Advantages of formate dehydrogenase reaction for efficient NAD⁺ quantification in biological samples

The medical significance of NAD⁺-dependent metabolic regulation acquires increasing attention, demanding rapid and clinically feasible quantification of NAD⁺ in complex biological samples. Here we describe the usage of formate dehydrogenase for a straightforward and highly specific fluorometric assay of NAD⁺ in tissue extracts, not requiring chromatographic separation of nucleotides. The assay employs the irreversible reaction of formate oxidation coupled to NAD⁺ reduction, catalyzed by the enzyme which has high affinity and specificity to NAD⁺, and is stable under a variety of conditions. The assay reliably quantifies NAD⁺ in the methanol extracts of the rat brain cortex and mitochondria.