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

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

  • Anal Biochem. 2020 Sep 15;605:113830. doi: 10.1016/j.ab.2020.113830.
Maša Vidmar Golja 1,  Jurij Trontelj 2,  Ksenija Geršak 3,  Irena Mlinarič-Raščan 4,  Alenka Šmid 5
Affiliations

Affiliations

  • 1 University Medical Centre Ljubljana, Department of Obstetrics and Gynecology, Research Unit, Šlajmerjeva 3, 1000, Ljubljana, Slovenia; University of Ljubljana, Faculty of Pharmacy, The Chair of Clinical Biochemistry, Aškerčeva cesta 7, 1000, Ljubljana, Slovenia.
  • 2 University of Ljubljana, Faculty of Pharmacy, The Chair of Biopharmaceutics and Pharmacokinetics, Aškerčeva cesta 7, 1000, Ljubljana, Slovenia.
  • 3 University Medical Centre Ljubljana, Department of Obstetrics and Gynecology, Research Unit, Šlajmerjeva 3, 1000, Ljubljana, Slovenia; University of Ljubljana, Faculty of Medicine, Vrazov Trg 2, Ljubljana, Slovenia.
  • 4 University of Ljubljana, Faculty of Pharmacy, The Chair of Clinical Biochemistry, Aškerčeva cesta 7, 1000, Ljubljana, Slovenia.
  • 5 University of Ljubljana, Faculty of Pharmacy, The Chair of Clinical Biochemistry, Aškerčeva cesta 7, 1000, Ljubljana, Slovenia. Electronic address: alenka.smid@ffa.uni-lj.si.
PMID: 32717185 DOI: 10.1016/j.ab.2020.113830
Abstract

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.

Keywords

5-Methyltetrahydrofolate; Cell lysate; Folate markers; Folic acid; Homocysteine; LC-MS/MS.

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