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

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 assa...

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Bibliographic Details
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(+)
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Summary: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.
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ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2020.113797