Biosynthesis of L-5-methyltetrahydrofolate by genetically engineered Escherichia coli.

L-5-Methyltetrahydrofolate (L-5-MTHF) is the only biologically active form of folate in the human body. Production of L-5-MTHF by using microbes is an emerging consideration for green synthesis. However, microbes naturally produce only a small amount of L-5-MTHF. Here, Escherichia coli BL21(DE3) was engineered to increase the production of L-5-MTHF by overexpressing the intrinsic genes of dihydrofolate reductase and methylenetetrahydrofolate (methylene-THF) reductase, introducing the genes encoding formate-THF ligase, formyl-THF cyclohydrolase and methylene-THF dehydrogenase from the one-carbon metabolic pathway of Methylobacterium extorquens or Clostridium autoethanogenum and disrupting the gene of methionine synthase involved in the consumption and synthesis inhibition of the target product. Thus, upon its native pathway, an additional pathway for L-5-MTHF synthesis was developed in E. coli, which was further analysed and confirmed by qRT-PCR, enzyme assays and metabolite determination. After optimizing the conditions of induction time, temperature, cell density and concentration of IPTG and supplementing exogenous substances (folic acid, sodium formate and glucose) to the culture, the highest yield of 527.84 µg g-1 of dry cell weight for L-5-MTHF was obtained, which was about 11.8 folds of that of the original strain. This study paves the way for further metabolic engineering to improve the biosynthesis of L-5-MTHF in E. coli.

Effect of germination on the main chemical compounds and 5-methyltetrahydrofolate metabolism of different quinoa varieties.

This study determined the content of macronutrients and micronutrients to investigate the nutritional value and health benefits of six varieties of quinoa seeds and sprouts. Germination markedly increased the contents of proteins, reducing sugars, free amino acids, vitamins, and phytochemicals such as phenolic and carotenoid compounds, with variation among different quinoa varieties. Relatively high levels of 5-methyltetrahydrofolate (5-MTHF) were found in 6-day-old quinoa sprouts, especially in the LL-1 variety (1747.25 µg/100 g DW), followed by QL-2 sprouts (1501.67 µg/100 g DW). Furthermore, we examined the relative expression of genes involved in the folate biosynthetic pathway during QL-2 germination. The expression of the ADCS gene was upregulated 28.31-fold in 6-day-old sprouts, greatly facilitating folate synthesis. Pterin synthesis genes regulate the biosynthesis and further accumulation of folate by controlling pterin metabolic flux. Overall, the 6-day-old sprouts were recommended as a functional food with nutritional value and health benefits in dietary supplements.

Absorption and Tissue Distribution of Folate Forms in Rats: Indications for Specific Folate Form Supplementation during Pregnancy.

Food fortification and folic acid supplementation during pregnancy have been implemented as strategies to prevent fetal malformations during pregnancy. However, with the emergence of conditions where folate metabolism and transport are disrupted, such as folate receptor alpha autoantibody (FRαAb)-induced folate deficiency, it is critical to find a folate form that is effective and safe for pharmacologic dosing for prolonged periods. Therefore, in this study, we explored the absorption and tissue distribution of folic acid (PGA), 5-methyl-tetrahydrofolate (MTHF), l-folinic acid (levofolinate), and d,l-folinic acid (Leucovorin) in adult rats. During absorption, all forms are converted to MTHF while some unconverted folate form is transported into the blood, especially PGA. The study confirms the rapid distribution of absorbed folate to the placenta and fetus. FRαAb administered, also accumulates rapidly in the placenta and blocks folate transport to the fetus and high folate concentrations are needed to circumvent or overcome the blocking of FRα. In the presence of FRαAb, both Leucovorin and levofolinate are absorbed and distributed to tissues better than the other forms. However, only 50% of the leucovorin is metabolically active whereas levofolinate is fully active and generates higher tetrahydrofolate (THF). Because levofolinate can readily incorporate into the folate cycle without needing methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) in the first pass and is relatively stable, it should be the folate form of choice during pregnancy, other disorders where large daily doses of folate are needed, and food fortification.

