Highly sensitive analytical method for the accurate determination of 5-methyltetrahydrofolic acid monoglutamate in various volumes of human plasma using isotope dilution ultra-high performance liquid chromatography-mass spectrometry.

One predominant and bioactive folate vitamer circulating in the blood is 5-methyltetrahydrofolate (5-Me-THF). In this study, a method for the accurate determination of 5-Me-THF in human plasma samples of various volumes was established using isotope dilution ultra-high performance liquid chromatography-mass spectrometry (ID-UPLC-MS). For this purpose, 500 µL of homogeneous human plasma was initially employed, and the 5-Me-THF and the 13C5-5-Me-THF standard solutions prepared using 1% ascorbic acid in water gave the calibration solution and spiking sample. The desired amount of 13C5-5-Me-THF standard solution was spiked into the sample followed by sample pretreatment. The method was validated for its repeatability, reproducibility, recovery, and limits of detection and quantification. Subsequently, it was applied to smaller volumes of human plasma samples (i.e., 50 and 10 µL), the results of which corresponded well with those obtained using 500 µL. The feasibility of the method was further confirmed using 10 µL of a standard reference material, SRM 3949, which is a human serum sample containing three different levels of 5-Me-THF. The established ID-UPLC-MS method was successfully applied to various volumes of human plasma or serum ranging from 500 to 10 µL, which exhibited particularly good sensitivity in addition to reliable results for the quantification of 5-Me-THF. Our method therefore expands on the ability to obtain accurate quantitative results for 5-Me-THF using small volumes of blood.

Relationship of several serum folate forms with the risk of mortality: A prospective cohort study.

OBJECTIVE: We aim to examine the relation of several folate forms (5-methyltetrahydrofolate [5-mTHF], unmetabolized folic acid [UMFA], non-methyl folate, and MeFox [pyrazino-s-triazine derivative of 4α-hydroxy-5-methyltetrahydrofolate]) with the risk of mortality. METHODS: Using data from National Health and Nutrition Examination Survey 2011-2014, a total of 10,661 people with folate forms data were recruited. Death information was obtained from the National Death Index until 2015. Cox proportional hazards regression models were developed to evaluate the relationship between folate forms and mortality. RESULTS: During 2.99 years of follow-up, 344 (2.6%) deaths occurred. Overall, significantly higher risks of all-cause mortality were found in participants with higher level of serum 5-mTHF (≥51.3 nmol/L [quartile 4] vs. 23.9-51.3 nmol/L [quartile 2-3]; HR, 1.61; 95% CI: 1.03-2.53), UMFA (≥1.1 nmol/L [quartile 4] vs. <1.1 nmol/L [quartile 1-3]; HR, 1.55; 95% CI: 1.15-2.09), non-methyl folate (≥1.7 nmol/L [quartile 4] vs. 1.2-1.7 nmol/L [quartile 3]; HR, 1.62; 95% CI: 1.06-2.48), or MeFox (≥2.5 nmol/L [quartile 4] vs. <2.5 nmol/L [quartile 1-3]; HR, 1.54; 95% CI: 1.11-2.12). In addition, there was an increased risk of all-cause mortality for those with low level of serum 5-mTHF (<23.9 nmol/L [quartile 1] vs. 23.9-51.3 nmol/L [quartile 2-3]; HR, 1.66; 95% CI: 1.12-2.47). Most importantly, none of any folate forms significantly modified the association between other folate forms and mortality (all P for interactions >0.05). CONCLUSION: Higher levels of serum folate forms (5-mTHF, UMFA, non-methyl folate, and MeFox) were associated with higher risk of mortality while 5-mTHF insufficiency also showed a negative impact on mortality. Our findings emphasized the importance of monitoring the folate forms concentrations and may help counsel future related clinical trials.

Cross-sectional association between vitamin B12 status and probable postpartum depression in Indian women.

