Folate bioavailability and health

Since its discovery in 1931 by Lucy Wills, and its first isolation in 1941 by Mitchell, Snell and Williams, our understanding of the fascinating world of folic acid and one-carbon metabolism, and its role in health and disease, has come a long way. However, there is still much to do in perfecting methods to measure folate bioavailability, and status, with a high degree of precision and accuracy. Future examination of the relationships of common gene polymorphims involved in folate bioavailability (folate polyglutamate deconjugation and carrier-mediated absorption) and one-carbon metabolism (methylation cycle, folate cycle and DNA synthesis/repair) to folate status, morbidity, mortality and longevity, need to be considered concurrently rather than as a series of individual associations, as has been the usual practice. This revised version was published online in June 2006 with corrections to the Cover Date.     

A mathematical model of the folate cycle: new insights into folate homeostasis

A mathematical model is developed for the folate cycle based on standard biochemical kinetics. We use the model to provide new insights into several different mechanisms of folate homeostasis. The model reproduces the known pool sizes of folate substrates and the fluxes through each of the loops of the folate cycle and has the qualitative behavior observed in a variety of experimental studies. Vitamin B(12) deficiency, modeled as a reduction in the V(max) of the methionine synthase reaction, results in a secondary folate deficiency via the accumulation of folate as 5-methyltetrahydrofolate (the "methyl trap"). One form of homeostasis is revealed by the fact that a 100-fold up-regulation of thymidylate synthase and dihydrofolate reductase (known to occur at the G(1)/S transition) dramatically increases pyrimidine production without affecting the other reactions of the folate cycle. The model also predicts that an almost total inhibition of dihydrofolate reductase is required to significantly inhibit the thymidylate synthase reaction, consistent with experimental and clinical studies on the effects of methotrexate. Sensitivity to variation in enzymatic parameters tends to be local in the cycle and inversely proportional to the number of reactions that interconvert two folate substrates. Another form of homeostasis is a consequence of the nonenzymatic binding of folate substrates to folate enzymes. Without folate binding, the velocities of the reactions decrease approximately linearly as total folate is decreased. In the presence of folate binding and allosteric inhibition, the velocities show a remarkable constancy as total folate is decreased.     

The effect of folic acid and/or methionine deficiency on deoxyribonucleotide pools and cell cycle distribution in mitogen-stimulated rat lymphocytes

Abstract. Folate deficiency will induce abnormal deoxynucleoside triphosphate (dNTP) metabolism because folate-derived one-carbon groups are essential for de novo synthesis of purines and the pyrimidine, thymidylate. Under conditions of methionine deprivation, a functional folate deficiency for deoxynucleoside triphosphate synthesis is induced as a result of the irreversible diversion of available folates toward endogenous methionine resynthesis from homocysteine. The purpose of the present study was to examine the effect of nutritional folate and/or methionine deprivation in vitro on intracellular dNTP pools as related to DNA synthesis activity and cell cycle progression. Primary cultures of mitogen-stimulated rat splenic T-cells were incubated in complete RPMI 1640 medium or in custom-prepared RPMI 1640 medium lacking in folic acid and/or methionine. Parallel cultures, initiated from the same cell suspension, were analysed for deoxyribonucleotide pool levels and for cell proliferation. The distribution of cells within the cell cycle was quantified by dual parameter flow cytometric bromodeoxyuridine/propidium iodide DNA analysis which allows more accurate definition of DNA synthesizing S-phase cells than the traditional DNA-specific staining with propidium iodide alone. Relative to cells cultured in complete RPMI 1640 media, the cells cultured in media deficient in folate, methionine or in both nutrients manifested increases in the deoxythymidylate pool and an apparent depletion of the deoxyguanosine triphosphate pool. Both adenosine triphosphate and nicotinamide adenine diphosphate levels were significantly reduced with single or combined deficiencies of folate and methionine. These nucleotide pool alterations were associated with a decrease in the proportion of cells actively synthesizing DNA and an increase in cells in G₂+ M phase of the cell cycle. Folate deprivation in the presence of adequate methionine produced a moderate decrease in DNA synthesizing cells over the 68 h incubation. However, methionine deprivation, in the presence or absence of folate, severely compromised DNA synthesis activity. These results are consistent with the established ‘methyl trap’ diversion of available folates towards the resynthesis of methionine from homocysteine and away from nucleotide synthesis. The data confirm the metabolic interdependence of folic acid and methionine and emphasize the pivotal role of methionine on the availability of folate one-carbon groups for deoxynucleotide synthesis. The decrease in DNA synthesis activity under nutrient conditions that negatively affect nucleotide biosynthesis suggest a possible role for abnormal dNTP metabolism in the regulation of cell cycle progression and DNA synthesis.     

