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

Vidmar Golja, Maša (author), Trontelj, Jurij (author), Geršak, Ksenija (author), Mlinarič-Raščan, Irena (author), Šmid, Alenka (author)

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.

• Language: English
• Keywords: LC-MS/MS, folic acid, 5-methyltetrahydrofolate, homocysteine, cell lysate, folate markers
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy; MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2020
• Number of pages: 12 str.
• Numbering: Vol. 605, art. 113830
• PID: 20.500.12556/RUL-138399
• UDC: 577.164.1:616.074
• ISSN on article: 0003-2697
• DOI: 10.1016/j.ab.2020.113830
• COBISS.SI-ID: 25786371
• Publication date in RUL: 19.07.2022

Record is a part of a journal

• Title: Analytical biochemistry
• Shortened title: Anal. biochem.
• Publisher: Elsevier
• ISSN: 0003-2697
• COBISS.SI-ID: 6506759

Licences

• License: CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
• Link: http://creativecommons.org/licenses/by-nc-nd/4.0/
• Description: The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.

Secondary language

• Language: Slovenian
• Keywords: homocistein, folna kislina, folatni markerji, vitamini