BACKGROUND: Vitamin B12 is an essential micronutrient for neurological function, as it leads to the regeneration of methionine from homocysteine, which is precursor of biologically active molecule S-Adenosyl Methionine (SAM). Pregnancy is a state of increased demand and delayed postpartum repletion of nutrients may predispose women to depression. METHODS: We included women who visited the hospital at 6-weeks postpartum for a regular checkup. Inclusion criteria were age (18-50 years), and willingness to donate venous sample for analysis. Exclusion criteria included previous history of mood disorders or antidepressant medication use, and any systemic illness like hypothyroidism, epilepsy, diabetes, and hypertension. Based on EPDS score of 10 as a cutoff, 217 women with probable postpartum depression (PPD) and equal number of age and BMI matched controls were included. Plasma total vitamin B12, holotranscobalamin (holotc), homocysteine (hcy), methyl malonic acid (MMA), 5-methyl tetrahydrofolate (THF), SAM and serotonin levels were estimated using commercially available ELISA kits. Combined B12 (cB12) score was calculated from study parameters. Multivariate analysis was performed to assess the risk of probable postpartum depression. RESULTS: Total vitamin B12 and combined B12 score were found to be significantly lower (p = 0.001) and MMA (p = 0.002) and 5-methyl THF (p < 0.001) levels were higher in women with probable depression than women without probable PPD. Women in the lowest vitamin B12 quartile had 4.53 times higher likelihood of probable postpartum depression (p < 0.001). Multivariate analysis demonstrated that decreasing vitamin B12 (OR = 0.394; 95% CI: 0.189-0.822) and cB12 (OR = 0.293; 95% CI: 0182-0.470) and increasing MMA (OR = 2.14; 95% CI: 1.63-2.83) and 5-methyl THF levels (OR = 3.29; 95% CI: 1.59-6.83) were significantly associated with the risk of probable PPD. CONCLUSION: Low vitamin B12 may contribute to depressive symptoms in vulnerable postpartum period.

Pharmacokinetics of Sodium and Calcium Salts of (6S)-5-Methyltetrahydrofolic Acid Compared to Folic Acid and Indirect Comparison of the Two Salts.

(6S)-5-Methyltetrahydrofolic acid ((6S)-5-Methyl-THF) salts and folic acid may differ in their abilities to raise plasma (6S)-5-Methyl-THF levels. We compared the area under the curve (AUC), Cmax, and Tmax of plasma (6S)-5-Methyl-THF after intakes of (6S)-5-Methyl-THF-Na salt (Arcofolin®) and folic acid. Moreover, we compared the AUCs after intakes of (6S)-5-Methyl-THF-Na and the calcium salt, (6S)-5-Methyl-THF-Ca, that were tested against folic acid in two independent studies. The study was randomized, double blind, and cross over. Twenty-four adults (12 men and 12 women) received a single oral dose of 436 µg (6S)-5-Methyl-THF-Na and an equimolar dose of folic acid (400 µg) on two kinetic days with two weeks washout period in between. The plasma concentrations of (6S)-5-Methyl-THF were measured at 9 time points between 0 and 8 h. We found that the AUC0-8 h of plasma (6S)-5-Methyl-THF (mean (SD) = 126.0 (33.6) vs. 56.0 (25.3) nmol/L*h) and Cmax (36.8 (10.8) vs. 11.1 (4.1) nmol/L) were higher after administration of (6S)-5-Methyl-THF-Na than after the administration of folic acid (p < 0.001 for both). These differences were present in men and women. Only administration of folic acid resulted in a transient increase in plasma unmetabolized folic acid (2.5 (2.0) nmol/L after 0.5 h and 4.7 (2.9) nmol/L after 1 h). Intake of (6S)-5-Methyl-THF-Na was safe. The ratios of the AUC0-8 h for (6S)-5-Methyl-THF-Na and (6S)-5-Methyl-THF-Ca to the corresponding folic acid reference group and the delta of these AUC0-8 h did not differ between the studies. In conclusion, a single oral dose of (6S)-5-Methyl-THF-Na caused higher AUC0-8 h and Cmax of plasma (6S)-5-Methyl-THF compared to folic acid. The Na- and Ca- salts of (6S)-5-Methyl-THF are not likely to differ in their pharmacokinetics. Further studies may investigate whether supplementation of the compounds for a longer time will lead to differences in circulating or intracellular/tissue folate concentrations.

A prospective birth cohort study on cord blood folate subtypes and risk of autism spectrum disorder.

