Search

Background North American health authorities recommend 0.4 mg/day folic acid before conception and throughout pregnancy to reduce the risk of neural tube defects. Folic acid is a synthetic form of folate that must be reduced by dihydrofolate reductase and then further metabolized. Recent evidence suggests that the maximal capacity for this process is limited and unmetabolized folic acid has been detected in the circulation. The biological effects of unmetabolized folic acid are unknown. A natural form of folate, (6S)-5-methyltetrahydrofolic acid (Metafolin®), may be a superior alternative because it does not need to be reduced in the small intestine. Metafolin® is currently used in some prenatal multivitamins; however, it has yet to be evaluated during pregnancy. Methods/design This double-blind, randomized trial will recruit 60 pregnant women aged 19–42 years. The women will receive either 0.6 mg/day folic acid or an equimolar dose (0.625 mg/day) of (6S)-5-methyltetrahydrofolic acid for 16 weeks. The trial will be initiated at 8–21 weeks’ gestation (after neural tube closure) to reduce the risk of harm should (6S)-5-methyltetrahydrofolic acid prove less effective. All women will also receive a prenatal multivitamin (not containing folate) to ensure adequacy of other nutrients. Baseline and endline blood samples will be collected to assess primary outcome measures, including serum folate, red blood cell folate and unmetabolized folic acid. The extent to which the change in primary outcomes from baseline to endline differs between treatment groups, controlling for baseline level, will be estimated using linear regression. Participants will have the option to continue supplementing until 1 week postpartum to provide a breastmilk and blood sample. Exploratory analyses will be completed to evaluate breastmilk and postpartum blood folate concentrations. Discussion This proof-of-concept trial is needed to obtain estimates of the effect of (6S)-5-methyltetrahydrofolic acid compared to folic acid on circulating biomarkers of folate status during pregnancy. These estimates will inform the design of a definitive trial which will be powered to assess whether (6S)-5-methyltetrahydrofolic acid is as effective as folic acid in raising blood folate concentrations during pregnancy. Ultimately, these findings will inform folate supplementation policies for pregnant women. Trial registration ClinicalTrials.gov, ID: NCT04022135. Registered on 14 July 2019.

A simple method for the verification of supplemental (6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid, “5-MTHF,” in nutritional products is described. Nutritional product samples are prepared for the liquid chromatographic/fluorescence detection (LC/FLD) determination of 5-MTHF by buffer dilution, 10-min centrifugation, and syringe filtration. Method performance has been defined by assessments of 5-MTHF linearity (r 2 averaged 0.9999 ± 0.0001, and all relative calibration errors averaged ≤0.6%, for ten consecutive six-point standard curves), intermediate precision (rsd = 1.1%, n = 9, for three products fortified at ∼1.73 μmol/kg = ∼795 μg/kg, tested on each of 3 days), accuracy (spike recovery average = 92.8 ± 1.0%, n = 9, for nutritional products spiked with 5-MTHF at ∼1.73 μmol/kg, or ∼795 μg/kg), and selectivity (absence of interference from reagent blanks, and from four compounds structurally related to 5-MTHF). The 5-MTHF recovery, as % of unheated controls, from a simulated heat treatment (20 min at 120 °C) averaged 99.1 ± 0.6%, n = 4. The limits of 5-MTHF detection (S/N = 3) and quantification (S/N = 10) were experimentally determined to be 10 μg/kg (∼0.020 μmol/kg) and 30 μg/kg (∼0.060 μmol/kg), respectively (

