Extraction and Quantitation of Nicotinamide Adenine Dinucleotide Redox Cofactors

AIMS: ) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms. RESULTS: , and NADPH concentrations in cells and mouse tissues were measured with this approach. INNOVATION: ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards. CONCLUSION: Extraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.