Developing a Sensitive Platform to Measure 5-Methyltetrahydrofolate in Subjects with MTHFR and PON1 Gene Polymorphisms.

Inadequate levels of 5-methyltetrahydrofolate (5-MTHF) and the T variant of MTHFR C677T have been suggested to be associated with an increased risk of developing mental illness, whereas the PON1 SNP variant provides a protective role. However, reports validating the methodology for plasma 5-MTHF levels in schizophrenia patients are limited. A sensitive LC−MS/MS system using an amide column and calibration curve was determined by dialyzed human plasma, and applied to schizophrenia patients and healthy controls in Taiwan, and the differences between the subgroups were discussed. This analysis system meets regulation criteria, and the lower limit of quantification for 5-MTHF levels was 4 nM from 200 µL plasma, within 7 min. The mean plasma 5-MTHF levels in schizophrenia patients (n = 34; 11.70 ± 10.37 nM) were lower than those in the healthy controls (n = 42; 22.67 ± 11.12 nM) significantly (p < 0.01). 5-MTHF concentrations were significantly lower in male carriers than in female carriers (18.30 ± 10.37 nM vs. 24.83 ± 11.01 nM, p < 0.05), especially in subjects who were MTHFR CT/PON1 Q allele carriers. In conclusion, this quantitative system, which employed sensitive and simple processing methods, was successfully applied, and identified that schizophrenic patients had significantly lower levels of 5-MTHF. Lower plasma 5-MTHF concentrations were observed in male subjects.

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.

The bioaccessibility of folate in breads and the stability of folate vitamers during in vitro digestion.

Both the liberation and stability of endogenous folate are relevant to the bioaccessibility of folate. Since folates are unstable, in addition to studying the natural folate content in foods, bioaccessibility should be considered. To understand folate changes during digestion, a mixture of standard folate compounds was subjected to a static in vitro gastrointestinal digestion assay. Next, different types of bread were analysed to study how food matrices influence folate bioaccessibility. Folates were identified and quantitated by a UHPLC-PDA/FL method. Folic acid and 10-formylfolic acid were stable throughout the digestion, and the conversions among formyl folates and 5,10-methenyltetrahydrofolate were triggered at the gastric phase. Tetrahydrofolate began to degrade during the oral phase and was lost completely during the gastric phase. During the intestinal phase, 5-methyltetrahydrofolate began to degrade and suffered a 60% loss. With bread matrices, folate conversions and the decrease of reduced folates were also common, but the extent of changes varied. Generally, rye breads had the highest (80-120%) bioaccessibility of folate, while oat breads had the lowest (31-102%). The high proportion of 5-methyltetrahydrofolate could result in low bioaccessibility because of its relatively low stability during digestion in bread matrices. An increase in 10-formylfolic acid content was observed for all the breads, but 10-formyldihydrofolate seemed to be more stable in rye breads than in oat and wheat breads. The results showed that folates undergo significant changes during digestion and that food matrices could be modified to affect these changes towards better folate bioaccessibility.

Red light enhances folate accumulation in wheat seedlings.

Red, white, blue, green, and yellow lights were applied to investigate their effects on folate accumulation in wheat seedlings. The different lights, especially red light, significantly increased the total folate content. Total folate showed maximum accumulation under 30 µmol/(m2·s) of red light, with an increase of 24% compared with the control (darkness). 5-Methyl-tetrahydrofolate (5-CH3-THF) was the dominant folate component, and was significantly increased by red light irradiation. In addition, under red light, the folate content of leaves was higher and more sensitive to light than that of endosperm or roots. Red light up-regulated the expression of guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1) and aminodeoxychorismate synthase(ADCS), enhanced the activity of GCH1 and ADCS, and increased the content of precursors of folate synthesis, including pterin and p-aminobenzoic acid (pABA). Hence, the increased folate accumulation promoted by light could be attributed to the increased content of folate synthesis precursors, the activity of key enzymes, and related gene expression.

Determination of the effect of germination on the folate content of the seeds of some legumes using HPTLC-mass spectrometry-multivariate image analysis.

