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

• Published in: Analytical biochemistry 2020-09, Vol.605, p.113830, Article 113830
• Main Authors: Vidmar Golja, Maša, Trontelj, Jurij, Geršak, Ksenija, Mlinarič-Raščan, Irena, Šmid, Alenka
• Format: Article
• Language: English
• Subjects: 5-Methyltetrahydrofolate; Cell Line; Cell lysate; Chromatography, High Pressure Liquid; Folate markers; Folic acid; Folic Acid - analysis; Homocysteine; Homocysteine - analysis; Humans; LC-MS/MS; Tandem Mass Spectrometry; Tetrahydrofolates - analysis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113830

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. [Display omitted] •A new LC-MS/MS method for quantification of intracellular folates was developed.•Clinically important FA, 5-Me-THF, and Hcy can be measured simultaneously.•The method uses isotopically labeled internal standards.•The method was validated and applied to lysates of human lymphoblastoid cell lines.