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

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

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

homocistein;folna kislina;folatni markerji;LC-MS/MS;folic acid;5-Methyltetrahydrofolate;homocysteine;cell lysate;folate markers;

Data

Language:	English
Year of publishing:	2020
Typology:	1.01 - Original Scientific Article
Organization:	UL FFA - Faculty of Pharmacy
UDC:	577.164.1:616.074
COBISS:	25786371
ISSN:	0003-2697
Views:	77
Downloads:	22
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	Slovenian
Secondary keywords:	Vitamini;
Type (COBISS):	Article
Pages:	str. 1-12
Issue:	ǂVol. ǂ605
Chronology:	2020
DOI:	10.1016/j.ab.2020.113830
ID:	15948762