Legumes are the main sources of folates which are not synthesized in the human body. The five folate species: 5-methyl tetrahydrofolate, tetrahydrofolate, pteroyl glutamate, 5-formyl tetrahydrofolate and 10-formyl tetrahydrofolate were quantitatively determined in legumes seeds and sprouts by a newly developed and validated high performance thin layer chromatography method. High resolution plate imaging hyphenated to mass spectrometry was exploited for fingerprint analysis of tested samples. Results indicated that germination of all seeds resulted in a 2.5-4 fold increase in the content of total folates as well as the individual vitamers. The total amount of folate reached a maximum on the fifth day in the case of black-eyed peas (861 µg/100 g Fresh Weight), white beans (755 µg/100 g FW) and brown lentils (681 µg/100 g FW). 5-CH3-H4 folate was found to be the most dominating folate species reaching its maximum content in day 5 sprouts of black-eyed peas (490 µg/100 g FW).

A novel electrochemical paper sensor for low-cost detection of 5-methyltetrahydrofolate in egg yolk.

An electrochemical deposition method was used to fabricate a gold nanoflower (AuNF) and carbon nanoparticle (CNP) modified carbon paper (CP) sensor (AuNFs-CNPs/CP) for the low-cost detection of 5-methyltetrahydrofolate (5-mTHF) in egg yolk. AuNF morphology and structures were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), revealing nanoflower sizes in the 50 to 200 nm range. AuNFs formed on the sensor were in the Au0. We evaluated 5-mTHF assay performance using cyclic voltammetry, differential pulse voltammetry and chronoamperometry. The AuNFs-CNPs/CP sensor detected 5-mTHF concentrations in the ranges from 1 to 5 mg L-1 and 1-20 µg L-1, with an excellent limit of detection of 1 µg L-1 and good selectivity toward 5-mTHF, when compared to other potentially interfering molecules in samples. The AuNFs-CNPs/CP sensor was also used to detect 5-mTHF in folate-rich, and was found to be twice than that of ordinary egg yolk.

Modelling folates reaction kinetics during cowpea seed germination in comparison with soaking.

Folate is a fundamental vitamin for metabolism in plants and humans. A modelling approach has been developed to characterize the reactivity of folates in cowpea seeds during germination at 30 °C, using a water-to-seed ratio of 1:1 (w/w). For this purpose, the concentrations of folic acid, 10-formylfolic acid, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate and tetrahydrofolate were determined in seeds during germination times up to 96 h. Two reaction models were sequentially built and adjusted to experimental data to describe changes in concentration in cowpea seed during two germination phases: before 14 h and after 48 h. Results showed intense enzymatic interconversion of all folate vitamers into 5-methyltetrahydrofolate before 14 h of germination and high enzymatic production of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate and tetrahydrofolate after 48 h of germination. This study suggests that a long germination process could be more beneficial than soaking to increase the production of bioavailable folates within the seed for human consumption.

Modified EMR-lipid method combined with HPLC-MS/MS to determine folates in egg yolks from laying hens supplemented with different amounts of folic acid.

Egg yolks are a good source of folates. However, the method for analyzing the naturally occurring folates in egg yolks is complicated and time-consuming. In this study, a simplified pre-treatment method followed by validated HPLC-MS/MS was developed to determine native folates in eggs from laying hens treated with different amounts of folic acid. The modified enhanced matrix removal -lipid method to purify samples showed good performance in lipid elimination, reduction of steps and time savings. According to experimental analysis, yolks contained total folate amounts ranging from 147 to 760 µg/100 g when laying hens' diet was supplemented with folic acid from 0 to 10 mg/kg. Four folate vitamers were detected in egg yolks: 5-methyltetrahydrofolate accounted for 91-98% of total folates, whereas folic acid, 5-formyltetrahydrofolate and 10-formylfolic acid together accounted for 2-9%. Therefore, laying hens efficiently converted folic acid in feed into 5-methyltetrahydrofolate in eggs with little folic acid deposition.

Simultaneous quantification of intracellular concentrations of clinically important metabolites of folate-homocysteine cycle by LC-MS/MS.

Inadequate folate status is detrimental to human development. Deficiency has been implicated in congenital birth defects and cancer, whereas excess has been linked to various negative neurocognitive development outcomes. We developed a method for translational studies involving lymphoblastoid cell models for studying role of folates in vital cell processes. We describe a simple, sensitive, and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of intracellular concentrations of clinically important metabolites of folate-homocysteine cycle; namely, folic acid (FA), 5-methyltetrahydrofolate (5-Me-THF), and homocysteine (Hcy). The method was validated for specificity, linearity, limits of quantification, repeatability, reproducibility, matrix effects, and stability. Method had a wide linear range between 0.341 and 71.053 ng Hcy/mg protein for Hcy, 0.004-0.526 ng FA/mg protein for FA and 0.003-0.526 ng 5-Me-THF/mg protein for 5-Me-THF. The method overcomes challenges associated with the quantification of endogenous molecules, poor stability, and extremely small amounts of the analytes. The method was successfully applied to evaluate the effects of FA and 5-Me-THF treatment of cells in vitro mimicking supplement therapy with various metabolically active species, and showed that 5-Me-THF is more effective than FA in increasing intracellular levels of the biologically active form of folate.

