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

• Published in: Analytical biochemistry Vol. 603; p. 113797
• Main Authors: Artiukhov, Artem V., Pometun, Anastasia A., Zubanova, Sofia A., Tishkov, Vladimir I., Bunik, Victoria I.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-08-2020
• Subjects: Animals; Brain Chemistry; Cerebral cortex; Enzymatic assay; Fluorometry - methods; Formate dehydrogenase; Formate Dehydrogenases - chemistry; Formate Dehydrogenases - metabolism; Methanol-acetic acid extract; Mitochondria - chemistry; NAD; NAD - analysis; NAD - chemistry; NAD - metabolism; Rats; Rats, Wistar; Sensitivity and Specificity; Tissue Extracts - analysis; NAD; Enzymatic assay; LDH; Cerebral cortex; Methanol-acetic acid extract; ADH; Formate dehydrogenase; FDH; OpaFDH; HPLC; NAD(+)
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113797

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.
•Formate dehydrogenase allows for accurate NAD+ assay from 5 to 60 pmol.•Formate dehydrogenase reaction is irreversible, rapid and artifact-prone.•Quantified NAD+ in the lipid-rich extracts of the rat brain is 75 nmol/g of tissue.