The Impact of Dietary Folate Intake on Reproductive Function in Premenopausal Women: A Prospective Cohort Study

Background

Folic acid is recommended to reproductive-aged women to prevent birth defects, though little is known about the effects of dietary intake on other reproductive outcomes. Improved pregnancy rates have been documented after folic acid supplement use, suggesting a possible link with ovulation, however research is limited. Our objective was to evaluate the association between dietary folate intake, hormone levels, and sporadic anovulation in healthy, regularly menstruating women.

Methodology/Principal Findings

The BioCycle study (2005–2007) prospectively followed 259 healthy women aged 18–44 years from the western New York region for up to 2 menstrual cycles. Total folate and specific sources of folate were assessed up to 4 times per cycle by 24-hour recall. Estradiol, progesterone, luteinizing hormone, and follicle-stimulating hormone were measured in serum up to 8 times per cycle, timed using fertility monitors. Anovulation was defined as a cycle with peak progesterone concentration ≤5 ng/mL and no LH peak in the mid/late luteal phase. Higher intake of dietary folate (in dietary equivalents) across tertiles had a marginally significant association with greater luteal progesterone levels (P trend 0.08). Higher intake of synthetic folate was significantly associated with higher luteal progesterone levels (P trend 0.05). Specifically, women in the 3^rd tertile of synthetic folate intake had, on average, 16.0% (95% CI, 0.5–33.8%) higher luteal progesterone levels compared to women in the 1^st tertile. Moreover, consumption of synthetic folate was significantly and inversely associated with anovulation such that women in the 3^rd tertile had a 64% (95% CI, 8–86%) decreased odds of anovulation compared to the women in the 1^st tertile (P trend 0.03).

Conclusions/Significance

These findings suggest that a diet high in synthetic folate may be associated with increased progesterone levels and lower risk of sporadic anovulation. Further study of the effect of dietary folate and folic acid supplement use on reproductive health is warranted.     

Mutation at the folate receptor 4 locus modulates gene expression profiles in the mouse uterus in response to periconceptional folate supplementation

Periconceptional supplementation of folic acid to the diet of women is considered a great success for a public health intervention. Higher folate status, either by supplementation, or via the mandatory fortification of grain products in the United States, has led to significant reduction in the incidence of neural tube defects. Besides birth defects, folate deficiency has been linked to a variety of morbidities, most notably to increased risk for cancer. However, recent evidence suggests that excess folate may be detrimental — for birth defect incidence or in the progression of cancer. How folate mediates beneficial or detrimental effects is not well understood. It is also unknown what molecular responses are elicited in women taking folate supplements, and thus experience a bolus of folate on top of the status achieved by fortification. To characterize the response to a periconceptional regimen of supplementation with folinic acid, we performed gene expression profiling experiments on uterus tissue of pregnant mice with either wildtype alleles or targeted disruption at the folate receptor 4 locus. We observed that, depending on the genetic background, folinic acid supplementation affects expression of genes that contribute to lipid metabolism, protein synthesis, mitochondrial function, cell cycle, and cell activation. The extent of the response is strongly modulated by the genetic background. Finally, we provide evidence that folinic acid supplementation in the mutant paradigm affects histone methylation status, a potential mechanism of gene regulation in this model.     

Mechanism of the antimicrobial drug trimethoprim revisited.

We tested the hypothesis that the mechanism of action of the antifolate drug trimethoprim is through accumulation of bacterial dihydrofolate resulting in depletion of tetrahydrofolate coenzymes required for purine and pyrimidine biosynthesis. The folate pool of a strain of Escherichia coli (NCIMB 8879) was prelabeled with the folate biosynthetic precursor [(3)H]-p-aminobenzoic acid before treatment with trimethoprim. Folates in untreated E. coli were present as tetrahydrofolate coenzymes. In trimethoprim-treated cells, however, a rapid transient accumulation of dihydrofolate occurred, followed by complete conversion of all forms of folate to cleaved catabolites (pteridines and para-aminobenzoylglutamate) and the stable nonreduced form of the vitamin, folic acid. Both para-aminobenzoylglutamate and folic acid were present in the cell in the form of polyglutamates. Removal of trimethoprim resulted in the reconversion of the accumulated folic acid to tetrahydrofolate cofactors for subsequent participation in the one-carbon cycle. Whereas irreversible catabolism is probably bactericidal, conversion to folic acid may constitute a bacteriostatic mechanism since, as we show, folic acid can be used by the bacteria and proliferation is resumed once trimethoprim is removed. Thus, the clinical effectiveness of this important drug may depend on the extent to which the processes of either catabolism or folic acid production occur in different bacteria or during different therapeutic regimes.     