BACKGROUND: We previously reported that extremely high concentrations of maternal plasma folate were associated with increased risk of autism spectrum disorder (ASD) in children. This study explored whether specific types of folate in cord blood have differential association with ASD. OBJECTIVES: In the Boston Birth Cohort (BBC), we assessed the association between cord blood unmetabolized folic acid (UMFA), 5-methyl tetrahydrofolate (THF), and total folate and a child's ASD risk. In a subset, we explored whether the association between UMFA and ASD risk can be affected by the dihydrofolate reductase (DHFR) genotype and cord plasma creatinine. We also examined prenatal correlates of cord UMFA concentrations. METHODS: This report included 567 BBC children (92 ASD, 475 neurotypical), who were recruited at birth and prospectively followed at the Boston Medical Center. ASD was defined from International Classification of Diseases (ICD)-9 and ICD-10 codes documented in electronic medical records. RESULTS: Children with cord UMFA in the highest, versus lowest quartile, had a greater ASD risk (adjusted OR, aORquartile4: 2.26; 95% CI: 1.08, 4.75). When stratified by race/ethnicity, the association was limited to 311 (45 ASD) Black children (aORquartile4: 9.85; 95% CI: 2.53, 38.31); a test of interaction between race/ethnicity and cord UMFA concentrations was significant (P = 0.007). The UMFA-ASD association in Black children slightly attenuated after adjusting for cord plasma creatinine (P = 0.05). There was no significant association between cord 5-methyl THF, total folate, DHFR genotype, and ASD risk. Cord total folate and maternal supplement intake during second trimester were associated with higher cord UMFA. CONCLUSIONS: Higher concentrations of cord UMFA, but not 5-methyl THF or total folate, were associated with a greater risk of ASD in Black children. This study in a preterm-birth-enriched cohort raises more questions than it could answer and underscores the need for additional investigations on the sources and role of cord UMFA in children's neurodevelopmental outcomes and underlying mechanisms.

Distribution of 5-Methyltetrahydrofolate and Folic Acid Levels in Maternal and Cord Blood Serum: Longitudinal Evaluation of Japanese Pregnant Women.

"Total" folate in blood has usually been measured to evaluate the folate status of pregnant women. However, folate is composed of many metabolites. The main substrate is 5-methyltetrahydrofolate (5-MTHF), with folic acid (FA) representing a very small component as an unmetabolized species in blood. We longitudinally evaluated 5-MTHF, FA and total homocysteine in maternal and cord blood from Japanese pregnant women. Subjects were 146 pregnant women who participated in the Chiba study of Mother and Child Health (C-MACH) prospective cohort study. Sera were obtained in early and late pregnancy, at delivery, and from cord blood. Species levels were measured by isotope-dilution mass spectrometry. Both 5-MTHF and FA levels were lower than reported levels from pregnant women in populations from countries with mandatory FA fortification. As gestational age progressed, serum 5-MTHF levels decreased, whereas serum FA levels were slightly reduced only at delivery compared to early pregnancy. A significant negative association between serum 5-MTHF and total homocysteine was shown at all examined times, but no associations with FA were evident. At delivery, cord 5-MTHF was significantly higher than maternal levels, while FA again showed no significant correlation. These results suggest that 5-MTHF is actively transported to the fetus through placental transporters and may reflect folate status during pregnancy as a physiologically important species.

A pilot study to assess the effect of a three-month vitamin supplementation containing L-methylfolate on systemic homocysteine plasma concentrations and retinal blood flow in patients with diabetes.

Purpose: The aim of the present study was to investigate the effect of a three-month dietary supplementation with a methylfolate formulation on homocysteine plasma concentrations and ocular blood flow parameters in patients with diabetes. Methods: Twenty-four patients with diabetes received a dietary supplement (Oculofolin, Aprofol AG, Switzerland) containing 900 µg L­methylfolate (levomefolate calcium or [6S]-5-methyltetrahydrofolic acid, calcium salt), methylcobalamin, and other ingredients for three consecutive months. The patients' plasma homocysteine concentration and retinal blood flow were assessed at baseline and after three months of folate intake. Retinal blood flow was measured using a custom-built dual-beam Doppler optical coherence tomography (OCT) system. In addition, flicker-induced retinal vasodilatation was assessed by means of a commercially available dynamic vessel analyzer (IMEDOS, Jena, Germany). Results: Supplementation was well tolerated by all patients. After three months, plasma homocysteine concentration significantly decreased from 14.2 ± 9.3 to 9.6 ± 6.6 µmol/L (p < 0.001). In addition, a tendency toward an increased total retinal blood flow from 36.8 ± 12.9 to 39.2 ± 10.8 µl/min was observed, but this effect did not reach the level of significance (p = 0.11). Supplementation had no effect on retinal vessel diameter or flicker-induced vasodilatation. Conclusions: The present data show that a three-month intake of a dietary supplement containing methylfolate can significantly reduce blood homocysteine levels in patients with diabetes. This is of importance because higher homocysteine plasma levels have been found to be associated with an increased risk of vascular associated systemic diseases and eye diseases. Whether systemic methylfolate supplementation affects retinal perfusion must be studied in a larger population.

