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Comput Struct Biotechnol J. 2013 May 27;4:e201301012. doi: 10.5936/csbj.201301012. eCollection 2013.

Targeted, LCMS-based Metabolomics for Quantitative Measurement of NAD(+) Metabolites.

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1
Department of Biochemistry ; Interdisciplinary Graduate Program in Genetics Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.

Abstract

Nicotinamide adenine dinucleotide (NAD(+)) is a coenzyme for hydride transfer reactions and a substrate for sirtuins and other NAD(+)-consuming enzymes. The abundance of NAD (+), NAD(+) biosynthetic intermediates, and related nucleotides reflects the metabolic state of cells and tissues. High performance liquid chromatography (HPLC) followed by ultraviolet-visible (UV-Vis) spectroscopic analysis of NAD(+) metabolites does not offer the specificity and sensitivity necessary for robust quantification of complex samples. Thus, we developed a targeted, quantitative assay of the NAD(+) metabolome with the use of HPLC coupled to mass spectrometry. Here we discuss NAD(+) metabolism as well as the technical challenges required for reliable quantification of the NAD(+) metabolites. The new method incorporates new separations and improves upon a previously published method that suffered from the problem of ionization suppression for particular compounds.

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