Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli.

5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 µg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 µg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 µg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

Role of folic acid in regulating gut microbiota and short-chain fatty acids based on an in vitro fermentation model.

Folic acid deficiency is common worldwide and is linked to an imbalance in gut microbiota. However, based on model animals used to study the utilization of folic acid by gut microbes, there are challenges of reproducibility and individual differences. In this study, an in vitro fecal slurry culture model of folic acid deficiency was established to investigate the effects of supplementation with 5-methyltetrahydrofolate (MTHF) and non-reduced folic acid (FA) on the modulation of gut microbiota. 16S rRNA sequencing results revealed that both FA (29.7%) and MTHF (27.9%) supplementation significantly reduced the relative abundance of Bacteroidetes compared with control case (34.3%). MTHF supplementation significantly improved the relative abundance of Firmicutes by 4.49%. Notably, compared with the control case, FA and MTHF supplementation promoted an increase in fecal levels of Lactobacillus, Bifidobacterium, and Pediococcus. Short-chain fatty acid (SCFA) analysis showed that folic acid supplementation decreased acetate levels and increased fermentative production of isobutyric acid. The in vitro fecal slurry culture model developed in this study can be utilized as a model of folic acid deficiency in humans to study the gut microbiota and demonstrate that exogenous folic acid affects the composition of the gut microbiota and the level of SCFAs. KEY POINTS: • Establishment of folic acid deficiency in an in vitro culture model. • Folic acid supplementation regulates intestinal microbes and SCFAs. • Connections between microbes and SCFAs after adding folic acid are built.

Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

CYP2U1: An emerging treatable neurometabolic disease with cerebral folate deficiency in 2 Chinese brothers.

With the rapid advancement of medical technologies in genomic and molecular medicine, the number of treatable neurometabolic diseases is quickly expanding. Spastic paraplegia 56 (SPG56), one of the severe autosomal recessive forms of neurodegenerative disorders caused by pathogenic variants in the CYP2U1 gene, has no reported specific targeted treatment yet. Here we report 2 Chinese brothers with CYP2U1 bi-allelic pathogenic variants with cerebral folate deficiency who were treated for over a decade with folinic acid supplement. Patients have remained stable under therapy.

HNRNPC mediated m6A methylation of 5-methyltetrahydrofolate-homocysteine methyltransferase and involved in the occurrence of RSA.

N6-methyladenosine methylated modification has been shown to play roles in recurrent spontaneous abortion. We aimed to explore role of heterogeneous nuclear ribonucleoprotein C in the occurrence of recurrent spontaneous abortion. We collected embryonic villous tissues from 3 patients with recurrent spontaneous abortion (RSA group) and 3 normal control pregnancy patients. Methylated RNA immunoprecipitation sequencing, RNA sequencing, methylated RNA immunoprecipitation quantitative PCR were conducted to detect the differentially expressed m6A methylation modification gene and regulatory gene in patients with recurrent spontaneous abortion. Methylated RNA immunoprecipitation sequencing and RNA sequencing results showed that the mRNA expression level of heterogeneous nuclear ribonucleoprotein C significantly decreased in RSA group and mRNA expression level of 5-methyltetrahydrofolate-homocysteine methyltransferase increased. Real-time quantitative PCR confirmed the differential expression of heterogeneous nuclear ribonucleoprotein C and 5-methyltetrahydrofolate-homocysteine methyltransferase. Methylated RNA immunoprecipitation quantitative PCR result showed that mRNA m6A modification level of 5-methyltetrahydrofolate-homocysteine methyltransferase decreased in RSA group. The results of western blotting, real-time quantitative PCR, immunofluorescence, matrigel invasion and wound healing assays indicated that heterogeneous nuclear ribonucleoprotein C might regulate the expression of 5-methyltetrahydrofolate-homocysteine methyltransferase by mediating m6A modification, thereby reducing the proliferation and migration of trophoblast cell line, ultimately leading to the occurrence of recurrent spontaneous abortion.