Folic Acid, Folinic Acid, 5 Methyl TetraHydroFolate Supplementation for Mutations That Affect Epigenesis through the Folate and One-Carbon Cycles.

Methylation is an essential biochemical mechanism that is central to the transmission of life, and crucially responsible for regulating gametogenesis and continued embryo development. The methylation of DNA and histones drives cell division and regulation of gene expression through epigenesis and imprinting. Brain development and its maturation also depend on correct lipid methylation, and continued neuronal function depends on biogenic amines that require methylation for their synthesis. All methylation processes are carried out via a methyltransferase enzyme and its unique co-factor S-adenosylmethionine (SAM); the transfer of a methyl group to a target molecule results in the release of SAH (SA homocysteine), and then homocysteine (Hcy). Both of these molecules are toxic, inhibiting methylation in a variety of ways, and Hcy recycling to methionine is imperative; this is achieved via the one carbon cycle, supported by the folates cycle. Folate deficiency causes hyperhomocysteinaemia, with several associated diseases; during early pregnancy, deficiency interferes with closure of the neural tube at the fourth week of gestation, and nutraceutical supplementation has been routinely prescribed to prevent neural tube defects, mainly involving B vitamins, Zn and folates. The two metabolic pathways are subject to single nucleotide polymorphisms that alter their activity/capacity, often severely, impairing specific physiological functions including fertility, brain and cardiac function. The impact of three types of nutraceutical supplements, folic acid (FA), folinic acid (FLA) and 5 Methyl THF (MTHF), will be discussed here, with their positive effects alongside potentially hazardous secondary effects. The issue surrounding FA and its association with UMFA (unmetabolized folic acid) syndrome is now a matter of concern, as UMFA is currently found in the umbilical cord of the fetus, and even in infants' blood. We will discuss its putative role in influencing the acquisition of epigenetic marks in the germline, acquired during embryogenesis, as well as the role of FA in the management of cancerous disease.

Methionine synthase supports tumour tetrahydrofolate pools.

Mammalian cells require activated folates to generate nucleotides for growth and division. The most abundant circulating folate species is 5-methyl tetrahydrofolate (5-methyl-THF), which is used to synthesize methionine from homocysteine via the cobalamin-dependent enzyme methionine synthase (MTR). Cobalamin deficiency traps folates as 5-methyl-THF. Here, we show using isotope tracing that MTR is only a minor source of methionine in cell culture, tissues or xenografted tumours. Instead, MTR is required for cells to avoid folate trapping and assimilate 5-methyl-THF into other folate species. Under conditions of physiological extracellular folates, genetic MTR knockout in tumour cells leads to folate trapping, purine synthesis stalling, nucleotide depletion and impaired growth in cell culture and as xenografts. These defects are rescued by free folate but not one-carbon unit supplementation. Thus, MTR plays a crucial role in liberating THF for use in one-carbon metabolism.

Regulation of the one carbon folate cycle as a shared metabolic signature of longevity.

The metabolome represents a complex network of biological events that reflects the physiologic state of the organism in health and disease. Additionally, specific metabolites and metabolic signaling pathways have been shown to modulate animal ageing, but whether there are convergent mechanisms uniting these processes remains elusive. Here, we used high resolution mass spectrometry to obtain the metabolomic profiles of canonical longevity pathways in C. elegans to identify metabolites regulating life span. By leveraging the metabolomic profiles across pathways, we found that one carbon metabolism and the folate cycle are pervasively regulated in common. We observed similar changes in long-lived mouse models of reduced insulin/IGF signaling. Genetic manipulation of pathway enzymes and supplementation with one carbon metabolites in C. elegans reveal that regulation of the folate cycle represents a shared causal mechanism of longevity and proteoprotection. Such interventions impact the methionine cycle, and reveal methionine restriction as an underlying mechanism. This comparative approach reveals key metabolic nodes to enhance healthy ageing.