Volumetric absorptive microsamplers (VAMS) have been developed recently as a promising tool for clinical blood sampling. Compared to dried blood spot samples analyzed by accurate stable isotope dilution assays (SIDAs), the new technique could provide further substantial miniaturizing of folate assays by eliminating hematocrit effects and uneven analyte distribution within the sample. Herein, we present a miniaturized SIDA coupled with LC-MS/MS measurement of 5-methyltetrahydrofolic acid as main folate vitamer in whole blood (WB) using [13C5]-5-methyltetrahydrofolic acid as internal standard. Elution and extraction of only 10.8 μL-dried WB were carried out by centrifugation followed by enzymatic treatment for polyglutamate deconjugation. Matrix separation was achieved by heating and centrifugation. To verify applicability, WB folate status of 11 volunteers was screened. Limits of detection and limits of quantitation were 9 and 26 nmol·L−1, respectively, which is sufficiently low for screening folate status. Recoveries were 97 (±5.8), 99 (±2.8), and 96 (±6.1)% for 800, 400, and 200 nmol L−1 5-methyltetrahydrofolic acid, respectively. Precision of the LC-MS/MS instrument and inter-assay precision trials revealed CVs of 8.1 and 3.5% (294 nmol L−1), respectively, thus confirming reproducible and precise quantitation. Compared to fresh WB, no significant degradation of 5-methyltetrahydrofolate was observed after 2.5 h of drying at room temperature. VAMS 5-CH3-H4folate was stable for at least 3 weeks at −20°C. In our pilot study, accurate and diagnostically conclusive determination of folate status was verified. Nevertheless, blood sampling should be performed by trained individuals to avoid substantial errors concerning the absorbed volume. Endogenous folate in rat serum and chicken pancreas caused a significant background especially at low blood 5-CH3-H4folate levels and, thus, for polyglutamate deconjugation, these background folates or alternative mixtures need to be removed. The superior feasibility of a minimized blood collection with VAMS allows further progress regarding time- and cost-effective methodologies in newborn or population screenings for 5-methyltetrahydrofolate status. Further steps toward minimization could include an automated assay coupled with UPLC-MS/MS.

Folates, such as [6S]-5-methyltetrahydrofolic acid, have been introduced in the market both in vitamin pills and in fortified foods. Their antioxidant activity has been evaluated, but stereoisomer influence on activity has not been proven. In this study, a comparison between [6S]-5-methyltetrahydrofolic acid (5-MTHF) and its racemate [6R,S] form was made by TEAC assay at different pHs, FRAP assay, and ORAC assay. The results showed that the [6S] form had higher antioxidant activity than its racemate form in the TEAC assay at all pHs, with similar values in the FRAP and ORAC assays. Results suggest that stereoisomeric difference could influence the antioxidant activity of 5-MTHF and hence should be taken into account when folates are added to foodstuffs to improve their nutritional value.

Fortification of foods with L-5-methyltetrahydrofolic acid (L-5-MTHF) is challenging due to low stability to environmental conditions that include exposure to pH, moisture, and temperature. The objective of the present study was to stabilize L-5-MTHF using microencapsulation technology. L-5-MTHF microcapsules constructed with different core-to-wall ratios of L-5-MTHF, both alone or in combination with sodium ascorbate, yielded high (>89%) recovery of L-5-MTHF. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis confirmed successful encapsulation of L-5-MTHF with high core-to-wall ratios. Microencapsulation of L-5-MTHF alone with a high core-to-wall ratio significantly (p < 0.05) improved the stability of L-5-MTHF over the course of bread baking, performed both in pilot plant and in commercial baking conditions. Breads made with fortified flour containing sodium ascorbate coencapsulated with L-5-MTHF had recoveries of L-5-MTHF that were 97% and 77%, respectively, for pilot plant and bakery breads. Co-encapsulating L-5-MTHF with ascorbate also significantly (p < 0.05) improved stability during storage, as compared to breads that contained free L-5-MTHF.

Our aim was to protect l-5-methyltetrahydrofolic acid (L-5-MTHF) from degradation throughout the baking and storage of a fortified white bread using microencapsulation. L-5-MTHF, with or without sodium ascorbate (ASC), was microencapsulated using skim milk powder (SMP) as the coating agent. Recoveries of L-5-MTHF in spray-dried materials were greater than 95 ± 5%. Microencapsulated L-5-MTHF was completely released from the skim milk coating material in simulated gastric fluid within the first 10 min at 37°C. Incorporation of SMP-L-5-MTHF or SMP-L-5-MTHF+ASC into bread gave recoveries of 81.3 ± 1.3% and 87.1 ± 1.2% (n=3), respectively, for L-5-MTHF immediately after bread baking. These treatments also showed significantly (p

