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.
COBISS.SI-ID: 25786371
Adequate levels of folates are essential for homeostasis of the organism, prevention of congenital malformations, and the salvage of predisposed disease states. They depend on genetic predisposition, and therefore, a pharmacogenetic approach to individualized supplementation or therapeutic intervention is necessary for an optimal outcome. The role of folates in vital cell processes was investigated by translational pharmacogenetics employing lymphoblastoid cell lines (LCLs). Depriving cells of folates led to reversible S-phase arrest. Since 5,10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in the biosynthesis of an active folate form, we evaluated the relevance of polymorphisms in the MTHFR gene on intracellular levels of bioactive metabolite, the 5-methyltetrahydrofolate (5-Me-THF). LCLs (n = 35) were divided into low- and normal-MTHFR activity groups based on their genotype. They were cultured in the presence of folic acid (FA) or 5-Me-THF. Based on the cells’ metabolic activity and intracellular 5-Me-THF levels, we conclude upplementation of FA is sucient to maintain adequate folate level in the normal MTHFR activity group, while low MTHFR activity cells require 5-Me-THF to overcome the metabolic defects caused by polymorphisms in their MTHFR genes. This finding was supported by the determination of intracellular levels of 5-Me-THF in cell lysates by LC-MS/MS. FA supplementation resulted in a 2.5-fold increase in 5-Me-THF in cells with normal MTHFR activity, but there was no increase after FA supplementation in low MTHFR activity cells. However, when LCLs were exposed to 5-Me-THF, a 10-fold increase in intracellular levels of this metabolite was determined. These findings indicate that patients undergoing folate supplementation to counteract anti-folate therapies, or patients with increased folate demand, would benefit from pharmacogenetics-based therapy choices.
COBISS.SI-ID: 28803331
The objective of this study was to identify new environmental and genetic risk factors for orofacial clefts that arise during early foetal development. In this retrospective, case%control, mother%child pair study, 172 orofacial clefts cases and 199 healthy controls, and their respective mothers, were genotyped for common variants in relevant genes obtained by text and database mining using STRING 10.0. Exposure to environmental risk factors was evaluated using questionnaires. Variant glycine N-methyltransferase (odds ratio (OR) 2.1, 95% confidence interval (95% CI) 1.0%4.4) and dihydrofolate reductase (OR 2.4, 95% CI 1.3%4.5) genotypes were identified as risk factors for cleft lip with or without cleft palate formation. Furthermore, synergy was detected between variant glycine N-methyltransferase and dihydrofolate reductase genotypes in promoting cleft lip with or without cleft palate formation (OR 7, 95% CI 2%23). This study is novel in finding that common glycine N-methyltransferase variant genotypes increase the risk of cleft lip with or without cleft palate.
COBISS.SI-ID: 4552561
Folate is vital for cell development and growth. It is involved in one-carbon transfer reactions essential for the synthesis of purines and pyrimidines. It also acts in conjunction with cobalamin (vitamin B12) as a fundamental cofactor in the remethylation cycle that converts homocysteine to methionine. A deficiency in folate or vitamin B12 can lead to elevated homocysteine level, which has been identified as an independent risk factor in several health-related conditions. Adequate folate levels are essential in women of childbearing age and in pregnant women, and folate deficiency is associated with several congenital malformations. Low folate levels can be caused by dietary deficiencies, a genetic predisposition or treatment with medicines that affect folate concentration. Women who are pregnant or of child-bearing age commonly use medicines, so it is important to identify the basic biochemical mechanisms by which medicines interfere with the folate-homocysteine-methionine pathway. This review focuses on prescription medicines associated with folate disruption. It also summarizes their undesirable/toxic effects. Recommendations regarding folate supplementation during medical therapy are also reviewed.
COBISS.SI-ID: 4642929