Symposium review: One-carbon metabolism and methyl donor nutrition in the dairy cow.

The present review focuses on methyl donor metabolism and nutrition in the periparturient and lactating dairy cow. Methyl donors are involved in one-carbon metabolism, which includes the folate and Met cycles. These cycles work in unison to support lipid, nucleotide, and protein synthesis, as well as methylation reactions and the maintenance of redox status. A key feature of one-carbon metabolism is the multi-step conversion of tetrahydrofolate to 5-methyltetrahyrofolate. Homocysteine and 5-methyltetrahyrofolate are utilized by vitamin B12-dependent Met synthase to couple the folate and Met cycles and generate Met. Methionine may also be remethylated from choline-derived betaine under the action of betaine hydroxymethyltransferase. Regardless, Met is converted within the Met cycle to S-adenosylmethionine, which is universally utilized in methyl-group transfer reactions including the synthesis of phosphatidylcholine. Homocysteine may also enter the transsulfuration pathway to generate glutathione or taurine for scavenging of reactive oxygen metabolites. In the transition cow, a high demand exists for compounds with a labile methyl group. Limited methyl group supply may contribute to inadequate hepatic phosphatidylcholine synthesis and hepatic triglyceride export, systemic oxidative stress, and compromised milk production. To minimize the perils associated with methyl donor deficiency, the peripartum cow relies on de novo methylneogenesis from tetrahydrofolate. In addition, dietary supplementation of rumen-protected folic acid, vitamin B12, Met, choline, and betaine are potential nutritional approaches to target one-carbon pools and improve methyl donor balance in transition cows. Such strategies have merit considering research demonstrating their ability to improve milk production efficiency, milk protein synthesis, hepatic health, and immune response. This review aims to summarize the current understanding of folic acid, vitamin B12, Met, choline, and betaine utilization in the dairy cow. Methyl donor co-supplementation, fatty acid feeding strategies that may optimize methyl donor supplementation efficacy, and potential epigenetic mechanisms are also considered.

A review of the effects of the anesthetic gas nitrous oxide on the immune system; a starting point for future experiences.

Nitrous oxide is a common gas used as an anesthetic agent and analgesic medication in operating rooms. The gas inhibits vitamin B12 dependent-methionine synthase, which converts L-homocysteine and 5-methyltetrahydrofolate to L-methionine and tetrahydrofolate, respectively, via a methylation process. The immune system has been frequently reported to be suppressed in anesthetized subjects during the postoperative period. Although previous reviews have focused on the pathophysiologic role of nitrous oxide, none of them has considered the harmful effects of nitrous oxide on the Defense system of the host. In this article, the authors review current studies on the effects of nitrous oxide on the immune system of both patients undergoing surgery and occupational exposure, as well as preclinical studies. Moreover, this paper opens a new horizon for future studies in the context of underlying mechanisms of nitrous oxide actions on the immune system.

Demographic, Physiologic, and Lifestyle Characteristics Observed with Serum Total Folate Differ Among Folate Forms: Cross-Sectional Data from Fasting Samples in the NHANES 2011-2016.