Among the folate compounds, 5-methyltetrahydrofolic acid (5-CH3THF) is regarded as one of the most bioactive forms of folate. It is regarded as the better source of folate to humans as compared to folic acid, a synthetic form of folate, which is used for fortifying foods to prevent the incidence of neural tube defects in the new born babies. The use of 5-CH3THF as an alternative fortificant, in place of folic acid, has been explored by various researchers. However, fortification of 5-CH3THF is problematic due to its lower stability. This study investigated the stability of microencapsulated 5-CH3THF in biscuits baked at various temperatures and times as well as changes in their physical properties. Microcapsule with pectin and alginate ratio of 80:20, prepared by spray drying, gave the highest retention (68.6%) of the 5-CH3THF, therefore, chosen for fortification. The encapsulated and unencapsulated 5-CH3THF were mixed separately with flour and biscuit ingredients and baked at 180℃, 200 and 220℃, each for 5, 9 and 12 min. The inclusion of encapsulated and unencapsulated 5-CH3THF in the biscuit formulation and subsequent baking at various temperatures and times resulted in retention of 5-CH3THF from 19.1% to 1.7%. Microencapsulation of 5-CH3THF slightly improved the retention of 5-CH3THF over unencapsuated biscuits at 180℃ for 5 min, but almost no such effect was achieved under baking temperatures of 200℃ and 220℃. Physical analysis showed darker colour, harder texture and lower moisture content for biscuits baked at higher test temperatures. It seems intense heating condition that caused “over baking” of the biscuit likely to be responsible for the loss of the vitamin as well as less desirable physical properties of the biscuits.

The effect of beta-mercaptoethanol (0-2%) addition to thermally and/or pressure-treated samples on [6S]-5-methyltetrahydrofolate was studied. Degradation of [6S]-5-methyltetrahydrofolate during both thermal and pressure treatments was mainly caused by oxidation, and the oxidized folates could be partly/completely reduced by beta-mercaptoethanol. The addition of beta-mercaptoethanol (2%) to the thermally and pressure-treated samples overestimated the "actual" stability of [6S]-5-methyltetrahydrofolate and misled the obtained kinetic information.

Oral administration of pyrimethamine (PYR) 3.6 mg/kgBW/day to the pregnant rats from day 11 to day 15 of gestation caused malformations in all the fetuses, but concomitant intraperitoneal administration of folic acid (ip FA) 50 mg/kg/day inhibited the incidence of malformations by 25%. On the other hand, the same dose of oral FA (in feed) had an opposite effect on PYR teratogenesis; the incidences of malformed fetuses were 0% in the PYR 1.6 group, but 100% in the PYR with oral FA group. The plasma levels of 5-methyltetrahydrofolic acid (5MF), a principal transfer form of folate, were measured after single oral administration of PYR with or without FA (ip or oral) in non-pregnant female rats. The area under the plasma concentration-time curve (AUC) after the concomitant administration of PYR with ip FA was larger than that after PYR alone. This result indicated that the available 5MF for the peripheral tissues was increased by the concomitant administration of ip FA. The AUC after the concomitant administration of PYR with oral FA was smaller than that after PYR alone. These results suggested that ip FA increased the available 5MF to ameliorate, but oral FA decreased the availability to aggravate the PYR teratogenesis.

Folic acid (pteroylmonoglutamic acid) has historically been used as the reference folate in human intervention studies assessing the relative bioavailability of dietary folate. Recent studies using labelled folates indicated different plasma response kinetics to folic acid than to natural (food) folates, thus obviously precluding its use in single-dose experiments. Since differences in tissue distribution and site of biotransformation were hypothesised, the question is whether folic acid remains suitable as a reference folate for longer-term intervention studies, where the relative bioavailability of natural (food) folate is assessed based on changes in folate status. Healthy adults aged 18–65 years (n163) completed a 16-week placebo-controlled intervention study in which the relative bioavailability of increased folate intake (453 nmol/d) from folate-rich foods was assessed by comparing changes in plasma and erythrocyte folate concentration with changes induced by an equal reference dose of supplemental (6S)-5-methyltetrahydrofolic acid or folic acid. The relative increase in plasma folate concentration in the food group was 31 % when compared with that induced by folic acid, but 39 % when compared with (6S)-5-methyltetrahydrofolic acid. The relative increase in erythrocyte folate concentration in the food group when compared with that induced by folic acid was 43 %, and 40 % when compared with (6S)-5-methyltetrahydrofolic acid. When recent published observations were additionally taken into account it was concluded that, in principle, folic acid should not be used as the reference folate when attempting to estimate relative natural (food) folate bioavailability in longer-term human intervention studies. Using (6S)-5-methyltetrahydrofolic acid as the reference folate would avoid future results' validity being questioned.

