Folates and Folic Acid: From Fundamental Research Toward Sustainable Health

Folates are of paramount importance in one-carbon metabolism of most organisms. Plants and microorganisms are able to synthesize folates de novo, making them the main dietary source for humans and animals, which are dependent on food or feed supplies for folates. Folate deficiency is an increasing problem in the developing, as well as in the developed regions of the world, affecting millions of people. Different strategies, such as food fortification and folic acid supplementation, remain far from accessible for the poor rural populations in developing countries. Increasing knowledge concerning folate biosynthesis, transport and catabolism does not only deepen our insight on the regulation of folate metabolism but also provides the keys towards folate enhancement through metabolic engineering in bacteria, as well as in plants. Recently, promising results were obtained using such an approach, but further fundamental research is a prerequisite to develop a practicable solution to fight folate deficiency. In parallel, progress in the development and improvement of folate analysis has been made. Here, we provide the state-of-the-art of folate biosynthesis, catabolism, and salvage. Finally, we report on progress in folate biofortification and discuss the agroeconomical aspect of biofortified crop plants.     

Formation of folates by microorganisms: towards the biotechnological production of this vitamin

Folates (vitamin B₉) are essential micronutrients which function as cofactors in one-carbon transfer reactions involved in the synthesis of nucleotides and amino acids. Folate deficiency is associated with important diseases such as cancer, anemia, cardiovascular diseases, or neural tube defects. Epidemiological data show that folate deficiency is still highly prevalent in many populations. Hence, food fortification with synthetic folic acid (i.e., folic acid supplementation) has become mandatory in many developed countries. However, folate biofortification of staple crops and dairy products as well as folate bioproduction using metabolically engineered microorganisms are promising alternatives to folic acid supplementation. Here, we review the current strategies aimed at overproducing folates in microorganisms, in view to implement an economic feasible process for the biotechnological production of the vitamin.

     

Australian survey on community knowledge and attitudes regarding the fortification of food with folic acid

BACKGROUND: In response to the proposal that mandatory fortification be introduced in Australia to reduce the incidence of NTDs, the purpose of this study was to establish, for the adult community, baseline data on knowledge, attitudes, and behaviors towards the fortification of foods with folic acid. METHODS: A cross-sectional, Computer Assisted Telephone Interviewing survey with respondents recruited randomly from the electronic version of the Western Australian telephone directory was used. One thousand members of the community aged 18 years or older were interviewed for the study. RESULTS: Interviews were conducted in September 2006, with a response rate of 76%. Half the respondents were aware folate is currently added to some breakfast cereals (59%) and some breads (53%), but awareness of other food types that may be voluntarily fortified with folate was much lower. Only 13% of respondents were concerned about folate being added to bread, 10% believed folate should not be added to foods, and 9% said they would avoid foods with added folate. Generally, around half the respondents were uncertain about the benefits of fortifying foods with folate and around a quarter were uncertain about any risks or concerns with this practice. CONCLUSIONS: The community appears no more concerned about the fortification of bread with folate than they are about other existing fortification programs in Australia, and appear more likely to support rather than oppose the fortification of foods with folate, particularly if they are informed of the benefits of such a program.     

Folic acid fortification: why not vitamin B12 also?

Folic acid fortification of cereal grains was introduced in many countries to prevent neural tube defect occurrence. The metabolism of folic acid and vitamin B12 intersect during the transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine catalyzed by B12-dependent methioine synthase. Regeneration of tetrahydrofolate via this reaction makes it available for synthesis of nucleotide precursors. Thus either folate or vitamin B12 deficiency can result in impaired cell division and anemia. Exposure to extra folic acid through fortification may be detrimental to those with vitamin B12 deficiency. Among participants of National Health And Nutrition Examination Survey with low vitamin B12 status, high serum folate (>59 nmol/L) was associated with higher prevalence of anemia and cognitive impairment when compared with normal serum folate. We also observed an increase in the plasma concentrations of total homocysteine and methylmalonic acid (MMA), two functional indicators of vitamin B12 status, with increase in plasma folate under low vitamin B12 status. These data strongly imply that high plasma folate is associated with the exacerbation of both the biochemical and clinical status of vitamin B12 deficiency. Hence any food fortification policy that includes folic acid should also include vitamin B12.     