Experiments on the biological activities of various fractions of the folic acid antagonist "X-methyl" folic acid

A crude synthetic preparation called crude "X-methyl" folate has previously been shown to function as a folate antagonist for rats and chicks. This product has been shown to contain two folate antagonists: 9-methyl folate, present as 6% by weight of the product and which has low activity as a folate antagonist for Streptococcus faecalis, and pyrrofolic acid, a compound present in small amounts (0.04%), but having high anti-folate biological activity for S. faecalis. These experiments deal with the antifolate activity of these two fractions for the rat as measured by their effects on histidine oxidation. Rats were fed a purified diet based on 20% vitamin-free casein and containing 1.0% sulfasuxidine. When this diet was supplemented with a marginal amount of folic acid (0.3 mg per kg diet), the addition of 4 g of crude antagonist decreased histidine oxidation and decreased liver folate levels. The addition of 240 mg of pure 9-methyl folic acid (amount of 9-methyl folic acid in 4 g of crude) produced similar decreases in histidine oxidation and liver folate levels. A concentrate of pyrrofolic acid (equivalent to 4 g of crude) free of 9-methyl folic acid produced no decrease in histidine oxidation and minimal changes in liver folate. This indicates that the folate antagonist activity observed previously with animals is probably due to the 9-methyl folic acid component rather than to the pyrrofolic acid activity.     

Culture cycle dependence of folate interconversion and related enzymes in Euglena gracilis

Folates are involved in one-carbon metabolism in which one-carbon groups of increasing reduction state (formyl, methylene and methyl) are cyclically accepted and donated by the coenzyme form of folic acid, tetrahydrofolic acid. The latter originates by reduction of dihydrofolic acid, the coenzymatically inactive form. Euglena culture cycle dependence of folate distribution in oxidized, formyl and methyl forms and of enzyme activities for folate interconversion were studied. Distribution levels of all the components examined varied widely during the culture, and many of these changes occurred in the logarithmic phase of growth. In the phase of folate synthesis, there was an appreciable delay in the conversion of oxidized to reduced forms and of formyl to methyl forms. This delay appeared to be correlated with the level of corresponding enzymes. The methyl folate peak coincided with the highest level of total cell folates, at which point a severe repression of folate synthesis began. During the last phase of exponential growth, when cell folate content was reduced to one-fifth and folates had shorter glutamate chains, the level of coenzymatically inactive and inhibitory oxidized forms increased again. The reduced efficiency of the system and the change in growth rate are discussed. The activity patterns of dihydrofolate reductase and methylene tetrahydrofolate reductase were markedly different. The peak in methylene tetrahydrofolate reductase activity coincided with the absence of oxidized folates. A regulation of folate synthesis by the level of methyl folates and of methylene tetrahydrofolate reductase synthesis by the level of oxidized forms is proposed.     

Effects of cultivation conditions on folate production by lactic acid bacteria

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.     

Enzymology of the acetyl-CoA pathway of CO2 fixation

We know of three routes that organisms have evolved to synthesize complex organic molecules from CO2: the Calvin cycle, the reverse tricarboxylic acid cycle, and the reductive acetyl-CoA pathway. This review describes the enzymatic steps involved in the acetyl-CoA pathway, also called the Wood pathway, which is the major mechanism of CO2 fixation under anaerobic conditions. The acetyl-CoA pathway is also able to form acetyl-CoA from carbon monoxide. There are two parts to the acetyl-CoA pathway: (1) reduction of CO2 to methyltetrahydrofolate (methyl-H4folate) and (2) synthesis of acetyl-CoA from methyl-H4folate, a carboxyl donor such as CO or CO2, and CoA. This pathway is unique in that the major intermediates are enzyme-bound and are often organometallic complexes. Our current understanding of the pathway is based on radioactive and stable isotope tracer studies, purification of the component enzymes (some extremely oxygen sensitive), and identification of the enzyme-bound intermediates by chromatographic, spectroscopic, and electrochemical techniques. This review describes the remarkable series of enzymatic steps involved in acetyl-CoA formation by this pathway that is a key component of the global carbon cycle.     

