NAD+

Mechanism of action in research models

Redox carrier function: NAD+ accepts hydride ions (H⁻) from metabolic intermediates (GAPDH-catalysed glyceraldehyde-3-phosphate oxidation; α-ketoglutarate dehydrogenase and malate dehydrogenase in the TCA cycle; mitochondrial NADH-ubiquinone oxidoreductase Complex I) to form NADH, which then donates electrons to the mitochondrial electron-transport chain driving oxidative phosphorylation. The NAD+/NADH ratio is a key indicator of cellular energy status studied extensively in metabolic research.

Sirtuin substrate: NAD+-dependent deacylases (SIRT1-7) cleave acyl modifications from lysine residues on histones, transcription factors, and metabolic enzymes, releasing nicotinamide and O-acyl-ADP-ribose. Key substrates studied include SIRT1-mediated deacetylation of PGC-1α (mitochondrial biogenesis), FOXO3 (stress resistance), and NF-κB (inflammation); SIRT3-mediated deacetylation of mitochondrial enzymes; and SIRT6-mediated deacetylation of H3K9/H3K56 (genome stability, telomere maintenance, DNA repair).

PARP substrate: PARP-1 and PARP-2 are activated by DNA strand breaks and use NAD+ to synthesise poly(ADP-ribose) chains on target proteins, facilitating damage-sensor recruitment and repair-scaffold assembly. Under excessive DNA damage, PARP hyperactivation can deplete cellular NAD+ to near zero — a cell-death mechanism (parthanatos) studied in neurodegeneration, ischaemia-reperfusion, and genotoxicity models.

CD38 / cADPR signalling: CD38 is an NAD+ glycohydrolase producing cyclic ADP-ribose (cADPR) and ADPR from NAD+, contributing to Ca²⁺ mobilisation from the endoplasmic reticulum. CD38 expression increases with age and is studied as a regulator of age-related NAD+ decline.

Research background and peer-reviewed literature

The centrality of NAD+ to ageing biology was established by Guarente and colleagues, whose work characterising Sir2 (yeast SIRT1 orthologue) as an NAD+-dependent deacetylase extended lifespan in budding yeast and defined the NAD+/SIRT1 axis as a longevity pathway conserved through evolution. Imai et al. published landmark research in Cell demonstrating that NAD+ levels decline significantly with age in mice and that NAD+ precursor supplementation restores SIRT1 activity and reverses multiple ageing-associated metabolic deficiencies. Cantó et al. characterised the AMPK-SIRT1-PGC-1α axis: AMPK increases NAD+ via NAMPT activation, activating SIRT1 to deacetylate PGC-1α, driving mitochondrial biogenesis.

Reconstitution and storage protocol

NAD+ is highly water-soluble and hygroscopic. Handle with care.

1. Equilibrate sealed vial to room temperature in a dry environment before opening — moisture absorption degrades NAD+.
2. Dissolve in sterile distilled water or PBS (pH 7.0–7.4). NAD+ is unstable at high pH (>8.5); use near-neutral diluent. For acidic applications, dilute in 10 mM HEPES buffer pH 7.0.
3. Typical research concentrations: 1–10 mM stock for cell-culture dilution to 100 μM–1 mM working concentrations. Extracellular NAD+ in published cell-culture research typically ranges 100 nM–500 μM.
4. Sterilise by 0.22 μm filtration; do not autoclave (heat degrades NAD+).

Storage: lyophilized at −20 °C, sealed with desiccant, light-protected (stable 24+ months if dry). In solution at −80 °C; avoid freeze-thaw. NAD+ hydrolyses relatively rapidly at room temperature compared to peptides — prepare fresh working solutions daily for cell-viability assays requiring precise concentrations.