Investigations Using Albumin Binders to Modify the Tissue Distribution Profile of Radiopharmaceuticals Exemplified with Folate Radioconjugates.

Introducing an albumin-binding entity into otherwise short-lived radiopharmaceuticals can be an effective means to improve their pharmacokinetic properties due to enhanced blood residence time. In the current study, DOTA-derivatized albumin binders based on 4-(p-iodophenyl)butanoate (DOTA-ALB-1 and DOTA-ALB-3) and 5-(p-iodophenyl)pentanoate entities (DOTA-ALB-24 and DOTA-ALB-25) without and with a hydrophobic 4-(aminomethyl)benzoic acid (AMBA) linker unit, respectively, were synthesized and labeled with lutetium-177 for in vitro and in vivo comparison. Overall, [177Lu]Lu-DOTA-ALB-1 demonstrated ~3-fold stronger in vitro albumin-binding affinity and a longer blood residence time (T50%IA ~8 h) than [177Lu]Lu-DOTA-ALB-24 (T50%IA ~0.8 h). Introducing an AMBA linker enhanced the albumin-binding affinity, resulting in a T50%IA of ~24 h for [177Lu]Lu-DOTA-ALB-3 and ~2 h for [177Lu]Lu-DOTA-ALB-25. The same albumin binders without or with the AMBA linker were incorporated into 6R- and 6S-5-methyltetrahydrofolate-based DOTA-conjugates (177Lu-RedFols). Biodistribution studies in mice performed with both diastereoisomers of [177Lu]Lu-RedFol-1 and [177Lu]Lu-RedFol-3, which comprised the 4-(p-iodophenyl)butanoate moiety, demonstrated a slower accumulation in KB tumors than those of [177Lu]Lu-RedFol-24 and [177Lu]Lu-RedFol-25 with the 5-(p-iodophenyl)pentanoate entity. In all cases, the tumor uptake was high (30-45% IA/g) 24 h after injection. Both diastereoisomers of [177Lu]Lu-RedFol-1 and [177Lu]Lu-RedFol-3 demonstrated high blood retention (3.8-8.7% IA/g, 24 h p.i.) and a 2- to 4-fold lower kidney uptake than the corresponding diastereoisomers of [177Lu]Lu-RedFol-24 and [177Lu]Lu-RedFol-25, which were more rapidly cleared from the blood (<0.2% IA/g, 24 h after injection). Kidney retention of the 6S-diastereoisomers of all 177Lu-RedFols was consistently higher than that of the respective 6R-diastereoisomers, irrespective of the albumin binder and linker unit used. It was demonstrated that the blood clearance data obtained with 177Lu-DOTA-ALBs had predictive value for the blood retention times of the respective folate radioconjugates. The use of these albumin-binding entities without or with an AMBA linker may serve for fine-tuning the blood retention of folate radioconjugates and also other radiopharmaceuticals and, hence, optimize their tissue distribution profiles. Dosimetry estimations based on patient data obtained with one of the most promising folate radioconjugates will be crucial to identify the dose-limiting organ, which will allow for selecting the most suitable folate radioconjugate for therapeutic purposes.

Serum 5-Methyltetrahydrofolate Status Is Associated with One-Carbon Metabolism-Related Metabolite Concentrations and Enzyme Activity Indicators in Young Women.

Maintaining optimal one-carbon metabolism (OCM) is essential for health and pregnancy. In this cross-sectional study, folate status was assessed based on 5-methyltetrahydrofolate (5-MTHF) levels, and the association between 5-MTHF and OCM-related metabolites was investigated in 227 female Japanese university students aged 18-25 years. The participants were divided into high and low 5-MTHF groups based on their folate status. Serum samples of the participants were collected while they were fasting, and 18 OCM-related metabolites were measured using stable-isotope dilution liquid chromatography-electrospray tandem mass spectrometry. The association between serum 5-MTHF and OCM-related metabolite concentrations was assessed using Spearman's rank correlation coefficient. Serum 5-MTHF concentrations were negatively correlated with total homocysteine (tHcy) concentrations and positively correlated with S-adenosylmethionine (SAM) and total cysteine (tCys) concentrations. Serum 5-MTHF concentrations demonstrated a stronger negative correlation with tHcy/tCys than with tHcy alone. The negative correlation between betaine and tHcy concentrations was stronger in the low 5-MTHF group than in the high 5-MTHF group. The 5-MTHF status could be linked to Hcy flux into the transsulfuration pathway via SAM. Therefore, the tHcy/tCys ratio may be a more sensitive indicator of the 5-MTHF status than tHcy alone. Furthermore, a low 5-MTHF status can enhance Hcy metabolism via betaine.