The relationship between pyrimethamine (PYR) teratogenesis and the plasma level of 5-methyltetrahydrofolic acid (5MF), known as an active form of folate in plasma, was studied in rats using an HPLC-ECD method for 5MF determination. The rat received, for 5 days, in-feed PYR with or without folic acid (FA), which was previously reported as a potentiator of PYR teratogenesis. PYR alone caused a dose-dependent decrease in the plasma 5MF level after the 5 day dosings. A potentiated decrease in the 5MF level was observed after the concomitant dosing of PYR with FA. The concomitant dosing of PYR 1.6 mg/kgBW/day (subteratogenic dose of PYR alone) with FA 50 mg/kg/day, which was 100% teratogenic dose in our previous report, decreased the 5MF level more than that after the dosing of PYR 3.6 mg/kg/day alone (100% teratogenic dose of PYR alone). The sequential changes of the plasma 5MF level after the single oral dosing of PYR 3.6 mg/kg with or without oral FA 50 mg/kg also revealed a strong potentiative effect of oral FA on the 5MF-level-lowering effect of PYR. These results indicate that a decrease in the plasma 5MF level may be related to PYR teratogenesis.

Dairy products are a potential matrix for folate fortification to enhance folate consumption in the Western world. Milk folate-binding proteins (FBP) are especially interesting because they seem to be involved in folate bioavailability. In this study, folate bioaccessibility was investigated using a dynamic computer-controlled gastrointestinal model [TNO gastrointestinal model (TIM)]. We used both ultrahigh temperature (UHT)-processed milk and pasteurized milk, differing in endogenous FBP concentrations and fortified with folic acid or 5-methyltetrahydrofolate (5-CH3-H4folate). To study FBP stability during gastrointestinal passage and the effect of additional FBP on folate bioaccessibility, FBP-fortified UHT and pasteurized milk products were also tested. Folate bioaccessibility and FBP stability were measured by taking samples along the compartments of the gastrointestinal model and measuring their folate and FBP concentrations. Folate bioaccessibility from folic acid-fortified milk products without additional FBP was 58-61%. This was lower (P < 0.05) than that of the 5-CH3-H4folate-fortified milk products (71%). Addition of FBP reduced (P < 0.05) folate bioaccessibility from folic acid-fortified milk (44-51%) but not from 5-CH3-H4folate-fortified milk products (72%). The residual FBP levels in the folic acid- and 5-CH3-H4folate-fortified milk products after gastrointestinal passage were 13-16% and 0-1%, respectively, of the starting amounts subjected to TIM. In conclusion, milk seems to be a suitable carrier for folate, because both folic acid and 5-CH3-H4folate are easily released from the matrix and available for absorption. However, our results suggest that folic acid remains partly bound to FBP during passage through the small intestine, which reduces the bioaccessibility of folic acid from milk in this model. Chemicals/CAS: 5 methyltetrahydrofolic acid, 134-35-0; folic acid, 59-30-3, 6484-89-5; 5-methyltetrahydrofolate, 134-35-0; Folic Acid, 59-30-3; Tetrahydrofolates

5-Methyl-5,6,7,8-tetrahydrofolic (5-methylTHF) acid can be measured in human serum and whole blood by fluorescence after deproteinization with perchloric acid and separation by high-performance liquid chromatography (HPLC). The technique is sensitive, rapid, and correlates with existing methods for total folates.

