphysiological amounts of folate (2 microM) routinely used in tissue culture. In addition, we elucidate for the first time the contribution of these aspects to consequent phenotype changes in epithelial cells. These results provide a strong rationale for studying the effects of folate manipulation on the prostate in vivo, where cells might be more sensitive to changes in folate status resulting from folate supplementation or antifolate therapeutic approaches.


 

Folic Acid Supplementation Mitigates Alzheimer's Disease by Reducing Inflammation: A Randomized Controlled Trial.
            (Chen et al., 2016) Download
Background/Aims. Low serum folate levels can alter inflammatory reactions. Both phenomena have been linked to Alzheimer's disease (AD), but the effect of folic acid on AD itself is unclear. We quantified folate supplementation's effect on inflammation and cognitive function in patients with AD over the course of 6 months. Methods. Patients newly diagnosed with AD (age > 60 years; n = 121; mild to severe; international criteria) and being treated with donepezil were randomly assigned into two groups with (intervention group) or without (control group) supplemental treatment with folic acid (1.25 mg/d) for 6 months. The Mini-Mental State Examination (MMSE) was administered to all patients at baseline and follow-up, and blood samples were taken before and after treatment. We quantified serum folate, amyloid beta (Aβ), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), plasma homocysteine (Hcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and the mRNA levels of presenilin (PS), IL-6, and TNFα in leukocytes. Data were analyzed using a repeated-measures mixed model. Results. The mean MMSE was slightly increased in the intervention group compared to that in the control group (P < 0.05). Posttreatment, plasma SAM and SAM/SAH levels were significantly higher (P < 0.05), while Aβ 40, PS1-mRNA, and TNFα-mRNA levels were lower in the intervention group than in the control group (P < 0.05). The Aβ 42/Aβ 40 ratio was also higher in the intervention group (P < 0.05). Conclusions. Folic acid is beneficial in patients with AD. Inflammation may play an important role in the interaction between folic acid and AD. This trial is registered with clinical trial registration number ChiCTR-TRC-13003246.

No reliable evidence that folate is harmful in B-12 deficiency.
            (Dickinson, 1995) Download
Before liver and B-12 therapy for pernicious anaemia were introduced, neurological deterioration was often very rapid, whether or not folic acid had been given.


 

Serum homocysteine and folate concentrations among a US cohort of adolescents before and after folic acid fortification.
            (Enquobahrie et al., 2012) Download
OBJECTIVE:  We assessed serum homocysteine (tHcy) and folate concentrations among US adolescents before and after fortification of cereal-grain products with folic acid, and associations with demographic, behavioural and physiological factors. DESIGN:  Observational study conducted among participants of a randomized trial. SETTING:  The Child and Adolescent Trial for Cardiovascular Health (CATCH) study. SUBJECTS:  Adolescents (n 2445) in grades 8 (pre-fortification, mean age 14 years) and 12 (post-fortification, mean age 18 years). RESULTS:  Average serum concentrations of tHcy, folate and vitamin B6 increased by 17 %, 16 % and 14 %, respectively, while serum concentrations of vitamin B12 decreased by 11 % post-fortification. Folic acid fortification provided, on average, an additional intake of 118 μg folate/d. Male sex (P < 0.0001) and white race (P = 0.0008) were associated with significantly greater increases in tHcy concentration, while increases in BMI (P = 0.006) and serum folate concentration (P < 0.0001) were associated with significant decreases in tHcy concentration. Female sex (P < 0.0001), non-smoking (P < 0.0001), use of multivitamins (P < 0.0001) and higher dietary intake of folate (P = 0.001) were associated with significantly greater increases in serum folate concentrations. From grade 8 to grade 12, the upward age trend in serum tHcy concentration was uninterrupted in its course (P > 0.50); whereas serum folic acid concentration showed a downward trend that incurred a discrete jump upward (17 % higher; P < 0.0001) with fortification. These trends differed significantly for males v. females (P < 0.001 for interaction). CONCLUSIONS:  Fortification had a significant impact on improving folate status but not serum tHcy concentrations among US adolescents.