Serum Folate Forms Are Stable during Repeated Analysis in the Presence of Ascorbic Acid and during Frozen Sample Storage.

BACKGROUND: Serum folate forms, and particularly tetrahydrofolate, are sensitive to oxidation. METHODS: Using a repeated measures design, we investigated the stability of folate forms in convenience samples with added ascorbic acid (AA; 5 g/L) analyzed initially and after variable (approximately 1-33 weeks) storage time at -70 °C. We examined the recovery of tetrahydrofolate added at different spiking levels to serum with and without AA (5 g/L). We also assessed the long-term frozen storage stability of folate forms. RESULTS: Repeat analysis produced consistent results with the initial analysis; the mean relative change (95% CI; Lin's concordance correlation between initial and repeat result; sample size) was 0.08% (-0.24% to 0.39%; r c = 0.999; n = 301) for 5-methyltetrahydrofolate, 4.23% (2.44%-6.05%; r c = 0.984; n = 211) for pyrazino-s-triazine derivative of 4α-hydroxy-5-methyltetrahydrofolate (MeFox), -0.22% (-1.90% to 1.49%; r c = 0.986; n = 214) for folic acid, and 1.49% (-2.71% to 5.88%; r c = 0.889; n = 81) for tetrahydrofolate. Linear regression testing for a time trend indicated an estimated average percent change of less than ±5% for samples retested after 4 months: 5-methyltetrahydrofolate P trend = 0.0007, folic acid P trend < 0.0001, MeFox P trend = 0.38, and tetrahydrofolate P trend = 0.0256. The mean ± SD tetrahydrofolate spiking recovery was 96.7% ± 9.4% for serum with added AA, but <50% for serum without added AA. We observed ≤10% loss for most serum folate forms during 4 years of storage at -70 °C. CONCLUSIONS: Serum containing added AA showed acceptable stability of folate forms during repeat analysis from the same vial within 4 months, complete spiking recovery of tetrahydrofolate during sample processing, and long-term frozen storage stability of folate forms.

Handling and Analysis of 5-Formyl-, 5,10-Methenyl-, 10-Formyl-, 5-Formimno-, and 5,10-Methylenetetrahydrofolates.

As the principal one-carbon carriers in mammalian biology, tetrahydrofolates are crucial for normal and malignant cells to synthesize and repair DNA and are the target of extensive research, including metabolomics analysis. The susceptibility of tetrahydrofolates to oxidization, as well as the propensity of substituted tetrahydrofolates to chemical degradation, mandates the use of carefully controlled experimental conditions to ensure their integrity. Analytical protocols for LC analysis along with handling and storage conditions for 5-formyl-, 5,10-methenyl-,10-formyl-, 5-formimno-, and 5,10-methylenetetrahydrofolate are described.

Simultaneous extraction and determination of mono-/polyglutamyl folates using high-performance liquid chromatography-tandem mass spectrometry and its applications in starchy crops.

Folates are typically present in polyglutamyl form in organisms. In traditional extraction methods, polyglutamyl folates are hydrolyzed to monoglutamates, sacrificing valuable information. To advance folate metabolism research, we developed an accurate, sensitive, and reproducible extraction method for polyglutamyl folate species in maize, the main crop in most parts of the world. Twelve folates, including six polyglutamyl folates, were simultaneously determined in maize for the first time using high-performance liquid chromatography-tandem mass spectrometry. The glutamation states of the folates were protected by boiling, which inactivated the native conjugases. α-Amylase and protease were added to obtain better recoveries and decrease difficulties in centrifugation and filtration. The recoveries (n = 5) of six polyglutamyl folates were between 80.5 and 101%. All calibration curves showed good linear regression (r2 ≥ 0.994) within the working range. The instrumental limits of detection and quantitation ranged from 0.070 to 2.4 ng/mL and 0.22 to 8.0 ng/mL, respectively. Intra- and inter-day precision was below 7.81% and 11.9%, respectively (n = 5). Using this method, changes in poly- and monoglutamyl folates during maize germination were determined for the first time. The results suggest that folates were largely synthesized as germination initiated, and 5-methyltetrahydrofolate was the most abundant species. Tetraglutamyl 5-methyltetrahydrofolate contributed more than 50% of the 5-methyltetrahydrofolate species. Inverse changes in contents of 5,10-methenyltetrahydrofolate, and 10-formyl folic acid, monoglutamate, and diglutamate of 5-formyltetrahydrofolate were also observed, indicating potential regulation. Additionally, polyglutamyl folates in sweet potatoes were determined using this method, indicating its applications in starchy crops.