Uptake of 5-methyltetrahydrofolic acid, and serum and red cell folate levels were measured in peripheral blood samples from seven mammalian species. Velocity of uptake of 5-methyltetrahydrofolic acid, at a concentration of 1 microM, increased from 0.001 pmole/10(9) red cells/minute (guinea pig) to 0.7 pmole/10(9) red cells/minute (pig) in the sequence: guinea pig less than sheep less than dog less than man less than rabbit less than rat less than pig. Michaelis constants (Km) were similar for human, dog and sheep erythrocytes (0.5--0.7 microM) but higher in red cells from rabbit, rat and pig (1.7--3.5 microM). Serum and red cell folate levels were comparable in human and dog samples; both levels were higher in rabbit and rat blood. In contrast, red cell folate levels were much lower in pig, sheep and guinea pig peripheral blood. These studies provide evidence that folate compounds are transported by an erythrocyte membrane carrier which is separate from the known permeation system for anions, since the species sequence for the latter is entirely different. They also suggest that some mammalian species, such as the dog, may be more relevant to man for studies of blood folate metabolism than several other species.

We have identified, by TLC, the product from the reaction between 5-methyltetrahydrofolic acid (5-MTHF) and β-phenylethylamine (βφEA) in rat brain extracts as methylene-β-phenylethylimine (MβφEI), a Schiff-base compound produced when formaldehyde, enzymatically formed from 5-MTHF, condenses with the amine. The formation of MβφEI in various brain regions, ranging from 517 ± 56 pmol formed per mg protein per hour in corpus striatum to 118 ± 9 pmol formed in hippocampus, is significantly correlated with that of 1,2,3,4-tetrahydro-β-carboline formed from 5-MTHF and tryptamine (r = 0.88; p

Thermal kinetic data (rate constants, k, and activation energies, Ea) for 5-Methyltetrahydrofolic acid (5-CH3-H4PteGlu) were determined in citrate buffers (pH 3-6) and in model food systems between 100° and 140°C. As pH increased from 3.0 to 6.0, the rate constants decreased as the temperature increased from 100° to 130°C, the rate constants increased. Ea values were 19.0, 17.0, 19.7 and 19.8 kcal/mole at pH 3, 4, 5 and 6, respectively. In the model food systems, the Ea values (kcal/mole) were 7.85 in apple juice and 10.6 in tomato juice. When dissolved oxygen content was reduced to 5.3 ppm, the stability of the 5-CH3H4PteGlu was increased substantially.

A sensitive method based on high-performance liquid chromatographyâtandem mass spectrometry (LCâMS/MS) has been developed for the simultaneous determination of folic acid (FA) and its active metabolite, 5-methyltetrahydrofolic acid (5-M-THF), in human plasma. The analytes were extracted from plasma with methanol solution containing 10Â mg/mL of 2-mercaptoethanol and 0.025% (v/v) ammonium hydroxide. FA and 5-M-THF were more stable after the addition of 2-mercaptoethanol and ammonium hydroxide in the sample preparation procedures of this study than they were in the previously published methods. Chromatographic separation was performed on a Hedera ODS-2 column using a gradient elution system of acetonitrile and 1Â mM ammonium acetate buffer solution containing 0.6% formic acid as mobile phase. LCâMS/MS was carried out with an ESI ion-source and operated in the multiple reaction monitoring (MRM) mode. The assay was linear over the concentration ranges of 0.249â19.9Â ng/mL for FA, and 5.05â50.5Â ng/mL for 5-M-THF. The developed LCâMS/MS method offers increased sensitivity for quantification of FA and 5-M-THF in human plasma and was applicable to a pharmacokinetic study of FA and 5-M-THF. Keywords: Folic acid, 5-Methyltetrahydrofolic acid, 2-Mercaptoethanol, LCâMS/MS, Pharmacokinetics

Serum folate, pteroylglutamic acid standards, and N-5-methyltetrahydrofolate standards extract differently during sample preparation for radiometric assay. B/B0 values for each material were determined before and after an alkaline extraction procedure. Serum folate most closely resembled N-5-methyltetrahydrofolate standards in that it exhibited a slightly greater ability to displace iodinated folate derivative in competition for beta-lactoglobulin binder. Pteroylglutamic acid standards displaced folate derivative less well, so that serum folate values were lower than when N-5-methyltetrahydrofolate was used as the standard material.

We measured the effects of external pH on influx, efflux and net steady-state levels for 14CH3H4PteGlu1. Initial rates of uptake were inversely proportional to external pH. Lowering the pH of the suspending medium increased influx by enhancing the affinity of the carrier: the apparent Km values at pH 6, 7.2 and 8.1 were 0.14, 0.25 and 0.63 microM respectively. In contrast, Vmax was independent of external pH. Efflux rate constants at pH 6, 7.5 and 8.5 were 0.034, 0.059 and 0.067/min respectively. consequently, lowering the external pH increased steady-state levels of 14CH3H4PteGlu1.