Nutrition throughout life: folate.
            (McNulty et al., 2012) Download
Scientific evidence supports a number of roles for folate in maintaining health from early life to old age. Folate is required for one-carbon metabolism, including the remethylation of homocysteine to methionine; thus elevated plasma homocysteine reflects functional folate deficiency. Optimal folate status has an established role in preventing NTD and there is strong evidence indicating that it also has a role in the primary prevention of stroke. The most important genetic determinant of homocysteine in the general population is the common 677C → T variant in the gene encoding the folate-metabolising enzyme, MTHFR; homozygous individuals (TT genotype) have reduced enzyme activity and elevated plasma homocysteine concentrations. Meta-analyses indicate that the TT genotype carries a 14 to 21 % increased risk of CVD, but there is considerable geographic variation in the extent of excess CVD risk. A novel interaction between this folate polymorphism and riboflavin (a co-factor for MTHFR) has recently been identified. Intervention with supplemental riboflavin targeted specifically at individuals with the MTHFR 677TT genotype was shown to result in significant lowering of blood pressure in hypertensive people and in patients with CVD. This review considers the established and emerging roles for folate throughout the lifecycle, and some public health issues related to optimising folate status.

Folic acid supplementation during pregnancy induces sex-specific changes in methylation and expression of placental 11beta-hydroxysteroid dehydrogenase 2 in rats.
            (Penailillo et al., 2015) Download
In the placenta, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) limits fetal glucocorticoid exposure and its inhibition has been associated to low birth weight. Its expression, encoded by the HSD11B2 gene is regulated by DNA methylation. We hypothesized that maternal diets supplemented with folic acid (FA) during pregnancy modify the expression of placental HSD11B2 through gene methylation. Wistar rats were fed with high (8 mg/kg) or normal low (1mg/kg, control) levels of FA during pregnancy. Concentrations of mRNA and protein in placentas were determined by qRT-PCR and Western blot respectively. Methylation in five CpG sites of the placental HSD11B2 promoter (-378 to -275) was analyzed by bacterial cloning and subsequent sequencing. In the FA-supplemented group, mRNA and protein levels of 11beta-HSD2 decreased by 58% and increased by 89%, respectively, only in placentas attached to males. In controls, most CpG sites were not methylated except for the CpG2 site which was 80% methylated. CpG2 methylation level increased under the FA treatment; however, only in placentas attached to females was this increase significant (113%). This change was not related to HSD11B2 expression. Fetal weight of females from FA- supplemented mothers was 6% higher than females from control mothers. In conclusion, this is the first study reporting that FA over supplementation during pregnancy modifies the placental HSD11B2 gene expression and methylation in a sex-dependent manner, suggesting that maternal diets with high content of FA can induce early sex-specific responses, which may lead to long-term consequences for the offspring.

Unmetabolized folic acid is detected in nearly all serum samples from US children, adolescents, and adults.
            (Pfeiffer et al., 2015) Download
BACKGROUND:  Serum total folate consists mainly of 5-methyltetrahydrofolate (5-methylTHF). Unmetabolized folic acid (UMFA) may occur in persons consuming folic acid-fortified foods or supplements. OBJECTIVES:  We describe serum 5-methylTHF and UMFA concentrations in the US population ≥1 y of age by demographic variables and fasting time, stratified by folic acid-containing dietary supplement use. We also evaluate factors associated with UMFA concentrations >1 nmol/L. METHODS:  Serum samples from the cross-sectional NHANES 2007-2008 were measured for 5-methylTHF (n = 2734) and UMFA (n = 2707) by HPLC-tandem mass spectrometry. RESULTS:  In supplement users compared with nonusers, we found significantly higher geometric mean concentrations of 5-methylTHF (48.4 and 30.7 nmol/L, respectively) and UMFA (1.54 and 0.794 nmol/L, respectively). UMFA concentrations were detectable (>0.3 nmol/L) in >95% of supplement users and nonusers, regardless of demographic or fasting characteristics; concentrations differed significantly by age and fasting time, but not by sex and race-ethnicity, both in supplement users and nonusers. The prevalence of UMFA concentrations >1 nmol/L was 33.2% overall and 21.0% in fasting (≥8 h) adults (≥20 y of age). Using multiple logistic regression analysis, UMFA concentrations >1 nmol/L were associated with being older, non-Hispanic black, nonfasting (<8 h), having smaller body surface area, higher total folic acid intake (diet and supplements), and higher red blood cell folate concentrations. In fasting adults, a decrease in the mean daily alcohol consumption was also associated with increased odds of UMFA concentrations >1 nmol/L. CONCLUSIONS:  UMFA detection was nearly ubiquitous, and concentrations >1 nmol/L were largely but not entirely explained by fasting status and by total folic acid intake from diet and supplements. These new UMFA data in US persons ≥1 y of age provide much-needed information on this vitamer in a fortified population with relatively high use of dietary supplements.