The evolution of folate supplementation - from one size for all to personalized, precision, poly-paths.

Folate is a crucial nutrient that supports physiological functions. Low folate levels is a risk factor for several diseases, including cardiovascular diseases and neural tube defects. The most used folate supplement is folic acid, a synthetic oxidative form, and folic acid grain fortification is a success story of public health. However, the metabolic conversion of folic acid to bioactive tetrahydrofolate requires several enzymes and cofactors. Therefore, these factors influence its bioavailability and efficacy. In contrast, 5-methyltetrahydrofolate is used directly and participates in one-carbon metabolism, and the use of 5-methyltetrahydrofolate as an alternative folate supplement has increased. The metabolism of 5-methyltetrahydrofolate is primarily dependent on the transmembrane transporter, reduced folate carrier (RFC), and the RFC gene SLC19A1 variant is a functional polymorphism that affects folate status indexes. Recent studies demonstrated that the expression of RFC and cystathionine ß-synthase, another enzyme required for homocysteine clearance, increases significantly by supplementation with calcitriol (vitamin D3), suggesting that calcitriol intake promotes the bioavailability of folate and has synergistic effects in homocysteine clearance. The advancements in biomedical and cohort studies and clinical trials have enhanced our understanding of the critical roles of folate and the regulation of one-carbon metabolism. We anticipate that the field of folate supplementation is poised to evolve from one size for all to personalized, precision, poly-paths (3Ps), which is a critical measure to meet individual needs, maximize health benefits, and minimize side effects.

Characterization of folic acid, 5-methyltetrahydrofolate and synthetic folinic acid in the high-affinity folate transporters: impact on pregnancy and development.

Folates are B vitamins that are essential for several molecular, cellular, and biological processes, including nucleotide synthesis, methylation, and methionine cycling. The physiological impacts of these processes on health also extend to cell proliferation, folate deficiency anemia, and reduction of the risk of birth defects during pregnancy. The primary objective of this study was to characterize the binding affinities of different folate forms, folic acid (FA), 5-methyltetrahydrofolate (5MTHF), and folinic acid, to the folate receptors α and ß, and to the bovine milk folate binding protein. These three dietary forms of folate are found in enriched grains (FA), various fruits and leafy vegetables (folinic acid), and red blood cells (5MTHF). Methods: The half maximal inhibitory concentration values and binding curves of each of these folates for each receptor were determined. Results: Our results indicated that FA had the highest affinity for all folate receptors, followed by 5MTHF, and lastly, by folinic acid, examined by several orders of magnitudes. Conclusion: These data are expected to provide new insights into the therapeutic applications of the different forms of folate in a variety of diseases.

An efficient LC-MS/MS method for simultaneous determination of four substances in the Hcy metabolism pathway.

Aim: This study aimed to develop and validate an efficient LC-MS/MS method for the simultaneous determination of Hcy, Cys, Met and 5-methyltetrahydrofolate in human serum and to apply this method to patients with coronary artery disease. Methodology and results: Serum samples were prepared by reduction with dithiothreitol followed by protein precipitation. The analytical run time was 2.2 min. The linearity was good in the range of 2-100 µmol/l-1 for Hcy and Met, 10-500 µmol/l-1 for Cys, and 1-50 ng/ml-1 for 5-methyltetrahydrofolate. Conclusion: An accurate and precise method that was rapid, robust and with high-throughput for the routine clinical monitoring of patients with coronary artery disease was developed.