BACKGROUND: Serum folate forms were measured in the US population during recent NHANES to assess folate status. OBJECTIVE: We describe post-folic acid-fortification concentrations of serum folate forms in the fasting US population ≥1 y from the NHANES 2011-2016. METHODS: We measured 5 biologically active folates and 1 oxidation product (MeFox) of 5-methyltetrahydrofolate (5-methyl-THF). We calculated geometric means of 5-methyl-THF, unmetabolized folic acid (UMFA), nonmethyl folate (sum of tetrahydrofolate, 5-formyltetrahydrofolate, and 5,10-methenyltetrahydrofolate), total folate (sum of above biomarkers), and MeFox by demographic, physiologic, and lifestyle variables; estimated the magnitude of variables on biomarker concentrations after covariate adjustment; and determined the prevalence of UMFA >2 nmol/L. RESULTS: After demographic adjustment, age, sex, and race-Hispanic origin were significantly associated with most folate forms. MeFox increased with age, while 5-methyl-THF, UMFA, and nonmethyl folate displayed U-shaped age patterns. Compared with non-Hispanic whites, non-Hispanic blacks had 23% lower predicted 5-methyl-THF but comparable UMFA; non-Hispanic Asians had comparable 5-methyl-THF but 28% lower UMFA; Hispanics, non-Hispanic Asians, and non-Hispanic blacks had ∼20% lower MeFox. After additional physiologic and lifestyle adjustment, predicted UMFA and MeFox concentrations were 43% and 112% higher, respectively, in adults with chronic kidney disease and 17% and 15% lower, respectively, in adults consuming daily 1-<2 alcoholic beverages; 5-methyl-THF concentrations were 20% lower in adult smokers. The prevalence of UMFA >2 nmol/L was highest in persons aged ≥70 y (9.01%) and lowest in those aged 12-19 y (1.14%). During 2011-2014, the prevalence was 10.6% in users and 2.22% in nonusers of folic acid-containing supplements. CONCLUSIONS: In fasting persons ≥1 y, the demographic, physiologic, and lifestyle characteristics observed with serum total folate differed among folate forms, suggesting biological and/or genetic influences on folate metabolism. High UMFA was mostly observed in supplement users and older persons.

Histidine catabolism is a major determinant of methotrexate sensitivity.

The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase1, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis2. Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production3. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials4, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR-Cas9-based screen6,7. This screen yielded FTCD, which encodes an enzyme-formimidoyltransferase cyclodeaminase-that is required for the catabolism of the amino acid histidine8, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.

Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy?

Homeostasis around vascular endothelium is a function of the equilibrium between the bioavailability of nitric oxide (NO) and oxidizing reactive oxygen species (ROS). Within the vascular endothelium, NO enhances vasodilatation, reduces platelet aggression and adhesion (anti-thrombotic), prevents smooth muscle proliferation, inhibits adhesion of leukocytes and expression of pro-inflammatory cytokines genes (anti-inflammatory), and counters the oxidation of low density lipoprotein (LDL) cholesterol. A shift in the equilibrium that favours NO deficiency and ROS formation leads to endothelial dysfunction and cardiovascular disease. The synthesis of NO is catalysed by nitric oxide synthase and co-factored by tetrahydrobiopterin (BH4), nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN). The focus of this review is on endothelial nitric oxide synthase (eNOS), although we recognize that the other nitric oxide synthases may contribute as well. Levels of homocysteine and the active metabolite of folate, 5-methyltetrahydrofolate (5-MTHF), play a determining role in circulating levels of nitric oxide. We review endothelial nitric oxide bioavailabilty in relation to endothelial dysfunction as well as the therapeutic strategies involving the nitric oxide synthesis pathway. Although folate supplementation improves endothelial function, results from large clinical trials and meta-analyses on palpable clinical endpoints have been inconsistent. There are however, encouraging results from animal and clinical studies of supplementation with the co-factor for nitric oxide synthesis, BH4, though its tendency to be oxidized to dihydrobiopterin (BH2) remains problematic. Understanding how to maintain a high ratio of BH4 to BH2 appears to be the key that will likely unlock the therapeutic potential of nitric oxide synthesis pathway.

Folic Acid Impairs the Uptake of 5-Methyltetrahydrofolate in Human Umbilical Vascular Endothelial Cells.