Is natural (6S)-5-methyltetrahydrofolic acid as effective as synthetic folic acid in increasing serum and red blood cell folate concentrations during pregnancy? A proof-of-concept pilot study.

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

Determination of plasma homocysteine with a UHPLC-MS/MS method: Application to analyze the correlation between plasma homocysteine and whole blood 5-methyltetrahydrofolate in healthy volunteers.

An ultra-high performance liquid chromatography tandem mass spectrometry method was developed for determination of homocysteine (HCY) in human plasma. The HCY was derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate and isolated using solid-phase extraction. Derivatization, isolation and detection procedures were optimized. Satisfactory linearity was obtained with determination coefficients (r2 ) >0.999. The intra- and inter-day precisions were in the interval of 1.2-5.1% and accuracy was within ±7%. Mean recoveries were close to 100%. The limit of detection and the limit of quantification were 0.46 and 1.38 µmol/L, respectively. The method was then applied to investigate the relationship between plasma HCY and whole blood 5-methyltetrahydrofolate levels in healthy volunteers. The results revealed that the plasma level of HCY was significantly negatively correlated to whole blood 5-methyltetrahydrofolate in volunteers.

Rare variants and biological pathways identified in treatment-refractory depression.

Individuals diagnosed with major depressive disorder not responding to at least two adequate treatments are defined as treatment-refractory major depressive disorder (TR-MDD). Some TR-MDD patients have altered metabolic phenotypes that may be pharmacologically reversed. The characterization of these phenotypes and their underlying etiologies is paramount, particularly their genetic components. In this study, TR-MDD patients (n = 124) were recruited and metabolites were quantified in their cerebrospinal fluid (CSF) and peripheral blood. Three sub-categories of deficiencies were examined, namely 5-methyltetrahydrofolte (in CSF; n = 13), tetrahydrobiopterin (in CSF; n = 11), and abnormal acylcarnitine profiles (in peripheral blood; n = 8). Whole exome sequencing was performed on genomic DNA from the entire TR-MDD cohort and exonic variant allele frequencies for cases were compared to a control cohort (1:5 matching on ancestry). Low frequency, damaging alleles were identified and used for in silico pathway analyses. Three association signals for TR-MDD approached genome-wide significance on chromosomes 22, 7, and 3. Three risk-associated variants from a prior depression study were replicated. Relevant biological pathways were identified that contained an enrichment of rare, damaging variants in central nervous system (CNS)-specific pathways, including neurotransmitter receptors, potassium channels, and synapse transmission. Some TR-MDD patients had rare variants in genes that were previously associated with other psychiatric disorders, psychiatric endophenotypes, CNS structural defects, and CNS-related cellular and molecular functions. Exome analysis of metabolically phenotyped TR-MDD patients has identified potentially functional gene pathways and low frequency, deleterious gene variants for further investigation. Further studies in larger cohorts of biochemically phenotyped TR-MDD patients are desirable to extend and confirm these findings.

One-carbon metabolites, B vitamins and associations with systemic inflammation and angiogenesis biomarkers among colorectal cancer patients: results from the ColoCare Study.

