Targeted Metabolomics: The LC-MS/MS Based Quantification of the Metabolites Involved in the Methylation Biochemical Pathways.

Georgia Ntasi,Emmanuel Mikros,Anthony Tsarbopoulos,Evagelos Gikas

doi:10.3390/metabo11070416

Abstract

Biochemical methylation reactions mediate the transfer of the methyl group regulating vital biochemical reactions implicated in various diseases as well as the methylation of DNA regulating the replication processes occurring in living organisms. As a finite number of methyl carriers are involved in the methyl transfer, their quantification could aid towards the assessment of an organism’s methylation potential. An Hydrophilic Interaction Chromatography-Liquid Chromatography Multiple Reaction Monitoring (HILIC-LC-MRM) mass spectrometry (MS) methodology was developed and validated according to Food & Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) for the simultaneous determination of nine metabolites i.e., B12, folic acid, 5-methyltetrahydrofolate, S-adenosylmethionine, S-adenosylhomocysteine, betaine, phosphocholine, N,N-dimethylglycine, and deoxythymidine monophosphate in human blood plasma. The sample pretreatment was based on a single step Solid-phase extraction (SPE) methodology using C18 cartridges. The methodology was found to accurately quantitate the analytes under investigation according to the corresponding dynamic range proposed in the literature for each analyte. The applicability of the method was assessed using blood donor samples and its applicability demonstrated by the assessment of their basal levels, which were shown to agree with the established basal levels. The methodology can be used for diagnostic purposes as well as for epigenetic screening.

Highlights

Methylation is central to the biochemical regulation of folic acid (B9) [8,9,10] and vitamin B12 [11,12] levels, which are responsible for the synthesis of nucleic acids and neurotransmitters, any dysregulation causes a variety of nervous system diseases [13]
We have developed a new methodology for the quantification of eight key methylation metabolites
This is in our opinion contradictory as there is no chance of discovering individuals without endogenous levels of these metabolites

Summary

Introduction

The methylation cycle can be thought of as consisting of five major biochemical pathways, their interconnection being summarized as follows: (a) the tetrahydrobiopterin cycle which involves the conversion of 7,8-dihydrobiopterin (BH2) to 5,6,7,8-tetrahydrobiopterin (BH4) with the transport of the methyl group (Me) to 5-Me-tetrahydrofolate (5-Me-THF). Through the action of methylenetetrahydrofolate reductase (involving the synthesis of tryptophan from serotonin and tyrosine from dopamine) [3]; (b) the folate cycle—where. B12 [4]; (c) the methionine cycle with the cycling of Me from methionine to S- adenosylmethionine (SAM) concomitant with S-adenosyl-homocysteine (SAH) to homocysteine by accepting Me through B12 [5]; (d) the trans-sulfuration cycle with the disposition of Me to cystathionine towards cysteine formation [6]; and (e) the urea cycle as arginine is converted to citrulline through the action of BH4 on endothelial nitric oxide synthase eNOS [7]. SAH shows clinical importance as a cardiovascular disease biomarker [24], being involved in the establishment of neurodegenerative diseases such as Alzheimer’s [25]

Methods

Results

Conclusion