Increased Sphingomyelin and Free Sialic Acid in Cerebrospinal Fluid of Kearns-Sayre Syndrome: New Findings Using Untargeted Metabolomics.

BACKGROUND: Kearns-Sayre syndrome (KSS) is caused by duplications and/or deletions of mitochondrial DNA (mtDNA) and is typically diagnosed based on a classic triad of symptoms with chronic progressive external ophthalmoplegia (CPEO), retinitis pigmentosa, and onset before age 20 years. The present study aimed to diagnose two patients, on suspicion of KSS. METHODS: One of the patients went through a diagnostic odyssey, with normal results from several mtDNA analyses, both in blood and muscle, before the diagnosis was confirmed genetically. RESULTS: Two patients presented increased tau protein and low 5-methyltetrahydrofolate (5-MTHF) levels in the cerebrospinal fluid (CSF). Untargeted metabolomics on CSF samples also showed an increase in the levels of free sialic acid and sphingomyelin C16:0 (d18:1/C16:0), compared with four control groups (patients with mitochondrial disorders, nonmitochondrial disorders, low 5-MTHF, or increased tau proteins). CONCLUSIONS: It is the first time that elevated sphingomyelin C16:0 (d18:1/C16:0) and tau protein in KSS are reported. Using an untargeted metabolomics approach and standard laboratory methods, the study could shed new light on metabolism in KSS to better understand its complexity. In addition, the findings may suggest the combination of elevated free sialic acid, sphingomyelin C16:0 (d18:1/C16:0), and tau protein as well as low 5-MTHF as new biomarkers in the diagnostics of KSS.

Infants' Folate Markers and Postnatal Growth in the First 4 Months of Life in Relation to Breastmilk and Maternal Plasma Folate.

BACKGROUND: Human milk is the sole source of folate in exclusively breastfed infants. We investigated whether human milk folate or maternal plasma folate are associated with infants' folate status and postnatal growth in the first 4 months of life. METHODS: Exclusively breastfed infants (n = 120) were recruited at age < 1 month (baseline). Blood samples were available at baseline and at the age of 4 months. Plasma and breastmilk samples were available from the mothers at 8 weeks postpartum. The concentrations of (6S)-5-methyltetrahydrofolate (5-MTHF) and different folate status markers were measured in samples of the infants and their mothers. The z-scores of weight, height, and head circumference of the infants were measured five times between baseline and 4 months. RESULTS: Women with 5-MTHF concentrations in breastmilk <39.9 nmol/L (median) had higher plasma 5-MTHF compared to those with milk 5-MTHF concentrations >39.9 nmol/L (mean (SD) plasma 5-MTHF = 23.3 (16.5) vs. 16.6 (11.9) nmol/L; p = 0.015). At the age of 4 months, infants of women who were higher suppliers of 5-MTHF in breastmilk had higher plasma folate than those of low-supplier women (39.2 (16.1) vs. 37.4 (22.4) nmol/L; adjusted p = 0.049). The concentrations of breastmilk 5-MTHF and maternal plasma folate were not associated with infants' longitudinal anthropometric measurements between baseline and 4 months. CONCLUSIONS: Higher 5-MTHF in breastmilk was associated with higher folate status in the infants and the depletion of folate in maternal circulation. No associations were seen between maternal or breastmilk folate and infants' anthropometrics. Adaptive mechanisms might counteract the effect of low milk folate on infant development.

Effects of folate on telomere length and chromosome stability of human fibroblasts and melanoma cells in vitro: a comparison of folic acid and 5-methyltetrahydrofolate.