Apparent folate deficiency in iron-deficiency anaemia.
            (Roberts et al., 1971) Download
Examination of 50 patients with iron-deficient hypochromic anaemia showed evidence suggesting a high incidence of folate depletion. The peripheral blood films of 44 patients (88%) showed more than 3% of neutrophils with five nuclear lobes, 35 patients (70%) had a high mean neutrophil lobe count, 20 patients (40%) had hypersegmented neutrophils and 19 patients (38%) had giant metamyelocytes in the bone marrow. The serum folate was below 3 ng/ml in 12 patients and 3–6 ng/ml in 18. Red-cell folate was subnormal in 15%, and 45% had a positive Figlu test. Correlation between various tests for folate deficiency was not found, apart from a correlation between red-cell folate levels and morphological changes in the neutrophils. The haemoglobin rise following intravenous iron therapy was smaller when the serum folate level was low. There was probably a similar relationship to the red-cell folate level, but the numbers tested were small. The presence of neutrophil multilobing in the peripheral blood film and giant metamyelocytes in the bone marrow did not influence the response to iron therapy, neither did an abnormal Figlu excretion. Following intravenous iron therapy, both neutrophil multilobing and marrow giant metamyelocytes were significantly reduced in number. This therapy did not significantly alter Figlu excretion measured 6 weeks after treatment, but both serum and red-cell folate levels fell. Intravenous iron therapy did not produce any significant changes in the serum vitamin B12 levels. The part that iron deficiency itself may play in causing apparent folate depletion is discussed.


Folate, folic acid and 5-methyltetrahydrofolate are not the same thing.
            (Scaglione and Panzavolta, 2014) Download
1. Folate, an essential micronutrient, is a critical cofactor in one-carbon metabolism. Mammals cannot synthesize folate and depend on supplementation to maintain normal levels. Low folate status may be caused by low dietary intake, poor absorption of ingested folate and alteration of folate metabolism due to genetic defects or drug interactions. 2. Folate deficiency has been linked with an increased risk of neural tube defects, cardiovascular disease, cancer and cognitive dysfunction. Most countries have established recommended intakes of folate through folic acid supplements or fortified foods. External supplementation of folate may occur as folic acid, folinic acid or 5-methyltetrahydrofolate (5-MTHF). 3. Naturally occurring 5-MTHF has important advantages over synthetic folic acid - it is well absorbed even when gastrointestinal pH is altered and its bioavailability is not affected by metabolic defects. Using 5-MTHF instead of folic acid reduces the potential for masking haematological symptoms of vitamin B12 deficiency, reduces interactions with drugs that inhibit dihydrofolate reductase and overcomes metabolic defects caused by methylenetetrahydrofolate reductase polymorphism. Use of 5-MTHF also prevents the potential negative effects of unconverted folic acid in the peripheral circulation. 4. We review the evidence for the use of 5-MTHF in preventing folate deficiency.

The methyl folate trap. A physiological response in man to prevent methyl group deficiency in kwashiorkor (methionine deficiency) and an explanation for folic-acid induced exacerbation of subacute combined degeneration in pernicious anaemia.
            (Scott and Weir, 1981) Download
It is suggested that in man the methyl folate trap is a normal physiological response to impending methyl group deficiency resulting from a very low supply of methionine. This decreases cellular S-adenosyl-methionine (SAM), which puts at risk important methylation reactions, including those required to maintain myelin. In order to protect these methylation reactions, the cell has evolved two mechanisms to maintain supplies of methionine and SAM as a first priority. (a) Decreased SAM causes the folate co-factors to be directed through the cycle involving 5-methyl-tetrahydrofolate (5-methyl-THF) and methionine synthetase and away from the cycles that produce purines and pyrimidines for DNA synthesis. This enhances the remethylation of homocysteine to methionine and SAM. In addition, by restricting DNA biosynthesis and with it cell, division, competition for methionine for protein synthesis is reduced. Thus, whatever methionine is available is conserved for the vital methylation reactions in the nerves, brain, and elsewhere. (b) 5-methyl-THF, the form in which almost all folate is transported in human plasma, must react with intracellular homocysteine before it can be retained by the cell as a polyglutamate. Since homocysteine is derived entirely from methionine, methionine deficiency will cause intracellular folate deficiency, and the rate of mitosis of rapidly dividing cells will be reduced. although these two processes have evolved as a response to methionine deficiency, they also occur in B12 deficiency, which the cell mistakenly interprets as lack of methionine. the resulting response is inappropriate and gives rise to a potentially lethal anaemia. In these circumstances the methylation reactions are also partly protected by the reduced rate of cell division. This explains why administration of folic acid, which induces cell division and use of methionine in protein synthesis, impairs methylation of myelin and precipitates or exacerbates subacute combined degeneration (SCD). During folate deficiency methionine biosynthesis is also diminished. As in methionine deficiency, the body responds to decreasing availability of SAM by diverting folate away from DNA biosynthesis towards the remethylation of homocysteine to methionine and SAM. The selective use pf available folate to conserve methionine, together with the ability of nerve tissue to concentrate folate form the plasma, explains the absence of SCD in folate deficiency.