The use of a plant enzyme for rapid and sensitive analysis of naturally-occurring folates in food by liquid chromatography-tandem mass spectrometry.

A rapid, sensitive and reproducible method for analysis of naturally-occurring folates and folic acid in food has been developed and validated. A single-enzyme extraction step, in which a pure recombinant enzyme of plant origin (Arabidopsis thaliana) was used, enabled fast and reproducible deglutamylation during folate extraction within the incubation time of 1 h. Six commonly occurring folate forms (tetrahydrofolate, 5,10-methenyltetrahydrofolate, 10-formylfolic acid, 5-formyltetrahydrofolate, folic acid and 5-methyltetrahydrofolate) were detected and quantified in 9 min using liquid chromatography-tandem mass spectrometry (LC-MS/MS). 13C5-labeled 5-formyltetrahydrofolate, 13C5-labeled folic acid and 13C5-labeled 5-methyltetrahydrofolate were used as internal standards for the quantification. The method is described by a calibration curve (R2>0.99 and trueness 85-115%), a limit of quantification at 0.1 µg/100 g, trueness at 80-120% in spiked samples and certified reference materials, and a precision <10%. However, the precision in quantification of tetrahydrofolate was not within the acceptable limits due to the lack of use of the corresponding internal standard. An interconversion study of unstable formyl forms was performed which showed that 50% of 5,10-methenyltetrahydrofolate is converted to 5-formyltetrahydrofolate during the analysis. The developed LC-MS/MS method is a candidate for a future standard method for folate analysis in food.

Quantification of polyglutamyl 5-methyltetrahydrofolate, monoglutamyl folate vitamers, and total folates in different berries and berry juice by UHPLC-MS/MS.

Edible berries are good sources of several phytochemicals. However, the native folate levels in berries are not well known. The structure of native folates contains polyglutamyl chains, which reportedly jeopardize the bioavailability of native folates; further γ-glutamyl hydrolase (GGH) can deglutamylate polyglutamyl chains. In this study we use a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine the distribution of polyglutamyl 5-methyltetrahydrofolate (5-CH3THF-Glun), folate vitamers, and total folates in different berries and to monitor changes in their concentration during processing of berries into juice. The pre-boiling treatment was optimized during extraction to stabilize the native polyglutamyl folates profile and facilitate folate extraction, which can replace the traditional di-enzyme treatment process. Additionally, the efficiency of commercially available human recombinant GGH was tested and it was found that a 10 µg GGH/mL extract at a pH of 6 could completely deconjugate polyglutamyl folates into monoglutamyl folates when incubated for 30 min. Pure human recombinant GGH with a higher catalytic efficiency and stable enzymatic properties was better than traditional folate conjugase for this purpose. From experimental analysis, it could be inferred that strawberries and blackberries contained the highest amount of total folates (93-118 µg/100 g), while the total folate contents in blueberries were the lowest. Most of the investigated berries are good to excellent folate sources. This study is the first time that 5-CH3THF-Glun and distribution of folate vitamers in various berries are quantitated. Further, this is the first study to show the application of recombinant pure GGH for the deconjugation of polyglutamyl folates for folate vitamers and total folates analysis.

The contribution of minor folates to the total vitamin B9 content of Infant formula and clinical nutrition products.

In the present study an optimization of trienzyme treatment combining α-amylase, protease and γ-carboxy peptidase allowing complete sample preparation within a working day for the analysis of vitamin B9 (folate) in infant formula and adult/pediatric nutritional products is presented. The optimized sample preparation was applied to a set of samples representing most of the products in the marketplace. Results on Standard Reference Material 1849a were well in agreement with certified values. The main contributor to total folate was folic acid, 5-methyl-tetrahydrofolate was the only minor contributor in milk-based products. Soy-based formulas contained polyglutamates of 5-formyl-tetrahydrofolate. The relative contribution of polyglutamates to the total folate content remained low in the types of product included in this study. The results suggest that a simple di-enzyme treatment could be enough for these products, nevertheless, this should be carefully evaluated prior to making a decision on the use of tri- or di-enzyme treatment.