Telomere length (TL), which is maintained by human telomerase reverse transcriptase (hTERT; component of telomerase) and/or TRF1/TRF2 (core components of shelterin) via different mechanisms, is essential for chromosomal stability and cell survival. Folates comprise a group of essential B9 vitamin that involve in DNA synthesis and methylation. This study aimed to evaluate the effects of folic acid (FA) and 5-methyltetrahydrofolate (5-MeTHF) on TL, chromosome stability, and cell survival of telomerase-negative BJ and telomerase-positive A375 cells in vitro. BJ and A375 cells were cultured in modified medium with FA or 5-MeTHF (22.6 or 2260 nM) for 28 days. TL and mRNA expression were determined by RT-qPCR. Chromosome instability (CIN) and cell death were measured by CBMN-Cyt assay. Results showed that abnormal TL elongation was observed in FA- and 5-MeTHF-deficient BJ cells. The TL of A375 cells showed no obvious alterations under the FA-deficient condition but was significantly elongated under the 5-MeTHF-deficient condition. In both BJ and A375 cells, FA and 5-MeTHF deficiency caused decreased TRF1, TRF2, and hTERT expression, increased CIN and cell death; while a high concentration of 5-MeTHF induced elongated TL, elevated CIN, increased TRF1 and TRF2 expression, and decreased hTERT expression, when compared with the FA counterpart. These findings concluded that folate deficiency induced TL instability in both telomerase-negative and -positive cells, and FA was more efficient in maintaining TL and chromosome stability compared with 5-MeTHF.

Infant blood concentrations of folate markers and catabolites are modified by 5,10-methylenetetrahydrofolate reductase C677T genotype and dietary folate source.

BACKGROUND: Folate intake and polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene may affect folate metabolism in infants. OBJECTIVES: We investigated the association between infant's MTHFR C677T genotype, the dietary folate source, and concentrations of folate markers in the blood. METHODS: We studied 110 breastfed infants (reference) and 182 infants who were randomly assigned to receive infant formulas enriched with either 78 µg folic acid or 81 µg (6S)-5-methyltetrahydrofolate (5-MTHF) per 100 g milk powder for 12 wk. The blood samples were available at the ages of <1 mo (baseline) and 16 wk. MTHFR genotype and concentrations of folate markers and catabolites [i.e., para-aminobenzoylglutamate (pABG)] were analyzed. RESULTS: At baseline, carriers of the TT genotype (vs. CC) had lower mean (SD) concentrations (all in nmol/L) of red blood cell (RBC) folate [1194 (507) vs. 1440 (521), P = 0.033) and plasma pABG [5.7 (4.9) vs. 12.5 (8.1), P < 0.001] but higher plasma 5-MTHF [33.9 (16.8) vs. 24.0 (12.6), P < 0.001]. Irrespective of the genotype, infant formula with 5-MTHF (vs. folic acid) caused a significant increase in RBC folate concentration [1278 (466) vs. 947 (552), P < 0.001]. In breastfed infants, plasma concentrations of 5-MTHF and pABG increased significantly by 7.7 (20.5) and 6.4 (10.5), respectively, from baseline to 16 wk. Infant formula that complies with the present EU legislation for folate intake increased RBC folate and plasma pABG concentrations at 16 wk (P < 0.001) than formula-fed infants. At 16 wk, plasma pABG concentrations remained ∼50% lower in carriers of the TT (vs. the CC) genotype among all feeding groups. CONCLUSIONS: Folate intake from infant formula according to the present EU legislation increased RBC folate and plasma pABG concentrations in infants to a greater extent than breastfeeding, particularly in carriers of the TT genotype. However, this intake did not completely abolish the between-genotype differences in pABG. Whether these differences have any clinical relevance, however, remains unclear. This trial was registered at clinicaltrials.gov as NCT02437721.

A sensitive, robust and high-throughput isotope dilution LC-MS/MS method for quantifying three folate forms in serum.

This study aimed to establish an isotope dilution LC-MS/MS method for the determination of folic acid, 5-formyltetrahydrofolate and 5-methyltetrahydrofolate in human serum. This method was then used to quantify these three folate forms in the healthy adult population and supplement users. A stable 96-well solid-phase extraction system was used to prepare serum samples. The highly sensitive method was established using a Shimadzu LCMS-8060NX. The linearity was good in the range of 0.1-10 nmol/l for folic acid and 5-formyltetrahydrofolate and 1.0-100 nmol/l for 5-methyltetrahydrofolate. The accuracy and precision were good. The method was sensitive, robust and high-throughput and could be used for the routine clinical monitoring of these three folate forms in the Chinese population.

