Abstract
The pyridine nucleotides nicotineamide adenine dinucleotide (NAD) and nicotineamide adenine dinucleotide phosphate (NADP) are conserved coenzymes across all domains of life, and are involved in more than 200 different hydride transfer reactions supporting essential catabolic and anabolic functions. The intracellular levels of these metabolites, and the ratio of their oxidized to reduced forms regulate an extensive network of reactions ranging beyond metabolism. Hence, monitoring their intracellular levels provides information about, but not limited to, the metabolic state of a cell or tissue. Interconversion between oxidized and reduced forms, varying pH liability and varying intracellular concentrations of the different species leaves absolute quantification of the pyridine nucleotides analytically challenging. These polar metabolites are poorly retained on conventional reverseed-phase stationary phases without ion-pair reagents that contaminates the LC-system. Herein we demonstrate that zwitterionic HILIC-tandem mass spectroemtry can be applied to successfully resolve the pyridine nucleotides in biological extracts in a fast, robust and highly sensitive way. The presented method applies isotope dilution to compensate potential loss of these labile metabolites and is validated for low, medium and high biomass samples of two popular biological model systems; Escherichia coli and the human cell line JJN-3. High stability and rapid sample preparation without solvent removal allows for long sequence runs, making this method ideal for high-throughput analysis of biological extracts.
Highlights
The pyridine nucleotides nicotineamide adenine dinucleotide (NAD) and its phosphorylated analogue nicotineamide adenine dinucleotide phosphate (NADP) are conserved coenzymes across all domains of life
While NAD+ is consumed in ADP-ribosylation, a post-translational modification implicated in cellular processes such as DNA repair, cell signaling and apoptosis (Reviewed in [2]), NADPH serves an important role in the cellular defense against reactive oxygen species by regenerating the antioxidant glutathione
As hydrophilic interaction liquid chromatography (HILIC) was considered a promising strategy for retaining pyridine nucleotides without the use of contaminating ion-pair reagents, three different HILIC stationary phases were tested for the separation of target metabolites; BEH Amide and two zwitterionic stationary phases, ZIC-pHILIC and AdvanceBio MS Spent Media
Summary
The pyridine nucleotides nicotineamide adenine dinucleotide (NAD) and its phosphorylated analogue nicotineamide adenine dinucleotide phosphate (NADP) are conserved coenzymes across all domains of life. The oxidized forms NAD+ and NADP+ can undergo reversible reduction to form NADH and NADPH in an extensive network of hydride transfer reactions catalyzed by more than 200 different enzymes. Though structurally similar, both redox pairs have specialized metabolic functions. NADPH is a cofactor in anabolic reactions such as reductive biosynthesis of fatty acids and cholesterol Both pyridine nucleotides have important regulatory roles. Hydrolysis of NAD+ is linked to protein deacetylation by the sirtuin SIRT1 which targets proteins including a variety of metabolic enzymes, as reviewed in [1] This leavies the NAD+/NADH-ratio an important regulator of metabolic homeostasis. The physiochemical properties and high turnover of these metabolites makes them analytically challenging
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