Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.

TitleAssays for Determination of Cellular and Mitochondrial NAD+ and NADH Content.
Publication TypeJournal Article
Year of Publication2021
AuthorsYang Y, Sauve AA
JournalMethods Mol Biol
Volume2310
Pagination271-285
Date Published2021
ISSN1940-6029
KeywordsCell Fractionation, Cells, Cultured, Chromatography, High Pressure Liquid, Energy Metabolism, Indicator Dilution Techniques, Mass Spectrometry, Mitochondria, NAD, Spectrometry, Fluorescence
Abstract

NAD+ is a redox cofactor essential to the proper functioning of a variety of important metabolic pathways, including key steps in mitochondrial energy metabolism. In addition, it serves as a signaling substrate for enzymes such as sirtuins and the poly-ADP ribosyl-polymerase family of enzymes. Sirtuins, which are NAD+-dependent protein deacylases, harness changes in cellular NAD+ concentrations to produce changes in protein acylation status, thereby affecting downstream functions including energy metabolism, stress resistance, and cell survival. Thus, the availability of NAD+ in cells, or in specific organelles such as the mitochondrion, regulates downstream signaling and key biological processes. This concept has driven a need for researchers to easily and precisely measure NAD+ concentrations in biological samples. We herein describe several protocols for the measurement of NAD+ and NADH concentrations in tissues, cells, or subcellular compartments such as mitochondria. These protocols include a cycling assay that can quickly measure NAD+ or NADH levels using a plate reader equipped with fluorescence measurement capabilities. This plate assay relies only upon commercially available materials in addition to the biological samples of interest. In addition, we describe a protocol employing stable isotope-labeled NAD+ as an internal standard to determine biological NAD+ content by isotope-dilution methods. This method requires mass spectrometry to ratio endogenous NAD+ with exogenous isotope-labeled NAD+ to obtain quantification using HPLC and mass spectrometry.

DOI10.1007/978-1-0716-1433-4_15
Alternate JournalMethods Mol Biol
PubMed ID34096008
Grant ListR01 GM106072 / GM / NIGMS NIH HHS / United States