Plasma and red cell reference intervals of 5-methyltetrahydrofolate of healthy adults in whom biochemical functional deficiencies of folate and vitamin B 12 had been excluded.
            (Sobczyńska-Malefora et al., 2014) Download
5-Methyltetrahydrofolate (5-MTHF) is the predominant form of folate and a strong determinant of homocysteine concentrations. There is evidence that suboptimal 5-MTHF availability is a risk factor for cardiovascular disease independent of homocysteine. The analysis of folates remains challenging and is almost exclusively limited to the reporting of "total" folate rather than individual molecular forms. The purpose of this study was to establish the reference intervals of 5-MTHF in plasma and red cells of healthy adults who had been prescreened to exclude biochemical evidence of functional deficiency of folate and/or vitamin B12. Functional folate and vitamin B12 status was assessed by respective plasma measurements of homocysteine and methylmalonic acid in 144 healthy volunteers, aged 19-64 years. After the exclusion of 10 individuals, values for 134 subjects were used to establish the upper reference limits for homocysteine (13  μ mol/L females and 15  μ mol/L males) and methylmalonic acid (430 nmol/L). Subjects with values below these cutoffs were designated as folate and vitamin B12 replete and their plasma and red cell 5-MTHF reference intervals determined, N = 126: 6.6-39.9 nmol/L and 223-1041 nmol/L, respectively. The application of these intervals will assist in the evaluation of folate status and facilitate studies to evaluate the relationship of 5-MTHF to disease.


 

Folate receptor alpha defect causes cerebral folate transport deficiency: a treatable neurodegenerative disorder associated with disturbed myelin metabolism
            (Steinfeld et al., 2009) Download
Sufficient folate supplementation is essential for a multitude of biological processes and diverse organ systems. At least five distinct inherited disorders of folate transport and metabolism are presently known, all of which cause systemic folate deficiency. We identified an inherited brain-specific folate transport defect that is caused by mutations in the folate receptor 1 (FOLR1) gene coding for folate receptor alpha (FRalpha). Three patients carrying FOLR1 mutations developed progressive movement disturbance, psychomotor decline, and epilepsy and showed severely reduced folate concentrations in the cerebrospinal fluid (CSF). Brain magnetic resonance imaging (MRI) demonstrated profound hypomyelination, and MR-based in vivo metabolite analysis indicated a combined depletion of white-matter choline and inositol. Retroviral transfection of patient cells with either FRalpha or FRbeta could rescue folate binding. Furthermore, CSF folate concentrations, as well as glial choline and inositol depletion, were restored by folinic acid therapy and preceded clinical improvements. Our studies not only characterize a previously unknown and treatable disorder of early childhood, but also provide new insights into the folate metabolic pathways involved in postnatal myelination and brain development.

