Mark Lucock

Is folic acid the ultimate functional food component for disease prevention

We are entering a new era in preventive medicine, which focuses on diet as a means to health. Folate has received much attention as a vitamin that can protect against many diseases, but do we know enough about the long term effects of supplementation?

Folic acid fortification: a double-edged sword.

Purpose of reviewTo examine the impact of folic acid fortification, including its use as a functional food component, on human health. Recent findingsThere is a consensus view that folic acid supplementation has numerous health benefits, many of which are significant in their impact. However, emerging evidence suggests that increased population exposure to folic acid may also have a negative impact with respect to certain developmental and degenerative disorders. As examples, presently much attention is focused on the role of folic acid fortification augmenting colon cancer risk, whereas earlier in the life cycle, the vitamin may additionally influence insulin resistance. Without question, conditions that are influenced by folic acid are both diverse and many – from concerns relating to cognitive decline, breast cancer and vascular disease through to preconceptional issues where maternal folate levels might conceivably alter the phenotype of offspring via epimutations. SummaryThe highly complex and critical biological importance of folic acid-related molecular nutrition makes it a difficult micronutrient to deploy as a simple intervention at a population level – it has far too many biochemical spheres of influence to predict effects in a generalized way. Additionally, several gene variants and other nutrients are interactive factors. It is, therefore, hardly surprising that the scientific community does not have a true consensus view on whether mandatory fortification is appropriate as a population measure. This latter point not withstanding, any ultimate decisions on fortification should be well rooted in scientific fact rather than political expediency.

The responsiveness of plasma homocysteine to small increases in dietary folic acid : a primary care study

Objectives: To assess the long term effects of small increases in dietary folic acid on the concentration of plasma homocysteine, an independent risk factor for occlusive vascular disease, in a general population.Design: A randomized double-blind placebo-controlled intervention study.Subjects: One hundred and nineteen healthy volunteers, whose intake of fortified or supplemental folic acid was low, were recruited by letter from the patient register of a large inner-city group general practice.Methods: Volunteers were randomized to receive unfortified cereals, or cereals fortified with 200 μg of folic acid per portion, with or without other vitamins. Blood samples were taken presupplement and at 4, 8 and 24 weeks on treatment and analysed for plasma homocysteine, cysteine and vitamin B12 and serum and red cell folate. Ninety-four subjects completed the study providing blood samples on all four occasions.Results: There were no significant changes in any measured parameter in those eating unfortified cereals. Overall, folic acid fortification of cereals led to significant increases (P<0.001) in serum folate (66%), and red cell folate (24%), and a decrease in plasma homocysteine (10%; P<0.001). There were no changes in vitamin B12 or cysteine. The homocysteine decrease persisted until the end of the study and was primarily seen in those who initially had the highest plasma homocysteine or the lowest serum folate.Conclusions: If homocysteine is found to be a causative risk factor in occlusive vascular disease, food fortification with physiological levels of folic acid should have a significant impact on the prevalence of the disease in the general population.Sponsorship: We acknowledge, with thanks, financial support from the Kellogg Company of Great Britain.

The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: A possible molecular basis for the site-specific cancer risk modification

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer although it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one‐carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one‐carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA‐MB‐435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA‐MB‐435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA‐MB‐435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site‐specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.

Preliminary evidence for involvement of the folate gene polymorphism 19bp deletion-DHFR in occurrence of autism.

Folate has long been implicated in both the metabolism of neurotransmitter molecules, and as an agonist with a direct effect upon neuronal tissue. Folates mediate transfer of one-carbon units into major biosynthetic pathways. From a developmental perspective, the most important reactions are de novo methionine and thymine synthesis, critical for DNA expression and elaboration, respectively. Dihydrofolate reductase (DHFR) is the sole enzyme responsible for maintaining the reduced state of the vitamin needed for these two pathways. Here, we report that the 19bp-deletion polymorphism of DHFR acts independently (OR 2.69, 95% CI; 1.00-7.28, p<0.05) and in concert with related folate polymorphisms as a significant risk factor for autism. Possible consequences of this are discussed in the context of the interaction between folate and the glutamatergic nervous system, an area of promising candidate genes for contributing to autism.