B vitamins involved in one-carbon metabolism have been implicated in the development of inflammation- and angiogenesis-related chronic diseases, such as colorectal cancer (CRC). Yet, the role of one-carbon metabolism in inflammation and angiogenesis among CRC patients remains unclear. The objective of this study was to investigate associations of components of one-carbon metabolism with inflammation and angiogenesis biomarkers among newly diagnosed CRC patients (n 238) in the prospective ColoCare Study, Heidelberg. We cross-sectionally analysed associations between twelve B vitamins and one-carbon metabolites and ten inflammation and angiogenesis biomarkers from pre-surgery serum samples using multivariable linear regression models. We further explored associations among novel biomarkers in these pathways with Spearman partial correlation analyses. We hypothesised that pyridoxal-5'-phosphate (PLP) is inversely associated with inflammatory biomarkers. We observed that PLP was inversely associated with C-reactive protein (CRP) (r -0·33, Plinear < 0·0001), serum amyloid A (SAA) (r -0·23, Plinear = 0·003), IL-6 (r -0·39, Plinear < 0·0001), IL-8 (r -0·20, Plinear = 0·02) and TNFα (r -0·12, Plinear = 0·045). Similar findings were observed for 5-methyl-tetrahydrofolate and CRP (r -0·14), SAA (r -0·14) and TNFα (r -0·15) among CRC patients. Folate catabolite acetyl-para-aminobenzoylglutamic acid (pABG) was positively correlated with IL-6 (r 0·27, Plinear < 0·0001), and pABG was positively correlated with IL-8 (r 0·21, Plinear < 0·0001), indicating higher folate utilisation during inflammation. Our data support the hypothesis of inverse associations between PLP and inflammatory biomarkers among CRC patients. A better understanding of the role and inter-relation of PLP and other one-carbon metabolites with inflammatory processes among colorectal carcinogenesis and prognosis could identify targets for future dietary guidance for CRC patients.

[Simultaneous determination of leucovorin and 5-methyl-tetrahydrofolate diastereoisomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry coupled with ultrafiltration centrifugation-based pretreatment and its application to a pharmacokinetic study].

A simple, sensitive, and stable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the simultaneous determination of leucovorin and 5-methyltetrahydrofolate diastereomers in human plasma using methotrexate as the internal standard. The analytes and the internal standard were extracted from plasma samples by simple ultrafiltration centrifugation-based extraction. The separation was achieved on a chiral HSA column (150 mm×4 mm, 5 µm) using mobile phases containing 10 mmol pH 8.0 ammonium acetate and acetonitrile in gradient mode. The method showed good linearities in the ranges of 25-5000 µg/L and 12.5-3000 µg/L for leucovorin and 5-methyltetrahydrofolate diastereoisomers, respectively. The method was fully validated with respect to sensitivity, precision, accuracy, matrix effect, extraction recovery, and stability of analytes under various conditions. The method was successfully applied to a pharmacokinetic study of 125 mg/m2 6R,S-leucovorin and 62.5 mg/m2 6S-leucovorin. The results showed that the maximum observed concentrations (Cmax) of 6S-leucovorin and L-5-methyltetrahydrofolate were (3137.917±408.837) and (1679.633±244.132) µg/L, respectively, and the areas under the curve from the time of dosing to the last measurable concentration (AUC0-t) were (7504.883±1185.101) and (14001.214±2868.949) µg/L in the 125 mg/m2 6R,S-leucovorin dose group. The Cmax values of 6S-leucovorin and L-5-methyltetrahydrofolate were (3187.917±387.298) and (1739.204±224.755) µg/L, respectively, and AUC0-t values were (7426.664±854.825) and (14884.331±1843.353) µg/L in the 62.5 mg/m2 6S-leucovorin dose group. There were no significant diffe-rences in the main pharmacokinetic parameters between the two dose groups, and the pharmacokinetic characteristics as well as the rate and extent of absorption were consistent. This method can provide technical support for future bioequivalence studies of sodium leucovorin.

Fisher discriminant analysis for classification of autism spectrum disorders based on folate-related metabolism markers.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders with an increasing prevalence but lack reliable biomarkers for early diagnosis. The present study investigated 13 serological metabolites and 2 genetic variants related to folate metabolism in a total of 89 ASD cases and 89 matched controls. Fisher discriminant analysis was used to establish the classification model to recognize ASD cases and controls. Ten metabolites were significantly different between the groups, of which six metabolites were used as predictors to determine the discriminant prediction model: vitamin B12, 5-methylene-tetrahydrofolate, methonine, the ratio of S-adenosylmethionine/S-adenosylhomocysteine, methionine synthase and transcobalamin II. The model had statistical significance (lambda=0.520, χ2=113.103, df=6, P<.001) and correctly identified 84.3% of ASD and normal cohorts. The area under the receiver operating characteristic curve was 0.913, with a sensitivity of 86.5% and a specificity of 85.4%. Overall, the results indicated that folate-related metabolism contributed to predisposition of ASD and the combined detection of folate-related metabolism biomarkers could be effective in distinguishing ASD from healthy controls, and provide new insights for the early diagnosis of ASD in the future.