Case for mandatory fortification of food with folate in Australia, for the prevention of neural tube defects

BACKGROUND: Since the publication of randomized controlled trials demonstrating the prevention of neural tube defects (NTDs) with periconceptional folate, several Australian states have promoted an increase in periconceptional use of folic acid supplements. Since 1996, voluntary fortification of food with folate has been allowed in Australia and New Zealand for the purpose of preventing NTDs. METHODS: For this report, we synthesized published and unpublished data on folic acid supplement use, voluntary fortification, and trends in NTDs. RESULTS: There has been an increase in the proportion of women (up to 30-40%) taking periconceptional folic acid supplements in Australia, and many foods (mainly breakfast cereals) are fortified. Supplement use is strongly correlated with educational and socioeconomic status; consumption of voluntarily fortified foods is not. There has been a fall in NTDs of about 30% in the non-Aboriginal population, but no change has been seen in the Aboriginal population. CONCLUSIONS: These data support mandatory fortification of food as a more equitable approach to achieving sufficient folate intake in the periconceptional period for all women in Australia and New Zealand to prevent the majority of NTDs in their offspring. In May 2004, based on these and other considerations, the Australia and New Zealand Food Regulation Ministerial Council agreed that mandatory fortification of food with folate should be considered as a priority.     

High rates of neural tube defects in Ukraine

BACKGROUND: Oral consumption of synthetic folic acid can prevent neural tube defects (NTDs), which are some of the most severe congenital anomalies. The prevalence of NTDs in Ukraine and other countries of the former U.S.S.R. has not been well studied. We determined the prevalence of NTD-affected pregnancies in Northwestern Ukraine as background for policy decisions related to flour fortification in this country. METHODS: The Ukrainian-American Birth Defects Program was established in 1999 and conducts population- based surveillance of birth defects in several oblasts (states) of Ukraine. We determined the prevalence of NTDs in the Volyn and Rivne oblasts of Northwestern Ukraine for three years, 2000-2002. RESULTS: There were 75,928 births in the two oblasts in 2000-2002. There were 159 cases of NTDs among live births, stillbirths, and induced abortions. The prevalence of NTDs in the two oblasts in Northwestern Ukraine is 2.1 per 1000 births. CONCLUSIONS: The prevalence of NTD-affected pregnancies we found in Northwestern Ukraine is almost four times what it should be. This prevalence suggests that population folate deficiency is widespread in Ukraine. Universal folic acid fortification of flour milled in Ukraine is urgently needed to end this epidemic of birth defects. Such fortification would be expected to prevent folate deficiency anemia, heart attacks, and strokes.     

Critical evaluation of strategies for mineral fortification of staple food crops

Staple food crops, in particular cereal grains, are poor sources of key mineral nutrients. As a result, the world’s poorest people, generally those subsisting on a monotonous cereal diet, are also those most vulnerable to mineral deficiency diseases. Various strategies have been proposed to deal with micronutrient deficiencies including the provision of mineral supplements, the fortification of processed food, the biofortification of crop plants at source with mineral-rich fertilizers and the implementation of breeding programs and genetic engineering approaches to generate mineral-rich varieties of staple crops. This review provides a critical comparison of the strategies that have been developed to address deficiencies in five key mineral nutrients—iodine, iron, zinc, calcium and selenium—and discusses the most recent advances in genetic engineering to increase mineral levels and bioavailability in our most important staple food crops.     

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Women's Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994–1995), peri- (1996–1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.     

A Novel Approach to Evaluating the Iron and Folate Status of Women of Reproductive Age in Uzbekistan after 3 Years of Flour Fortification with Micronutrients

Background

The Uzbekistan 1996 Demographic Health Survey reported 60.4% of women of reproductive age (WRA) had low hemoglobin concentrations (<120 g/L), and anemia was an important public health problem. Fortification of wheat flour was identified as an appropriate intervention to address anemia due to the ubiquitous consumption of wheat flour. A National Flour Fortification Program (NFFP) was implemented in 2005.

Methodology/Principal Findings

After 3-years of the NFFP, a national survey using large country-lot quality assurance sampling was carried out to assess iron, folate, hemoglobin and inflammation status of WRA; the coverage and knowledge of the fortified first grade UzDonMakhsulot (UDM) flour/grey loaf program; and consumption habits of women to investigate the dietary factors associated with anemia. Estimated anemia prevalence was 34.4% (95% CI: 32.0, 36.7), iron depletion 47.5% (95% CI: 45.1, 49.9) and folate deficiency 28.8% (95% CI: 26.8, 30.8); the effect of inflammation was minimal (4% with CRP >5 mg/L). Severe anemia was more prevalent among folate deficient than iron depleted WRA. Presence of UDM first grade flour or the grey loaf was reported in 71.3% of households. Among WRA, 32.1% were aware of UDM fortification; only 3.7% mentioned the benefits of fortification and 12.5% understood causes of anemia. Consumption of heme iron-containing food (91%) and iron absorption enhancers (97%) was high, as was the consumption of iron absorption inhibitors (95%).