BACKGROUND: Adequate folate status supports endothelial structure and function. Folic acid (FA), an oxidized synthetic folate, which is present in the plasma of patients consuming fortified food or FA supplements, may impair cellular uptake of physiological, reduced folates. We studied the effect of FA on uptake of the dominant circulatory folate, 5-methyltetrahydrofolate (5MTHF) in endothelial cells. METHODS AND RESULTS: For short-term effects of FA, primary human umbilical vein endothelial cells (HUVECs) were maintained in growth medium containing 200 nM 5MTHF and preincubated with 20 nM FA 10 minutes before the 5MTHF uptake assessment. For long-term effects, HUVECs were cultured for 3 passages in growth medium containing either 200 nM 5MTHF, or a combination of 100 nM 5MTHF and 100 nM FA. 5MTHF uptake was assessed after exposing cells to 200 nM [C5]-5MTHF, after which intracellular [C5]-5MTHF was quantified using liquid chromatography/tandem mass spectrometry. Acute FA exposure caused a 57% reduction in 5MTHF uptake compared with control conditions (51 ± 12 vs. 22 ± 7 fmol·min·mg protein; P = 0.01). Long-term exposure to FA reduced 5MTHF uptake by 41% (51 ± 12 vs. 30 ± 11 fmol·min·mg protein; P = 0.05) and reduced total cellular 5MTHF levels by 47 ± 21% in HUVEC (P = 0.02). CONCLUSION: Unmetabolized FA, which appears in the plasma after consumption of fortified food or FA supplements, may impair uptake of 5MTHF, the dominant bioactive form of folate, in HUVEC.

Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism.

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

Unmetabolized Folic Acid, Tetrahydrofolate, and Colorectal Adenoma Risk.

In a randomized trial of folic acid supplementation for the prevention of colorectal adenomas, we previously found indications of increased risk during later treatment and follow-up. This could have been due to the unmetabolized folic acid (UFA) or natural reduced and methylated folates (mF) to which it is metabolized. In post hoc analyses, we measured mF (the sum of 5-methyl-tetrahydrofolate and 4-alfa-hydroxy-5-methyl-THF) and UFA concentrations in the serum of 924 participants. Using binomial regression models with a log link, we assessed the associations between plasma mF or UFA and adenoma occurrence. We found no association between plasma mF or UFA and overall adenoma risk. However, during later follow-up, the prespecified, composite endpoint of high-risk findings (advanced or multiple adenomas) was positively associated with plasma mF (Plinear trend = 0.009), with a 58% increased risk for participants in the upper versus lowest quartile. An irregular association was seen with plasma UFA, with suggestions of an inverse trend (Plinear trend=0.049). A modest, significant inverse association was also seen between mF and risk of serrated lesions, with a 39% lower risk for upper versus lower quartile participants (Plinear trend = 0.03). In conclusion, during the later follow-up period in which folic acid supplementation was previously seen to increase the risk of advanced and multiple adenomas, higher serum mF was associated with a higher risk of multiple and/or advanced adenomas, but no clear indication that UFA played a direct role. There were indications that higher mF was associated with reduced risk of serrated polyps. Cancer Prev Res; 10(8); 451-8. ©2017 AACR.

L-Methylfolate supplementation in a child with autism and methyltetrahydrofolate reductase, enzyme gene C677TT allele.

Errors in folate metabolism may play a role in the pathology of autism spectrum disorders because of increased vulnerability to oxidative stress. We report a case where L-methylfolate supplementation improved symptoms of aggression and disruptive behavior in a child with autism who tested positive for the C677TT allele of the methyltetrahydrofolate reductase enzyme gene. To our knowledge, this is the first report of L-methylfolate administration in this situation. Further controlled studies of L-methylfolate in this population are warranted.

Total folate and unmetabolized folic acid in the breast milk of a cross-section of Canadian women.