Folate concentrations and folic acid supplementation among women in their first trimester of pregnancy in a rural area with a high prevalence of neural tube defects in Shanxi, China

BACKGROUND: Although an information campaign concerning periconceptional folic acid supplementation was launched in 1998 in Shanxi Province, China, the prevalence of neural tube defects in rural areas was reported as high as 140 per 10,000 births in 2002. The blood folate concentrations and the practice of folic acid supplementation among pregnant women in rural areas of the province are described. METHODS: A total of 483 pregnant women (mean gestation, 8.1 weeks) in a rural area of Shanxi were interviewed. Nonfasting blood samples and information on folic acid supplementation were collected. Folate concentrations in plasma and erythrocytes were determined by a microbiological assay. RESULTS: The mean concentrations of plasma and erythrocyte folate for pregnant women was 10.4 nmol/liter and 375.8 nmol/liter, respectively. Deficiencies of plasma and erythrocyte folate were observed in 20.9% and 47.6% of women, respectively. Seasonal variations were noted in the prevalence of folate deficiency, with significantly lower plasma folate concentrations in spring and summer and lower erythrocyte folate concentrations in seasons other than summer. Among pregnant women, <10% reported having taken or currently taking folic acid, and virtually no women (0.6%) took folic acid as recommended. CONCLUSIONS: Women in rural areas had low plasma and erythrocyte folate levels, and folate deficiency was highly prevalent in the area. Few women followed the recommendations regarding folic acid supplementation, and the information campaign in Shanxi was unsuccessful. These findings suggest the urgent need for combined strategies in rural areas to fortify grain with folic acid and promote folic acid supplements for childbearing-age women.     

Repression of folate synthesis in the logarithmic phase of Euglena gracilis growth

Microbiological assay showed that in Euglena gracilis cultures the amount of cell folates reaches its maximum at the beginning of the culture cycle and rapidly and markedly decreases long before the cells reduce their duplication rate. [³H]Folic acid was a suitable precursor of Euglena folates (a full recovery of growth in sulfanilamide inhibited cultures was obtained by addition of folic acid), and a complete radiochromatographic profile of cell folates was obtained by separation on G-25 columns. This allowed the measurement of the rate of folate degradation, obtained from the rate of radioactivity disappearance in chromatographic patterns of extracts corresponding to increasing times of culture cycles. With the exception of the stationary phase, the process of folate degradation showed a first order kinetics with a rate constant of 5.4 + 10⁻⁴ min⁻¹ and a half-life of 21 h and 12 min. The rate of folate biosynthesis was calculated by adding the amount of degradation to the measured increase (or decrease) in cell folates. The specific rate reached its maximum (8.6 ng of folinic acid equivalent h⁻¹ for 10⁶ cells) as the culture entered the logarithmic phase of growth and rapidly decreased to about 1/20 of this value before leaving it. This indicates that the logarithmic phase of growth corresponds to a phase in which folate biosynthesis is strongly repressed.     

乳酸菌合成叶酸的研究进展

叶酸普遍存在于各类食物中,但由于叶酸的不稳定性以及饮食习惯的差异性,各国叶酸缺乏现象普遍存在。叶酸是参与核酸合成及细胞分裂分化的重要物质,叶酸缺乏引起机体功能的混乱,由此引发癌症等一系列的疾病。大部分乳酸菌是叶酸缺陷型菌株,但越来越多的研究表明其很多种类都具有叶酸合成能力。本文主要概述产叶酸乳酸菌的分类,乳酸菌生物合成叶酸的机制,以及利用乳酸菌进行的叶酸基因工程研究进展。     

Determination of blood folate using acid extraction and internally standardized gas chromatography-mass spectrometry detection

Whole blood folate level is a superior indicator of folate nutritional status than serum/plasma level. Problems with and lack of confidence in results of current whole blood folate assays have limited its popularity for assessing folate nutritional status. Here, an acid extraction GCMS detection method that measures total folate whole blood is presented. Folates are released from the matrix of whole blood and cleaved to para-aminobenzoic acid (pABA) by acid hydrolysis in the presence of [(13)C(6)]pABA as internal standard (IS). The hydrolysate is passed over a C18 resin to remove heme. The pABA isotopomers are ethyl esterified, isolated on C18 resin, and trifluoroacetylated. Following normal-phase HPLC separation, the isotopomers are silylated to their tBDMS derivatives. The abundance of these derivatives are measured at m/z 324 for [(13)C(6)]pABA as IS and m/z 318 for pABA from whole blood folate. Our method uses readily available chemicals and our results agree well with those using Lactobacillus casei, the current gold standard reference assay. The presence of folate analogs (methotrexate) or antibacterials (sulfonamines) does not affect our method. This feature makes it useful in monitoring folate status of patients undergoing chemotherapy. Before using our method, pABA supplements must be discontinued for a few days.     