Conclusions/Significance

The NFFP coincided with a substantial decline in the prevalence of anemia. Folate deficiency was a stronger predictor of severe anemia than iron depletion. However, the prevalence of iron depletion was high, suggesting that women are not eating enough iron or iron absorption is inhibited. Fortified products were prevalent throughout Uzbekistan, though UDM flour must be adequately fortified and monitored in the future. Knowledge of fortification and anemia was low, suggesting consumer education should be prioritized.     

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.     

Folate status and neural tube defects

Periconceptional folic acid supplementation prevents approximately 70% of neural tube defects (NTDs). While most women carrying affected fetuses do not have deficient blood folate levels, the risk of having an NTD affected child is inversely correlated with pregnancy red cell folate levels. Current research is focused on the discovery of genetic abnormalities in folate related enzymes which might explain the role of folate in NTD prevention. The first candidate gene to emerge was the C677T variant of 5,10-methylenetetrahydrofolate reductase. Normal subjects who are homozygous for the mutation (TT) have red cell folate status some 20% lower than expected. It is now established that the prevalence of the TT genotype is significantly higher among spina bifida cases and their parents. Nevertheless, our studies show that the variant does not account for the reduced blood folate levels in many NTD affected mothers. We conclude that low maternal folate status may in itself be the most important risk factor for NTDs and that food fortification may be the only population strategy of benefit in the effort to eliminate NTDs.     

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Women's Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994–1995), peri- (1996–1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.     

Intake and Biomarkers of Folate and Risk of Cancer Morbidity in Older Adults,NHANES 1999-2002 with Medicare Linkage

Background

After the 1998 mandatory folic acid fortification of enriched cereal-grain products in the U.S., safety concerns were raised that excess consumption of folic acid and high blood folate biomarkers detected in adults may increase the risk of certain types of cancer.

Methods

Baseline data from about 1400 participants in the National Health and Nutrition Examination Survey (NHANES) 1999–2002, aged ≥ 57 years were linked to Medicare and mortality files through December 31, 2007. Using cox proportional hazards regression models, we assessed associations between dietary folate equivalents, folate biomarkers, the presence of unmetabolized folic acid and, overall cancer incidence.

Results

With 8,114 person-years of follow-up (median follow-up, 6.3 years), about 125 cancer cases were identified. After adjusting for confounders, the hazard ratios of the highest quartile versus the second quartile of RBC folate and dietary folate equivalents were 0.54 (95% CI: 0.31–0.93) and 0.54 (95% CI: 0.30–0.95), respectively. Additionally, serum and RBC folate as continuous variables were inversely and significantly associated with cancer incidence (p<0.01). No significant associations were observed between the presence of unmetabolized folic acid, intake of naturally-occurring food folate or folic acid separately, and cancer incidence.

Conclusions

High total folate intake and biomarkers in older adults appear to be protective against cancer in post-folic acid fortification years. This study does not show a negative impact of current level of folic acid fortification on cancer risk. As this is one of the few studies to examine the association between unmetabolized folic acid and cancer outcome, a study including a larger nationwide representative sample of the U.S. population is needed.     

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。     

Effect of Folate Deficiency on Local Cerebral Glucose Utilization in the Rat

There is considerable debate on the role of folate in CNS function. Recent work indicates that folate deficiency may affect CNS serotonin metabolism, and clinical studies describe many consequences of such a deficiency. On the other hand some workers maintain that folate deficiency alone causes CNS abnormalities. We maintained rats, through dietary deprivation, at folate levels below 4 ng/ml for more than 6 weeks and showed that at that time both their liver and brain folate levels were significantly reduced. We then studied their local cerebral glucose utilization (LCGU) using the [14C]deoxyglucose technique. This method assesses cerebral function by measuring regional metabolic activity. We also determined LCGU in rats given the same diet but replenished with folate (folate control) and in others given free access to commercially available food (normal controls). Our results show that this degree of folate deficiency has no effect on cerebral function. This contrasts with the focal suppression of LCGU we previously reported in a model of vitamin B12 deficiency.     

Impact of folic acid food fortification on the birth prevalence of lipomyelomeningocele in Canada