Background: Folate requirements increase during pregnancy and lactation. It is recommended that women who could become pregnant, are pregnant, or are lactating consume a folic acid (FA)-containing supplement.Objectives: We sought to determine breast-milk total folate and unmetabolized folic acid (UMFA) contents and their relation with FA-supplement use and doses in a cohort of Canadian mothers who were enrolled in the MIREC (Maternal-Infant Research on Environmental Chemicals) study.Design: Breast-milk tetrahydrofolate (THF), 5-methyl-THF, 5-formyl-THF, 5,10-methenyl-THF, and UMFA were measured with the use of liquid chromatography-tandem mass spectrometry (n = 561). Total daily supplemental FA intake was based on self-reported FA-supplement use.Results: UMFA was detectable in the milk of 96.1% of the women. Total daily FA intake from supplements was associated with breast folate concentration and species. Breast-milk total folate was 18% higher (P < 0.001) in supplement users (n = 401) than in nonusers (n = 160), a difference driven by women consuming >400 µg FA/d (P ≤ 0.004). 5-Methyl-THF was 19% lower (P < 0.001) and UMFA was 126% higher (P < 0.001) in supplement users than in nonusers. Women who consumed >400 µg FA/d had proportionally lower 5-methyl-THF and higher UMFA than did women who consumed ≤400 µg FA/d.Conclusions: FA-supplement use was associated with modestly higher breast-milk total folate. Detectable breast-milk UMFA was nearly ubiquitous, including in women who did not consume an FA supplement. Breast-milk UMFA was proportionally higher than 5-methyl-THF in women who consumed >400 µg FA/d, thereby suggesting that higher doses exceed the physiologic capacity to metabolize FA and result in the preferential uptake of FA in breast milk. Therefore, FA-supplement doses >400 µg may not be warranted, especially in populations for whom FA fortification is mandatory.

Folate bioavailability from foods rich in folates assessed in a short term human study using stable isotope dilution assays.

Different sources of folate may have different bioavailability and hence may impact the standard definition of folate equivalents. In order to examine this, a short term human study was undertaken to evaluate the relative native folate bioavailabilities from spinach, Camembert cheese and wheat germs compared to pteroylmonoglutamic acid as the reference dose. The study had a single-centre, randomised, four-treatment, four-period, four-sequence, cross-over design, i.e. the four (food) items to be tested (referred to as treatments) were administered in sequences according to the Latin square, so that each experimental treatment occurred only once within each sequence and once within each study period. Each of the 24 subjects received the four experimental items separated by a 14-day equilibrium phase and received a pteroylmonoglutamic acid supplement for 14 days before the first testing and between the testings for saturation of body pools. Folates in test foods, plasma and urine samples were determined by stable isotope dilution assays, and in urine and plasma, the concentrations of 5-methyltetrahydrofolate were evaluated. Standard non-compartmental methods were applied to determine the biokinetic parameters C(max), t(max) and AUC from baseline corrected 5-methyltetrahydrofolate concentrations within the interval from 0 to 12 hours. The variability of AUC and C(max) was moderate for spinach and oral solution of pteroylmonoglutamic acid but high for Camembert cheese and very high for wheat germs. The median t(max) was lowest for spinach, though t(max) showed a high variability among all treatments. When comparing the ratio estimates of AUC and C(max) for the different test foods, highest bioavailability was found for spinach followed by that for wheat germs and Camembert cheese. The results underline the dependence of folate bioavailability on the type of food ingested. Therefore, the general assumption of 50% bioavailability as the rationale behind the definition of folate equivalents has to be questioned and requires further investigation.

A pharmacokinetic and pharmacodynamic investigation of Modufolin® compared to Isovorin® after single dose intravenous administration to patients with colon cancer: a randomized study.