Relationship between folate concentration and expression of folate-associated genes in tissue and plasma after intraoperative administration of leucovorin in patients with colorectal cancer.

PURPOSE: The aim of study was to investigate the relationship between folate concentration and expression of folate-associated genes in tumour, mucosa and plasma of patients with colorectal cancer, after intraoperative administration of bolus leucovorin (LV). METHODS: Eighty patients were randomized into four groups to receive 0, 60, 200, or 500 mg/m2 LV, respectively. Tissue and plasma folate concentrations were assessed by LC-MS/MS. Gene expression of ABCC3/MRP3, FPGS, GGH, MTHFD1L, SLC46A1/PCFT, and SLC19A1/RFC-1 was determined using quantitative PCR. RESULTS: The folate concentration in tumour increased with increasing dosage of LV. Half of the patients treated with 60 mg/m2 did not reach a level above the levels of untreated patients. A significant correlation between folate concentration in tumour and mucosa was found in untreated patients, and in the group treated with 60 mg/m2 LV. The 5-MTHF/LV ratio correlated negatively with folate concentration in mucosa, whereas a positive correlation was found in tumour of patients who received 200 or 500 mg/m2 LV. A positive correlation was found between folate concentration and expression of all genes, except MTHFD1L, in patients who received LV. There was a negative correlation between 5-MTHF concentration in plasma of untreated patients and expression of GGH and SLC46A1/PCFT in tumour. CONCLUSIONS: The results indicate the possibility of using the individual plasma 5-MTHF/LV ratio after LV injection as a surrogate marker for tissue folate concentration. Expression of several folate-associated genes is associated with folate concentration in tissue and plasma and may become useful when predicting response to LV treatment.

Laboratory assessment of folate (vitamin B9) status.

Folate (vitamin B9) plays a crucial role in fundamental cellular processes, including nucleic acid biosynthesis, methyl group biogenesis and amino acid metabolism. The detection and correction of folate deficiency prevents megaloblastic anaemia and reduces the risk of neural tube defects. Coexisting deficiencies of folate and vitamin B12 are associated with cognitive decline, depression and neuropathy. Folate deficiency and excess has also been implicated in some cancers. Excessive exposure to folic acid, a synthetic compound used in supplements and fortified foods, has also been linked to adverse health effects. Of at least three distinct laboratory markers of folate status, it is the total abundance of folate in serum/plasma that is used by the majority of laboratories. The analysis of folate in red cells is also commonly performed. Since the folate content of red cells is fixed during erythropoiesis, this marker is indicative of folate status over the preceding ~4 months. Poor stability, variation in polyglutamate chain length and unreliable extraction from red cells are factors that make the analysis of folate challenging. The clinical use of measuring specific folate species has also been explored. 5-Methyltetrahydrofolate, the main form of folate found in blood, is essential for the vitamin B12-dependent methionine synthase mediated remethylation of homocysteine to methionine. As such, homocysteine measurement reflects cellular folate and vitamin B12 use. When interpreting homocysteine results, age, sex and pregnancy, specific reference ranges should be applied. The evaluation of folate status using combined markers of abundance and cellular use has been adopted by some laboratories. In the presence of discordance between laboratory results and strong clinical features of deficiency, treatment should not be delayed. High folate status should be followed up with the assessment of vitamin B12 status, a review of previous results and reassessment of folic acid supplementation regime.

Harmonizing the Calibrator and Microorganism Used in the Folate Microbiological Assay Increases the Comparability of Serum and Whole-Blood Folate Results in a CDC Round-Robin Study.

