TARGETED, LCMS-BASED METABOLOMICS FOR QUANTITATIVE MEASUREMENT OF NAD+ METABOLITES

• Main Authors: Samuel AJ Trammell, Charles Brenner
• Format: Article
• Language: English
• Published: Elsevier 2013-01-01
• Series: Computational and Structural Biotechnology Journal
• Online Access: http://www.sciencedirect.com/science/article/pii/S2001037014600556

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.