Determination of Plasma Total Homocysteine and Cysteine Using HPLC with Fluorescence Detection and an Ammonium 7‐fluoro‐2,1,3‐benzoxadiazole‐4‐sulphonate (SBD‐F) Derivatization Protocol Optimized for Antioxidant Concentration, Derivatization Reagent Concentration, Temperature and Matrix pH

A sensitive HPLC-fluorescence method for determining total endogenous plasma homocysteine (Hcy), cysteine (Cys) and cysteinylglycine (Cys-Gly) following derivatization with ammonium 7-fluoro 2,1,3-benzoxadiazole-4-sulphonate (SBD-F) is described. Quantitation utilizes an internal standard, 2-mercaptoethylamine. The derivatization procedure has been optimized for concentration of SBD-F, reducing agent (tributylphosphine) and temperature. Findings indicate that values for plasma determinations vary according to the nature of the matrix in which calibration standards are made up. If quantitation is based on a peak height ratio, then standards should be made up in either pH 7.4 phosphate buffered saline or plasma taking into account the endogenous thiol concentration. These findings are based on calibration data, and 30 plasma samples quantified using thiol standards made up in plasma, pH 7.4 and pH 9.5 buffers. By defining how this matrix/pH effect influences thiol quantitation, it should be possible to make a more meaningful comparison of Hcy measurements between laboratories. The chromatographic separation was investigated at several mobile-phase pH values with the following conditions ascertained to be optimal: a mobile phase consisting of 5% (v/v) acetonitrile in 0.1 M KH2PO4, pH 2.15 was run at a flow rate of 0.5 mL/min. It was used in conjunction with a Supelco LC-18 base deactivated analytical column (150 x 4.6 cm i.d. 3 microM bonded silica). The internal standard and thiols were measured by fluorescence detection at 385 nm excitation and 515 nm emmission. Plasma levels are easily measured in a 100 microL volume. Storage for 2 months at -20 degrees C resulted in no deterioration of thiols. Furthermore, no difference in thiol levels was observed between bloods collected in lithium heparin and EDTA. Collected blood should, however, be separated as soon as possible to avoid red cell metabolism of Hcy which was observed in a case of hyperhomocysteinemia. Once derivatized, thiols are stable for at least one week at +4 degrees C.

In vivo characterization of the absorption and biotransformation of pteroylmonoglutamic acid in man: A model for future studies

HPLC-EC has been used to measure the appearance of 5-CH3-H4 folic acid in human plasma following oral administration of folic acid. The process was found to be saturable in accordance with Michaelis-Menten kinetics. The apparent Km for this enzyme system indicates that low doses of oral folic acid are rapidly converted into 5-CH3-H4 folic acid, an observation consistent with the needs of intestinal absorption of essential trace nutrients. The appearance of L. casei active folate in plasma was not rate-limited and showed a biphasic relationship to dose. Preparative HPLC combined with L. casei bioassay demonstrated that most of the L. casei active folate appearing in plasma following a 20,000-micrograms dose of folic acid was due to the unmodified vitamin, only 5.6% being due to 5-CH3-H4 folic acid and with no detectable contribution from 5-CHO-H4 folic acid. The absorption characteristics of the system seem consistent between and within subject(s). No relationship could be demonstrated between predose levels of plasma 5-CH3-H4 folic acid and total folate in erythrocytes, which reflect the status of transport and storage forms of the vitamin, respectively.

A critical role for B-vitamin nutrition in human developmental and evolutionary biology

Several molecular mechanisms that underpin genomic integrity and function are sensitive to B-vitamin status, and in particular, are responsive to the interaction between folate nutrition and folate dependent enzyme polymorphisms. Mechanisms that may be affected include maintenance of genomic CpG methylation patterns for regulated gene expression, and proficient synthesis of nucleotides to prevent DNA strand breakage. We review some of these important molecular mechanisms that underpin the role of folate and other B-vitamins in disease processes, and discuss how they explain why this vitamin may be an important factor in human evolution. We look at three particular phenomena that link folate status/genes to evolutionary pressures: 1) The evolution of pigmentation to avert the impact of UV light on labile folates needed for developmental processes. 2) The effect of C677T-MTHFR on foetal survival, and 3) the ability of folate to mask harmful developmental mutations.