Effects of folate on telomere length and chromosome stability of human fibroblasts and melanoma cells in vitro: a comparison of folic acid and 5-methyltetrahydrofolate.

Telomere length (TL), which is maintained by human telomerase reverse transcriptase (hTERT; component of telomerase) and/or TRF1/TRF2 (core components of shelterin) via different mechanisms, is essential for chromosomal stability and cell survival. Folates comprise a group of essential B9 vitamin that involve in DNA synthesis and methylation. This study aimed to evaluate the effects of folic acid (FA) and 5-methyltetrahydrofolate (5-MeTHF) on TL, chromosome stability, and cell survival of telomerase-negative BJ and telomerase-positive A375 cells in vitro. BJ and A375 cells were cultured in modified medium with FA or 5-MeTHF (22.6 or 2260 nM) for 28 days. TL and mRNA expression were determined by RT-qPCR. Chromosome instability (CIN) and cell death were measured by CBMN-Cyt assay. Results showed that abnormal TL elongation was observed in FA and 5-MeTHF deficient BJ cells. The TL of A375 cells showed no obvious alterations under the FA-deficient condition but was significantly elongated under the 5-MeTHF-deficient condition. In both BJ and A375 cells, FA and 5-MeTHF deficiency caused decreased TRF1, TRF2, and hTERT expression, increased CIN and cell death; while a high concentration of 5-MeTHF induced elongated TL, elevated CIN, increased TRF1 and TRF2 expression and decreased hTERT expression, when compared with the FA counterpart. These findings concluded that folate deficiency induced TL instability in both telomerase-negative and -positive cells, and FA was more efficient in maintaining TL and chromosome stability compared with 5-MeTHF.

Pre-analytical conditions influencing analysis of folate in dried plasma microsamples.

Introduction: Determination of folate insufficiency is of considerable interest given its importance in fetal development and red blood cell formation; however, access to blood tests may be limited due to the requirement for phlebotomy as well as controlled temperature shipping of blood specimens to laboratories for testing due to the inherent instability of folate and its vitamers. Methods: An LC-MS/MS test was developed and validated for the measurement of 5-methyltetrahydrofolate (5MTHF) in dried plasma specimens collected from fingerstick blood using a laminar flow blood separation device, as well as liquid venous plasma for comparison. Two pre-analytical factors investigated influencing the measurement of 5MTHF in dried plasma were hemolysis of the fingerstick blood during collection and storage/shipment of the dried plasma. Results: Although observed infrequently, hemolysis >10 % resulted in elevated 5MTHF measurements, but hemolysis >1 % resulted in elevated chloride measurements, which were necessary to normalize 5MTHF measurements for variation in volume of dried plasma specimens. Stability of 5MTHF was improved in dried plasma relative to liquid plasma at ambient temperatures, but not sufficiently to allow for uncontrolled temperature shipping despite controlling for humidity and light exposure. Shipping studies emulating ISTA procedure 7D were conducted with a reusable cold packaging solution. The packaging failed to stabilize 5MTHF in dried plasma specimens during a 2-day summer shipping evaluation, but did provide sufficient temperature control to stabilize 5MTHF during the overnight shipping evaluation. Conclusion: Our studies provide boundary conditions with respect to hemolysis, storage, and shipping for successful analysis of 5MTHF from dried plasma specimens.

Associations of different serum folate forms with indices of nonalcoholic fatty liver disease and advanced fibrosis.