Control of prostate cancer associated with withdrawal of a supplement containing folic acid, L-methyltetrahydrofolate and vitamin B12: a case report.
            (Tisman and Garcia, 2011) Download
INTRODUCTION: This is the first report of possible direct stimulation of hormone-resistant prostate cancer or interference of docetaxel cytotoxicity of prostate cancer in a patient with biochemical relapse of prostatic-specific antigen. This observation is of clinical and metabolic importance, especially at a time when more than 80 countries have fortified food supplies with folic acid and some contemplate further fortification with vitamin B12. CASE PRESENTATION: Our patient is a 71-year-old Caucasian man who had been diagnosed in 1997 with prostate cancer, stage T1c, and Gleason score 3+4 = 7. His primary treatment included intermittent androgen deprivation therapy including leuprolide + bicalutamide + deutasteride, ketoconazole + hydrocortisone, nilandrone and flutamide to resistance defined as biochemical relapse of PSA. While undergoing docetaxel therapy to treat a continually increasing prostate-specific antigen level, withdrawal of 10 daily doses of a supplement containing 500 mug of vitamin B12 as cyanocobalamin, as well as 400 mug of folic acid as pteroylglutamic acid and 400 mug of L-5-methyltetrahydrofolate for a combined total of 800 mug of mixed folates, was associated with a return to a normal serum prostatic-specific antigen level. CONCLUSION: This case report illustrates the importance of the effects of supplements containing large amounts of folic acid, L-5-methyltetrahydrofolate, and cyanocobalamin on the metabolism of prostate cancer cells directly and/or B vitamin interference with docetaxel efficacy. Physicians caring for patients with prostate cancer undergoing watchful waiting, hormone therapy, and/or chemotherapy should consider the possible acceleration of tumor growth and/or metastasis and the development of drug resistance associated with supplement ingestion. We describe several pathways of metabolic and epigenetic interactions that could affect the observed changes in serum levels of prostate-specific antigen.

Addition of bisulfite to folate and dihydrofolate.
            (Vonderschmitt et al., 1967) Download
At pH 6.5, bisulfite reacts with folate and dihydrofolate to form nucleophilic adducts whose absorption spectra are similar to the spectra of dihydrofolate and tetra-hydrofolate, respectively. Because of the unfavorable equilibrium, a large excess of bisulfite is necessary to achieve an appreciable amount of adduct formation. The lability toward oxygen prevents isolation of the dihydrofolate-bisulfite complex, but a 1:1 adduct of folate and bisulfite can be precipitated from solution and obtained in solid form. Equilibrium constants are calculated for the formation of the bisulfite adducts of the oxidized and dihydro forms of folate, 2-amino-4-hydroxypteridine, and 2-amino-4-hydroxy-6-methylpteridine. In each case, adduct formation occurs more readily with the dihydropteridine than with the oxidized form. These results are are discussed in relation to reactions catalyzed by dihydrofolate reductase.

Update and new concepts in vitamin responsive disorders of folate transport and metabolism.
            (Watkins and Rosenblatt, 2012) Download
Derivatives of folic acid are involved in transfer of one-carbon units in cellular metabolism, playing a role in synthesis of purines and thymidylate and in the remethylation of homocysteine to form methionine. Five inborn errors affecting folate transport and metabolism have been well studied: hereditary folate malabsorption, caused by mutations in the gene encoding the proton-coupled folate transporter (SLC46A1); glutamate formiminotransferase deficiency, caused by mutations in the FTCD gene; methylenetetrahydrofolate reductase deficiency, caused by mutations in the MTHFR gene; and functional methionine synthase deficiency, either as the result of mutations affecting methionine synthase itself (cblG, caused by mutations in the MTR gene) or affecting the accessory protein methionine synthase reductase (cblE, caused by mutations in the MTRR gene). Recently additional inborn errors have been identified. Cerebral folate deficiency is a clinically heterogeneous disorder, which in a few families is caused by mutations in the FOLR1 gene. Dihydrofolate reductase deficiency is characterized by megaloblastic anemia and cerebral folate deficiency, with variable neurological findings. It is caused by mutations in the DHFR gene. Deficiency in the trifunctional enzyme containing methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase activities, has been identified in a single patient with megaloblastic anemia, atypical hemolytic uremic syndrome and severe combined immune deficiency. It is caused by mutations in the MTHFD1 gene.