Background: Harmonizing critical reagents for the folate microbiological assay (MBA) may improve among-laboratory comparability. Objective: We assessed the comparability of the MBA for serum folate (S-FOL) and whole-blood folate (WB-FOL) in an international comparison study. Methods: Eight laboratories obtained a kit containing CDC microorganism inoculum (chloramphenicol-resistant Lactobacillus rhamnosus), CDC calibrator (5-methyltetrahydrofolate), and 23 serum and WB hemolysate samples each. Laboratories analyzed the samples in single measurement over 2 d using 4 conditions: in-house microorganism and in-house calibrator (IH-MO & IH-CAL), in-house microorganism and CDC calibrator (IH-MO & CDC-CAL), CDC microorganism and in-house calibrator (CDC-MO & IH-CAL), and CDC microorganism and CDC calibrator (CDC-MO & CDC-CAL). We calculated geometric mean concentrations for each laboratory and condition and compared data to the CDC MBA (target). Results: The among-laboratory arithmetic mean S-FOL concentrations for the 4 conditions were 30.2, 28.1, 30.0, and 29.9 (group 1, 5-methyltetrahydrofolate IH-CAL) compared with 35.3, 33.3, 33.6, and 30.7 nmol/L (group 2, folic acid IH-CAL), respectively; and 428, 405, 398, and 393 (group 1) compared with 469, 423, 477, and 418 nmol/L (group 2), respectively, for WB-FOL. Differences to the CDC MBA target values were smaller for group 1 (range across conditions; S-FOL: 9.9-21%; WB-FOL: 9.0-18%) compared with group 2 laboratories (S-FOL: 13-30%; WB-FOL: 16-32%) and smaller when CDC reagents were used compared with in-house reagents (S-FOL: 12% compared with 22%; WB-FOL: 13% compared with 25%). A linear mixed model estimated a small microorganism effect (S-FOL: 2.3%; WB-FOL: 2.3%) and a larger mean calibrator effect; folic acid compared with 5-methyltetrahydrofolate calibrator produced 12% higher S-FOL and 15% higher WB-FOL results. When laboratories used CDC reagents, the estimated among-laboratory variability was ∼10% for S-FOL and WB-FOL. Conclusion: Harmonizing the calibrator and microorganism for the folate MBA has the potential to improve the among-laboratory comparability in future surveys.

Low Dietary Folate Interacts with MTHFD1 Synthetase Deficiency in Mice, a Model for the R653Q Variant, to Increase Incidence of Developmental Delays and Defects.

Background: Suboptimal folate intake, a risk factor for birth defects, is common even in areas with folate fortification. A polymorphism in methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), R653Q (MTHFD1 c.1958 G > A), has also been associated with increased birth defect risk, likely through reduced purine synthesis. Objective: We aimed to determine if the interaction of MTHFD1 synthetase deficiency and low folate intake increases developmental abnormalities in a mouse model for MTHFD1 R653Q. Methods: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed control or low-folate diets (2 and 0.3 mg folic acid/kg diet, respectively) before mating and during pregnancy. Embryos and placentas were examined for anomalies at embryonic day 10.5. Maternal 1-carbon metabolites were measured in plasma and liver. Results: Delays and defects doubled in litters of Mthfd1S+/- females fed low-folate diets compared to wild-type females fed either diet, or Mthfd1S+/- females fed control diets [P values (defects): diet 0.003, maternal genotype 0.012, diet × maternal genotype 0.014]. These adverse outcomes were associated with placental dysmorphology. Intrauterine growth restriction was increased by embryonic Mthfd1S+/- genotype, folate deficiency, and interaction of maternal Mthfd1S+/- genotype with folate deficiency (P values: embryonic genotype 0.045, diet 0.0081, diet × maternal genotype 0.0019). Despite a 50% increase in methylenetetrahydrofolate reductase expression in low-folate maternal liver (P diet = 0.0007), methyltetrahydrofolate concentration decreased 70% (P diet <0.0001) and homocysteine concentration doubled in plasma (P diet = 0.0001); S-adenosylmethionine decreased 40% and S-adenosylhomocysteine increased 20% in low-folate maternal liver (P diet = 0.002 and 0.0002, respectively). Conclusions: MTHFD1 synthetase-deficient mice are more sensitive to low folate intake than wild-type mice during pregnancy. Reduced purine synthesis due to synthetase deficiency and altered methylation potential due to low folate may increase pregnancy complications. Further studies and individualized intake recommendations may be required for women homozygous for the MTHFD1 R653Q variant.

Helicobacter pylori moderates the association between 5-MTHF concentration and cognitive function in older adults.