5-Methyltetrahydrofolic acid is prepared from folic acid by sodium borohydride reduction, reac­ tion with formaldehyde and reduction to the methyl derivative by sodium borohydride. The crude product is precipitated as barium salt which after having been converted to the sodium salt by ion exchange is adsorbed to QAE-Sephadex and eluted by different acetic acid gradients in subsequent chromatographies. This method allows to process gram quantities on reasonably small columns

1. 5-Methyltetrahydrofolate appeared in the CSF when given intravenously to man. 2. The data suggest considerable circulation of folate between CSF and other body compartments.

The standard chemotherapeutic agent for the treatment of advanced colorectal cancer is fluorouracil (5-FU). Yet the overall response rate to this drug has remained at 20%, and its use has not significantly improved the survival of patients with large-bowel cancer [1].

Chromatographic conditions for the determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid are described, involving simple pretreatment of the sample. Electrochemical detection was used. The linear range of the method is more than 103. Recovery from plasma and spinal fluid is 100%, and the detection limit of the method is 2·10−9M, sufficient for the detection of endogenous plasma and spinal fluid levels. The detection conditions are discussed. Endogenous concentrations of the compound in plasma and spinal fluid were determined and correlated with a folate bioassay. Plasma concentrations have been shown after the administration of leucovorin which is used in anticancer therapy.

The products obtained on incubation of rat and rabbit lung with indoleamines and 5-methyltetrahydrofolic acid (MTHF) were found not to be the methylated compounds, as previously reported by others. Thin-layer chromatography of the basic extracts, as well as their acetylated derivatives, in solvent systems which adequately resolved very similar compounds, was employed to tentatively identify the products as tetrahydro-β-carboline derivatives. It is concluded that these products are formed by the condensation of the various indoleamines with formaldehyde generated enzymatically from MTHF, and that MTHF does not appear to be a significant methyl donor for biogenic amines.

Enzymes from chick and rat tissues catalyze the reaction of N-methyl tryptamine with 5-methyltetrahydrofolic acid to form 2,3,4,9-tetrahydro-2-methyl-1H-pyrido[3,4b] indole. N,N-Dimethyltryptamine was not formed. With tryptamine as substrate the product is 2,3,4,9-tetrahydro-1H-pyrido[3,4b] indole and not N-methyltryptamine. These pyridoindoles were not formed when S-adenosylmethionine was cosubstrare.

Contrary to previous reports, 5-methyltetrahydrofolic acid does not mediate the methylation of dopamine to epinine. Instead, this methyl donor is degraded enzymatically to formaldehyde, which condenses with dopamine to form a tetahydoisoquinoline derivative. The latter has chromotographic characteristics very similar to those of epinine, which likely accounts for the original misidentification of the product.

5-Methyltetrahydrofolate cannot be cleaved at the C9N10 bond by the zinc/HCl reductive or the permanganate oxidative cleavage methods. A new method has been developed to perform this cleavage, using peracetic acid in 50% trifluoroacetic acid; the cleavage is quantitative and nondestructive of γ-glutamyl peptide bonds.

Detailed kinetic studies of [6S] and [6RS] 5-methyltetrahydrofolic acid (5-CH3-H4folate) degradation during thermal (from 60 to 90 °C) and high pressure/thermal (from 30 to 45 °C; from 200 to 700 MPa) treatments were carried out. The results confirmed that the temperature and pressure induced degradation kinetics of [6S] 5-CH3-H4folate were identical (within 95% confidence interval) with those of [6RS] 5-CH3-H4folate. Under equal processing conditions, the estimated degradation rate constants (k), activation energy (Ea), and activation volume (Va) values of [6S] and [6RS] 5-CH3-H4folate were the same (95% confidence interval). The modified thermodynamic model proposed by Nguyen and co-workers (J. Agric. Food Chem. 2003, 51, 3352−3357) to describe the pressure and temperature dependence of the rate constant for folate degradation was reevaluated. Keywords: 5-CH3-H4folate; kinetics; degradation; temperature; pressure