Extraction and measurement of NAD(P)+ and NAD(P)H

Abstract

Nicotinamide adenine dinucleotides are critical redox-active substrates for countless catabolic and anabolic reactions. Ratios of NAD+ to NADH and NADP+ to NADPH are therefore considered key indicators of the overall intracellular redox potential and metabolic state. These ratios can be measured in bulk conditions using a highly sensitive enzyme cycling-based colorimetric assay (detection limit at or below 0.05 μM or 1 pmol) following a simple extraction procedure involving solutions of acid and base. Special considerations are necessary to avoid measurement artifacts caused by the presence of endogenous redox-active metabolites, such as phenazines made by diverse Pseudomonas species (see Chapter 25).