Targeted versus untargeted omics - the CAFSA story.

BACKGROUND: In 2009, untargeted metabolomics led to the delineation of a new clinico-biological entity called cerebellar ataxia with elevated cerebrospinal free sialic acid, or CAFSA. In order to elucidate CAFSA, we applied sequentially targeted and untargeted omic approaches. METHODS AND RESULTS: First, we studied five of the six CAFSA patients initially described. Besides increased CSF free sialic acid concentrations, three patients presented with markedly decreased 5-methyltetrahydrofolate (5-MTHF) CSF concentrations. Exome sequencing identified a homozygous POLG mutation in two affected sisters, but failed to identify a causative gene in the three sporadic patients with high sialic acid but low 5-MTHF. Using targeted mass spectrometry, we confirmed that free sialic acid was increased in the CSF of a third known POLG-mutated patient. We then pursued pathophysiological analyses of CAFSA using mass spectrometry-based metabolomics on CSF from two sporadic CAFSA patients as well as 95 patients with an unexplained encephalopathy and 39 controls. This led to the identification of a common metabotype between the two initial CAFSA patients and three additional patients, including one patient with Kearns-Sayre syndrome. Metabolites of the CSF metabotype were positioned in a reconstruction of the human metabolic network, which highlighted the proximity of the metabotype with acetyl-CoA and carnitine, two key metabolites regulating mitochondrial energy homeostasis. CONCLUSION: Our genetic and metabolomics analyses suggest that CAFSA is a heterogeneous entity related to mitochondrial DNA alterations either through POLG mutations or a mechanism similar to what is observed in Kearns-Sayre syndrome.

Identification and determination of naturally occurring folates in grains of rice (Oryza sativa L.) by UPLC-MS/MS analysis.

The genetic potential and biofortification of India-grown rice with bioavailable folate has not been studied yet. The objectives of this study were to determine the folates concentration in four cultivars of rice through UPLC-MS/MS. Total folate concentration in rice cultivars ranged from 11.0 to 51 µg/100 g with a mean of 26.0 µg/100 g. Among the four rice cultivars, the pigmented grain cultivar Nootripathu possesses two-fold rich sources of total folates than the other three non-pigmented grain cultivars. The average value of 100 g serving of rice grains could provide the amount of recommended daily allowance (% RDA) of dietary folates (6.5%) for adults, which ranged from 2.7-12.7%. Among the 5 individual forms of folates, 5-methyltetrahydrofolate was most abundant in rice cultivars followed by 10-Formylfolic acid and folic acid. The result of this study has been useful for biofortification of folates in rice.

Quantitative analysis of tetrahydrofolate metabolites from clostridium autoethanogenum.

INTRODUCTION: Quantification of tetrahydrofolates (THFs), important metabolites in the Wood-Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen. OBJECTIVE: To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors. METHODS: Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC-MS/MS was used for quantification. RESULTS: Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP. CONCLUSION: Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate.

The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells. Graphical abstract Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.

Development of a lateral flow dipstick immunoassay for evaluation of folate levels in maize.

Folates (vitamin B9) are essential for all organisms as cofactors for one-carbon metabolism. However, measurement of folates is technically complicated and time-consuming. In this study, we developed a dipstick immunoassay using a folate-specific monoclonal antibody (mAb), allowing rapid and low-cost detection of folates. The indicator range of the dipstick for 5-formylterahydrofolate (5-CHO-THF), 5-methyltetrahydrofolate (5-CH3-THF) and their polyglutamyl forms was 100-200 ng mL-1; moreover, no cross-reactivity was observed with tetrahydrofolate (THF) or 5,10-methenyltetrahydrofolate (5,10-CH=THF) at 500 ng mL-1, or with the folate precursors pterin-6-COOH, p-aminobenzoate (pABA), and L-glutamate, or with the folate analogues methotrexate and 10-formyltetrahydrofolate (10-CHO-THF) at up to 1000 ng mL-1. The dipstick immunoassay was tested in maize seeds; the results classified the seeds into those with low, moderate, and high levels of folates, and were in agreement with those of liquid chromatography-mass spectrometry. Thus, we conclude that the dipstick assay will provide a versatile tool to facilitate large-scale screening of maize rich in folates. Graphical Abstract The dipstick based immunoassay for analyzing folate level in maize.