Pharmacokinetic study on the utilisation of 5-methyltetrahydrofolate and folic acid in patients with coronary artery disease.
            (Willems et al., 2004) Download
1. Methylenetetrahydrofolate reductase (MTHFR) is a regulating enzyme in folate-dependant homocysteine remethylation, because it catalyses the reduction of 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate (5-MTHF). 2. Subjects homozygous for the 677C --> T mutation in the MTHFR enzyme suffer from an increased cardiovascular risk. It can be speculated that the direct administration of 5-MTHF instead of folic acid can facilitate the remethylation of homocysteine in methionine. 3. The aim of this study was to determine the pharmacokinetic properties of orally administered 6[R,S] 5-MTHF versus folic acid in cardiovascular patients with homozygosity for 677C --> T MTHFR. 4. This is an open-controlled, two-way, two-period randomised crossover study. Patients received a single oral dose of either 5 mg folic acid or 5 mg 5-MTHF in each period. The concentrations of the 6[S] 5-MTHF and 6[R] 5-MTHF diastereoisomers were determined in venous blood samples. 5. All pharmacokinetic parameters demonstrate that the bioavailability of 5-MTHF is higher compared to folic acid. The peak concentration of both isomers following the administration of 6[R,S] 5-MTHF is almost seven times higher compared to folic acid, irrespective of the patient's genotype. However, at 1 week after the administration of a single dosage 6[R,S] 5-MTHF, we detected 6[R] 5-MTHF following the administration of folic acid, indicating storage of this isomer in the body. 6. Our results demonstrate that oral 5-MTHF has a different pharmacokinetic profile with a higher bioavailability compared to folic acid, irrespective of the patient's genotype. Detrimental effects of the storage of high levels of the non-natural isomer 6[R] 5-MTHF cannot be excluded.

 


References

Chen, H, et al. (2016), ‘Folic Acid Supplementation Mitigates Alzheimer’s Disease by Reducing Inflammation: A Randomized Controlled Trial.’, Mediators Inflamm, 2016 5912146. PubMed: 27340344
Dickinson, CJ (1995), ‘No reliable evidence that folate is harmful in B-12 deficiency.’, BMJ, 311 (7010), 949. PubMed: 7580573
Enquobahrie, DA, et al. (2012), ‘Serum homocysteine and folate concentrations among a US cohort of adolescents before and after folic acid fortification.’, Public Health Nutr, 15 (10), 1818-26. PubMed: 22974678
McNulty, H, et al. (2012), ‘Nutrition throughout life: folate.’, Int J Vitam Nutr Res, 82 (5), 348-54. PubMed: 23798054
Penailillo, R, et al. (2015), ‘Folic acid supplementation during pregnancy induces sex-specific changes in methylation and expression of placental 11beta-hydroxysteroid dehydrogenase 2 in rats.’, PLoS One, 10 (3), e0121098. PubMed: 25793274
Pfeiffer, CM, et al. (2015), ‘Unmetabolized folic acid is detected in nearly all serum samples from US children, adolescents, and adults.’, J Nutr, 145 (3), 520-31. PubMed: 25733468
Roberts, PD, et al. (1971), ‘Apparent folate deficiency in iron-deficiency anaemia.’, Br J Haematol, 20 (2), 165-76. PubMed: 5548484
Scaglione, F and G Panzavolta (2014), ‘Folate, folic acid and 5-methyltetrahydrofolate are not the same thing.’, Xenobiotica, 44 (5), 480-88. PubMed: 24494987
Scott, JM and DG Weir (1981), ‘The methyl folate trap. A physiological response in man to prevent methyl group deficiency in kwashiorkor (methionine deficiency) and an explanation for folic-acid induced exacerbation of subacute combined degeneration in pernicious anaemia.’, Lancet, 2 (8242), 337-40. PubMed: 6115113
Sobczyńska-Malefora, A, et al. (2014), ‘Plasma and red cell reference intervals of 5-methyltetrahydrofolate of healthy adults in whom biochemical functional deficiencies of folate and vitamin B 12 had been excluded.’, Adv Hematol, 2014 465623. PubMed: 24527038
Steinfeld, R., et al. (2009), ‘Folate receptor alpha defect causes cerebral folate transport deficiency: a treatable neurodegenerative disorder associated with disturbed myelin metabolism’, Am J Hum Genet, 85 (3), 354-63. PubMed: 19732866
Tisman, G and A Garcia (2011), ‘Control of prostate cancer associated with withdrawal of a supplement containing folic acid, L-methyltetrahydrofolate and vitamin B12: a case report.’, J Med Case Rep, 5 413. PubMed: 21867542
Vonderschmitt, DJ, et al. (1967), ‘Addition of bisulfite to folate and dihydrofolate.’, Arch Biochem Biophys, 122 (2), 488-93. PubMed: 5235082
Watkins, D and DS Rosenblatt (2012), ‘Update and new concepts in vitamin responsive disorders of folate transport and metabolism.’, J Inherit Metab Dis, 35 (4), 665-70. PubMed: 22108709
Willems, FF, et al. (2004), ‘Pharmacokinetic study on the utilisation of 5-methyltetrahydrofolate and folic acid in patients with coronary artery disease.’, Br J Pharmacol, 141 (5), 825-30. PubMed: 14769778