PURPOSE: Leucovorin is commonly used as folate supplement in 5-fluorouracil-based chemotherapy, but needs to be converted to active 5,10-methylenetetrahydrofolate (methyleneTHF) intracellularly. This provides for interindividual differences. MethyleneTHF has recently been developed into the stable, distributable drug, Modufolin®. The aim was to compare the concentration of folate metabolites in tumor, mucosa, and plasma of patients with colon cancer after administration of Modufolin® or Isovorin® (levo-leucovorin). METHODS: Thirty-two patients scheduled for colon resection were randomized to receive Modufolin® or Isovorin® at dosage of 60 or 200 mg/m². The study drug was given as one i.v. bolus injection after anesthesia. Plasma was collected for pharmacokinetic (PK) analysis before, during, and after surgery. Tissue biopsies were collected at surgery. Folate metabolites were analyzed by LC-MS/MS. RESULTS: MethyleneTHF and THF concentrations were significantly higher in mucosa (p < 0.01, both dosages) and tumors (p < 0.01, 200 mg/m²) after Modufolin® as compared to Isovorin® administration. The results correlated with PK observations. The Modufolin® to Isovorin® C(max) ratio for methyleneTHF was 113 at 200 mg/m² and 52 at 60 mg/m²; the AUC(last) ratios were 17 and 9, respectively. The THF plasma concentrations were also higher after Modufolin® administration (C(max) ratio 23, AUC(last) ratio 13 at 200 mg/m²; C(max) ratio 15, AUC(last) ratio 11 at 60 mg/m²). CONCLUSION: Modufolin® administration resulted in significantly higher methyleneTHF levels than Isovorin® and may potentially increase the efficacy of 5-fluorouracil-based chemotherapy. The results encourage further evaluation of Modufolin® as a substitute to Isovorin® including the potential clinical benefits.

Folic acid handling by the human gut: implications for food fortification and supplementation.

BACKGROUND: Current thinking, which is based mainly on rodent studies, is that physiologic doses of folic acid (pterylmonoglutamic acid), such as dietary vitamin folates, are biotransformed in the intestinal mucosa and transferred to the portal vein as the natural circulating plasma folate, 5-methyltetrahydrofolic acid (5-MTHF) before entering the liver and the wider systemic blood supply. OBJECTIVE: We tested the assumption that, in humans, folic acid is biotransformed (reduced and methylated) to 5-MTHF in the intestinal mucosa. DESIGN: We conducted a crossover study in which we sampled portal and peripheral veins for labeled folate concentrations after oral ingestion with physiologic doses of stable-isotope-labeled folic acid or the reduced folate 5-formyltetrahydrofolic acid (5-FormylTHF) in 6 subjects with a transjugular intrahepatic porto systemic shunt (TIPSS) in situ. The TIPSS allowed blood samples to be taken from the portal vein. RESULTS: Fifteen minutes after a dose of folic acid, 80 ± 12% of labeled folate in the hepatic portal vein was unmodified folic acid. In contrast, after a dose of labeled 5-FormylTHF, only 4 ± 18% of labeled folate in the portal vein was unmodified 5-FormylTHF, and the rest had been converted to 5-MTHF after 15 min (postdose). CONCLUSIONS: The human gut appears to have a very efficient capacity to convert reduced dietary folates to 5-MTHF but limited ability to reduce folic acid. Therefore, large amounts of unmodified folic acid in the portal vein are probably attributable to an extremely limited mucosal cell dihydrofolate reductase (DHFR) capacity that is necessary to produce tetrahydrofolic acid before sequential methylation to 5-MTHF. This process would suggest that humans are reliant on the liver for folic acid reduction even though it has a low and highly variable DHFR activity. Therefore, chronic liver exposure to folic acid in humans may induce saturation, which would possibly explain reports of systemic circulation of unmetabolized folic acid.

Neural tube defects, folate, and immune modulation.

Periconceptional supplementation with folic acid has led to a significant worldwide reduction in the incidence of neural tube defects (NTDs). However, despite increasing awareness of the benefits of folic acid supplementation and the implementation of food fortification programs in many countries, NTDs continue to be a leading cause of perinatal morbidity and mortality worldwide. Furthermore, there exists a significant subgroup of women who appear to be resistant to the protective effects of folic acid supplementation. The following review addresses emerging clinical and experimental evidence for a role of the immune system in the etiopathogenesis of NTDs, with the aim of developing novel preventative strategies to further reduce the incidence of NTD-affected pregnancies. In particular, recent studies demonstrating novel roles and interactions between innate immune factors such as the complement cascade, neurulation, and folate metabolism are explored.