Optimisation of chromatographic conditions for the determination of folates in foods and biological tissues for nutritional and clinical work

Abstract Recent studies implicate folate metabolism in the aetiology of heart disease, neural tube defects, malignant transformation and affective disorders. The paper reports a rapid, isocratic HPLC separation of 11 folylmonoglutamate compounds which should prove useful when adapted to the varied needs of analysts researching these and other specific areas. Also reported are the separation, UV spectra including λmax values, fluorescence emission scans at an excitation wavelength of 295 nm and electrochemically derived hydrodynamic voltammograms with optimum oxidation voltages for p-aminobenzoylglutamate, tetrahydrofolate, 5-methyldihydrofolate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, dihydrofolate, pteroylmonoglutamate and 5,10-methylenetetrahydrofolate. In particular, 5-methyltetrahydrofolate, the main food folate and form of the vitamin found in plasma, can be measured easily by electrochemical detection using a low and highly selective voltage of 450 mV. This reduced folate is also readily detected fluorimetrically using an excitation wavelength of 295 nm and measuring emission at 365 nm. Electrochemical and fluorimetric detection offer equal sensitivity for 5-methyltetrahydrofolate measurement (300 pg on column). No other folate studied could be measured down to this level using fluorimetric detection under the described conditions. At pH 3.5, folate coenzyme λmax for UV detection varies between 267 and 300 nm with 5,10-methenyltetrahydrofolate giving maximum absorption at 355nm. UV measurement of 5-methyltetrahydrofolate is approaching an order of magnitude less sensitive than the former methods of detection. However, for in vitro studies, particularly in the form of a photodiode array, UV detection is a particularly useful tool. For cerebrospinal fluid, plasma, erythrocyte or food measurement of 5CH3H4PteGlu, electrochemical or fluorimetric detection is recommended; whilst for pharmacokinetic studies of plasma 5CHOH4PteGlu during methotrexate rescue therapy, electrochemical or UV detection is most appropriate. For analysis of plasma PteGlu following supplementation, or in food stuffs, UV detection offers the best measurement technique. The information presented should help address the major problem of trace folate analysis by HPLC, that is the need to combine high sensitivity with optimum selectivity in studying complex matrices such as physiological fluids, tissue preparations and food samples.

Contemporary Issues Surrounding Folic Acid Fortification Initiatives

The impact of folate on health and disease, particularly pregnancy complications and congenital malformations, has been extensively studied. Mandatory folic acid fortification therefore has been implemented in multiple countries, resulting in a reduction in the occurrence of neural tube defects. However, emerging evidence suggests increased folate intake may also be associated with unexpected adverse effects. This literature review focuses on contemporary issues of concern, and possible underlying mechanisms as well as giving consideration the future direction of mandatory folic acid fortification. Folate fortification has been associated with the presence of unmetabolized folic acid (PteGlu) in blood, masking of vitamin B12 deficiency, increased dosage for anti-cancer medication, photo-catalysis of PteGlu leading to potential genotoxicity, and a role in the pathoaetiology of colorectal cancer. Increased folate intake has also been associated with twin birth and insulin resistance in offspring, and altered epigenetic mechanisms of inheritance. Although limited data exists to elucidate potential mechanisms underlying these issues, elevated blood folate level due to the excess use of PteGlu without consideration of an individual’s specific phenotypic traits (e.g. genetic background and undiagnosed disease) may be relevant. Additionally, the accumulation of unmetabolized PteGlu may lead to inhibition of dihydrofolate reductase and other enzymes. Concerns notwithstanding, folic acid fortification has achieved enormous advances in public health. It therefore seems prudent to target and carefully monitor high risk groups, and to conduct well focused further research to better understand and to minimize any risk of mandatory folic acid fortification.