Reciprocal changes in the levels of functionally related folate enzymes during the culture cycle in human fibroblasts

The activities of 6 folate enzymes were measured in extracts of human diploid skin fibroblasts during the lag, log and stationary phases of the culture cycle. The levels of 4 folate enzymes involved in nucleic acid biosynthesis, $\text{viz.}$, folate reductase, serine hydroxymethyltransferase, thymidylate synthetase and 10-formyl-THF synthetase, increased from 2–20 fold during the log phase of growth. In contrast, the levels of 2 enzymes, $\text{viz.}$, methylene-THF reductase and 5-methyl-THF: homocysteine methyltransferase, involved in regulating the levels of 5-methyl-THF, the major tissue and serum folate compound, decreased 3–4 fold during log growth, returning to high levels again only after the cells had been in the stationary phase for 5 and 20 days respectively. This reciprocal pattern of change is consistent with the known or postulated functions of these folate enzymes.     

硒与叶酸在抗病毒治疗中的作用及其在植物中的应用前景分析

2020年,科学家在新型冠状病毒肺炎的治疗过程中发现,治疗效果与患者体内的硒和叶酸水平有一定相关性。这说明提高人体内硒和叶酸含量的膳食方案,可以有效地增加人体免疫能力来应对病毒性疾病的冲击。通过膳食保障人体硒和叶酸的摄入是植物营养强化的主要任务之一,但目前硒和叶酸的复合营养强化工作鲜见报道。综述了硒和叶酸这两种微量营养素在抗病毒治疗中的机制、以及硒和叶酸在植物中的应用前景,为提升营养品质、创制富含硒和叶酸的营养强化植物提供理论支撑。     

Effect of high levels of dietary folic acid on folate metabolism in vitamin B12 deficiency

The effect of administering high levels of folic acid to vitamin B12-deficient animals was studied. In B12 deficiency histidine oxidation is decreased. This is the result of both decreased liver folate levels and increases in the proportion of methyltetrahydrofolates. The purpose of this study was to determine if the addition of very high levels of folic acid to B12-deficient diets could increase liver folates and thereby restore histidine oxidation. Rats were fed a soy protein B12-deficient diet containing 10% pectin which has been shown previously to accelerate B12 depletion. When this diet was supplemented with B12 and folic acid, histidine oxidation was 5.4% in 2 h and the livers contained 3.49 micrograms of folate/g. In the absence of B12, the histidine oxidation rate was 0.34% and the liver folate level was 1.33 micrograms/g. When 200 mg/kg of folic acid was added to the B12-deficient diet there was no increase in histidine oxidation (0.35%) but the liver folates were increased to 3.68 micrograms which is about the same as that with B12 supplementation. The percentage tetrahydrofolate of the total liver folates was the same with and without a high level of dietary folic acid. Thus there was an increase in the absolute level of tetrahydrofolate without any increase in folate function as measured by histidine oxidation. Red cell folate levels were the same with and without B12, which is in contrast to the markedly lower liver folate levels in B12 deficiency. These data suggest a difference between B12 regulation of folate metabolism in the liver and in the bone marrow.     

Differential binding of folates by rat renal cortex brush border and basolateral membrane preparations

The binding of radioactive 5-methyltetrahydrofolate and folic acid was found to be greater in brush border than in basolateral membrane preparations of rat renal cortex. This appeared to be due to an increased amount of a specific folate binding protein in the brush border membrane preparations as compared to those of the basolateral membrane. The binding was saturable and inhibited by nonradioactive folic acid and, therefore, a specific, rather than nonspecific process. The Km's for folic acid binding in brush border and basolateral membrane preparations were similar and involved a single high-affinity binding site. In contrast, methotrexate was found to bind equally well to both brush border and basolateral membrane preparations. Moreover, folic acid binding was not inhibited by an equimolar amount of methotrexate. A folate binding protein could be extracted from either membrane preparation with 1% Triton X-100 and, to a lesser extent, with 0.6 M NaCl. These different extraction procedures resulted in different apparent molecular weights for folate binding protein (greater than 160,000 for Triton X-100-extracted samples and 40,000 for NaCl-extracted samples). The membrane preparation pellets remaining after NaCl extraction were able to rebind tritiated folic acid and also the 40,000-Da folate binding protein. On the other hand, membrane preparations extracted with Triton X-100 lost the ability to bind folic acid or the 40,000-Da folate binding protein. These differences in molecular weight and rebinding capacity may be explained by the existence of a receptor for folate binding protein which was extracted by Triton X-100, but not by NaCl. The greater concentration of folate binding protein in the renal tubule cell brush border membrane preparations as compared to those from basolateral membranes ascribes, for the first time, a functional role for folate binding protein in the renal reabsorption of folates which is required to prevent loss of folate in the urine and perhaps in the membrane transport of folates in general.