OBJECTIVE: We aim to examine the associations of different serum folate forms (total folate, 5-methyltetrahydrofolate [5-mTHF] and unmetabolized folic acid [UMFA]), with the prevalence of nonalcoholic fatty liver disease (NAFLD) and advanced fibrosis. METHODS: This cross-sectional analysis was conducted in 6610 participants aged ≥ 18 years from the 2011-2018 National Health and Nutrition Examination Survey (NHANES) database. NAFLD was defined as a United States fatty liver index (USFLI) ≥ 30. Advanced fibrosis was defined as a Fibrosis-4 score (FIB-4) > 3.25, a NAFLD Fibrosis Score (NFS) > 0.676, and a Hepamet Fibrosis Score (HFS) ≥ 0.47, respectively. RESULTS: The prevalence of NAFLD was 34.5%. Overall, serum total folate and 5-mTHF were inversely associated with the prevalence of NAFLD (both P for trend across quartiles <0.001). A similar trend was found for advanced fibrosis based on NFS and HFS (both P for trend across quartiles <0.05). However, a higher concentration of UMFA was significantly related to a higher prevalence of NAFLD (P for trend across quartiles =0.004). A similar relation was found for advanced fibrosis based on NFS (P for trend across quartiles =0.024). CONCLUSIONS: Higher concentrations of serum total folate and 5-mTHF were associated with a lower prevalence of NAFLD and advanced fibrosis, while a higher concentration of UMFA was related to a higher prevalence of NAFLD.

Evaluation of Cardiovascular Toxicity of Folic Acid and 6S-5-Methyltetrahydrofolate-Calcium in Early Embryonic Development.

Folic acid (FA) is a synthetic and highly stable version of folate, while 6S-5-methyltetrahydrofolate is the predominant form of dietary folate in circulation and is used as a crystalline form of calcium salt (MTHF-Ca). The current study aims to evaluate the toxicity and safety of FA and MTHF-Ca on embryonic development, with a focus on cardiovascular defects. We began to analyze the toxicity of FA and MTHF-Ca in zebrafish from four to seventy-two hours postfertilization and assessed the efficacy of FA and MTHF-Ca in a zebrafish angiogenesis model. We then analyzed the differently expressed genes in in vitro fertilized murine blastocysts cultured with FA and MTHF-Ca. By using gene-expression profiling, we identified a novel gene in mice that encodes an essential eukaryotic translation initiation factor (Eif1ad7). We further applied the morpholino-mediated gene-knockdown approach to explore whether the FA inhibition of this gene (eif1axb in zebrafish) caused cardiac development disorders, which we confirmed with qRT-PCR. We found that FA, but not MTHF-Ca, could inhibit angiogenesis in zebrafish and result in abnormal cardiovascular development, leading to embryonic death owing to the downregulation of eif1axb. MTHF-Ca, however, had no such cardiotoxicity, unlike FA. The current study thereby provides experimental evidence that FA, rather than MTHF-Ca, has cardiovascular toxicity in early embryonic development and suggests that excessive supplementation of FA in perinatal women may be related to the potential risk of cardiovascular disorders, such as congenital heart disease.

Folate vitamers in the Australian green plum: Through growth and ripening and across locations.

The green plum is a native fruit of Australia that grows on the tree Buchanania obovata. This study aimed to confirm the high level of folate in green plums by analyzing a large number of ripe samples from multiple locations and to understand how folate vitamers change as the fruit grows through maturity stages. This study analyzed green plums for five vitamers of folate, H4folate, 5-CH3-H4folate, 5-CHO-H4folate, 10-CHO-PteGlu, and PteGlu (folic acid) using a stable isotope dilution assay on a liquid chromatograph mass spectrometer (LC-MS). Green plums were tested from four locations, two harvests and five maturity stages. Another 11 ripe samples, each from different tree clumps from one location, were also tested as were ripe red-colored green plums. The results show the 5-CH3-H4folate in green plum increases and accumulates in the fruit through development, ripening and senescence. The ripe green plums contain between 82.4 ± 5.5 and 149.4 ± 10.7 µg/100 g Fresh Weight (FW). The red-colored green plums are even higher in folate, with total folate measured as 192.5 ± 7.0 and 293.7 ± 27.4 µg/100 g FW, and further analysis of them is suggested. There is some variation in amounts of folate between fruit from different locations and sets of trees, but all ripe green plums tested are considered good dietary sources of folate.