OBJECTIVE: To explore potential interactions between folate-cycle factors and Helicobacter pylori seropositivity in the prediction of cognitive function. METHODS: We used data obtained from the 1999-2000 continuous National Health and Nutrition Examination Survey produced by the United States' Centers for Disease Control and Prevention. Using Ordinary Least Squares regression, we tested for associations between multiple folate-cycle factors, Helicobacter pylori seropositivity, and cognitive function assessed by the digit symbol coding subtest of the Wechsler Adult Intelligence Scale-III. We then tested for interactions between each of the folate-cycle factors and Helicobacter pylori in the prediction of cognitive function. RESULTS: Although Helicobacter pylori seropositivity, 5-methyltetrahydrofolate, vitamin B-12, and homocysteine were not associated with performance on the digit symbol coding task, Helicobacter pylori seropositivity interacted with 5-methyltetrahydrofolate concentration to predict performance on the digit symbol coding task. The Helicobacter pylori seropositive group performed worse on the digit symbol coding task as 5-methyltetrahydrofolate concentration decreased. CONCLUSION: The interaction between Helicobacter pylori seropositivity and reduced folate-cycle factor 5-methyltetrahydrofolate might impair aspects of cognitive function.

Supplementation with [6S]-5-methyltetrahydrofolate or folic acid equally reduces serum homocysteine concentrations in older adults.

Folic acid (FA) supplementation reduces the elevated serum homocysteine (Hcy) concentrations. [6 S]-5-methyltetrahydrofolate ([6 S]-5-MTHF) is an alternative to FA due to possible advantages, that is, no masking cobalamin deficiency. The study aim was to evaluate the effectiveness of [6 S]-5-MTHF in relations to FA supplementation in reducing the serum Hcy. Healthy volunteers, aged 50-65, had normal serum folate and did not use supplements with B-vitamins for 6 months. Forty subjects were divided into two groups: receiving 400 µg/d FA or the equimolar amount of [6 S]-5-MTHF. Blood was collected at baseline and after 4 weeks. In both groups, a significant decrease in the mean Hcy level after intervention period was observed. Supplementation with [6 S]-5-MTHF was slightly less effective, but not significantly, in Hcy lowering than FA (p = .243 between the groups), that is, by 7.8% and 13.4%, respectively. The [6 S]-5-MTHF was shown to be an adequate alternative to FA in reducing Hcy concentrations.

Two International Round-Robin Studies Showed Good Comparability of 5-Methyltetrahydrofolate but Poor Comparability of Folic Acid Measured in Serum by Different High-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods.

Background: Serum folate methods produce different results. The comparability of HPLC-mass spectrometry (MS)/MS methods is not well documented.Objective: We conducted an international "round-robin" investigation to assess the comparability, precision, and accuracy of serum folate HPLC-MS/MS methods.Methods: The CDC laboratory, 7 laboratories with independently developed methods (group 1), and 6 laboratories with an adapted CDC method (group 2) analyzed folate forms in 6 serum pools and 6 calibrators from the CDC (duplicate analysis over 2 d) and in two 3-level reference materials (duplicate analysis).Results: All laboratories measured 5-methyltetrahydrofolate (5-methylTHF) and folic acid; some measured additional folate forms. The geometric mean (range) concentrations (nanomoles per liter) for 5-methylTHF in the 6 serum pools were 18.3 nmol/L (CDC), 13.8-28.9 nmol/L (group 1), and 16.8-18.6 nmol/L (group 2); for folic acid the concentrations were 3.42 nmol/L (CDC), 1.09-4.74 nmol/L (group 1), and 1.74-2.90 nmol/L (group 2). The median imprecision (CV) for 5-methylTHF was 4.1% (CDC), 4.6-11% (group 1), and 1.7-6.0% (group 2); for folic acid it was 6.9% (CDC), 4.9-20% (group 1), and 3.9-23% (group 2). The mean ± SD (range) recovery of 5-methylTHF spiked into serum was 98% ± 27% (59-138%) for group 1 and 98% ± 10% (82-111%) for group 2; for folic acid it was 93% ± 29% (67-198%) for group 1 and 81% ± 16% (64-102%) for group 2. The mean relative bias for 5-methylTHF compared with the reference material certificate value was 12% (CDC), -24% to 30% (group 1), and -0.6% to 16% (group 2); for folic acid it was 73% (CDC), -47% to 578% (group 1), and -3.3% to 67% (group 2).Conclusions: For 5-methylTHF, group 2 laboratories demonstrated better agreement and precision, less variable spiking recovery, and less bias by using a reference material. Laboratory performance for folic acid was highly variable and needs improvement. Certified reference materials for serum folate forms and total folate are needed to improve method accuracy.