BACKGROUND: Recent studies reported no reduction in the frequency of lipomeningomyelocele (LMMC) in Hawaii and Nova Scotia after the implementation of a folic acid food fortification policy in 1998, while a marked reduction in the prevalence of other NTDs was observed. This study was performed to assess the prevalence of LMMC in Canada in relation to the timing of food fortification. METHODS: The study population included livebirths, stillbirths, and terminations of pregnancies because of fetal anomaly to women residing in seven Canadian provinces, from 1993 to 2002. In each province, the ascertainment of NTD cases relied on multiple sources, and in addition all medical charts were reviewed. The study period was divided into pre‐, partial, and full fortification periods, based on results of red cell folate tests published in the literature. RESULTS: A total of 86 LMMC cases were recorded among approximately 1.9 million live births. The average birth prevalence rate was 0.05/1,000, ranging from a minimum of 0.01/1,000 in 2002 to a maximum of 0.08/1,000 in 1999. There was statistical heterogeneity between years (p = .01), but no pattern compatible with a decrease following fortification. Comparing the full fortification period with the prefortification period, there was a slight but not statistically significant decrease in LMMC birth prevalence. CONCLUSIONS: LMMC seems to be pathogenically distinct from myelomeningocele and more studies are needed to understand the embryologic mechanisms leading to this condition, and the environmental and genetic factors involved in its etiology. Birth Defects Research (Part A), 2008. © 2007 Wiley‐Liss, Inc.     

Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.)

Folate (vitamin B9) deficiency causes several health problems globally. However, folate biofortification of major staple crops is one alternative that can be used to improve vitamin intakes in populations at risk. We increased the folate levels in common bean by engineering the pteridine branch required for their biosynthesis. GTP cyclohydrolase I from Arabidopsis (AtGchI) was stably introduced into three common bean Pinto cultivars by particle bombardment. Seed‐specific overexpression of AtGCHI caused significant increases of up to 150‐fold in biosynthetic pteridines in the transformed lines. The pteridine boost enhanced folate levels in raw desiccated seeds by up to threefold (325 μg in a 100 g portion), which would represent 81% of the adult recommended daily allowance. Unexpectedly, the engineering also triggered a general increase in PABA levels, the other folate precursor. This was not observed in previous engineering studies and was probably caused by a feedforward mechanism that remains to be elucidated. Results from this work also show that common bean grains accumulate considerable amounts of oxidized pteridines that might represent products of folate degradation in desiccating seeds. Our study uncovers a probable different regulation of folate homoeostasis in these legume grains than that observed in other engineering works. Legumes are good sources of folates, and this work shows that they can be engineered to accumulate even greater amounts of folate that, when consumed, can improve folate status. Biofortification of common bean with folates and other micronutrients represents a promising strategy to improve the nutritional status of populations around the world.     

Iron Fortification of Banana by the Expression of Soybean Ferritin

Iron deficiency anemia is one of the serious ailments related to nutrition in the developing countries. Fruit and vegetable crops favor the bioavailability of iron. Banana is consumed as a staple food in the tropics. Iron-fortified bananas provide an effective means of controlling the iron deficiency. Embryogenic cells of banana cv. Rasthali (AAB) were transformed with soybean ferritin cDNA using two different expression cassettes pSF and pEFE-SF to express ferritin. Transgenic nature of the regenerated plants was confirmed by PCR. Transgenic plants were regenerated and analyzed through PCR and PCR-Southern analysis. The expression of ferritin was confirmed by RT-PCR. Iron and zinc levels in the transgenic and control plants were estimated by atomic absorption spectroscopy. A 6.32-fold increase in iron accumulation and a 4.58-fold increase in the zinc levels were noted in the leaves of transgenic plants. Thus, iron- and zinc-fortified bananas could be developed as a functional food to overcome the malnutrition-related iron deficiency. This is the first report on the iron and zinc fortification of banana.     

Low fruit consumption and folate deficiency are associated with LINE-1 hypomethylation in women of a cancer-free population

Several dietary agents, such as micronutrient and non-nutrient components, the so-called bioactive food components, have been shown to display anticancer properties and influence genetic processes. The most common epigenetic change is DNA methylation. Hypomethylation of long interspersed elements (LINE-1) has been associated with an increased risk of several cancers, although conflicting findings have also been observed. The aim of the present study was to test the hypothesis that a low adherence to the Mediterranean diet (MD) and folate deficiency may cause LINE-1 hypomethylation in blood leukocytes of healthy women, and thus genomic instability. One hundred and seventy-seven non-pregnant women were enrolled. Mediterranean diet score (MDS) and folate intake were calculated using a food frequency questionnaire. LINE-1 methylation level was measured by pyrosequencing analysis in three CpG sites of LINE-1 promoter. According to MDS, only 9.6 % of subjects achieved a high adherence to MD. Taking into account the use of supplements, there was a high prevalence of folate deficiency (73.4 %). Women whose consumption of fruit was below the median value (i.e., <201 gr/day) were 3.7 times more likely to display LINE-1 hypomethylation than women whose consumption was above the median value (OR 3.7; 95 % CI 1.4–9.5). Similarly, women with folate deficiency were 3.6 times more likely to display LINE-1 hypomethylation than women with no folate deficiency (OR 3.6; 95 % CI 1.1–12.1). A dietary pattern characterized by low fruit consumption and folate deficiency is associated with LINE-1 hypomethylation and with cancer risk.