NAD+: The Coenzyme Behind Cellular Redox Chemistry

NAD+ (nicotinamide adenine dinucleotide, oxidized form) is a coenzyme with the formula C21H27N7O14P2 and a molecular weight of 663.4 g/mol (PubChem CID 5892, CAS 53-84-9). It is not a peptide. It is a dinucleotide: two nucleotides, one carrying adenine and one carrying nicotinamide, joined through their phosphate groups. Every living cell uses it, first as a carrier of electrons in redox reactions and second as a substrate that several families of signaling enzymes break apart.

This profile sets out what is established about the molecule: its identity, how it was found, how cells make it and use it up, what the literature reports about its levels over a lifespan, what happens to it outside the cell, its regulatory position, and the handling points that matter when it arrives in a lab as a lyophilized powder.

Identity at a glance

The values below come from the PubChem record for NAD+ (CID 5892, listed there under the name nadide).

NAD+ is listed in the Anhydrolabs catalog as NAD+.

The "+" in the name refers to the positive charge on the nitrogen of the nicotinamide ring in the oxidized form. It is a formal charge on one atom, not the charge of the whole molecule: the two phosphate groups are negatively charged at neutral pH, so the molecule as a whole is an anion in solution.

How NAD+ was found

The molecule entered the literature in 1906, when Arthur Harden and William John Young showed that the fermenting activity of yeast extract depended on a heat-stable, low-molecular-weight factor that could be separated from the enzymes by dialysis. They called it a coferment. Hans von Euler-Chelpin later purified it and worked out that it was built from a sugar, phosphate, adenine and a second base, and the two shared the 1929 Nobel Prize in Chemistry for work on sugar fermentation and fermentative enzymes.

The function came in the 1930s, when Otto Warburg and colleagues showed that the nicotinamide ring is the part that accepts and gives up a hydride, which made the molecule a hydrogen carrier between enzymes. In 1937 Conrad Elvehjem identified nicotinic acid deficiency as the cause of pellagra, which tied a dietary vitamin to the supply of this coenzyme. The second role, as a substrate that enzymes consume, was established decades later with the discovery of poly(ADP-ribose) and then of the sirtuins.

Structure: two nucleotides and one reactive ring

NAD+ is made of adenosine monophosphate and nicotinamide mononucleotide, linked by a pyrophosphate bridge between the 5′ positions of their two ribose sugars. Three parts of that structure explain most of its chemistry.

• The nicotinamide ring. In NAD+ the ring is an aromatic pyridinium. It accepts a hydride ion (a proton and two electrons) at its C4 position to become NADH, in which the ring is no longer aromatic. The reaction is reversible, which is why one molecule can shuttle electrons many times.
• The glycosidic bond to nicotinamide. The bond between the nicotinamide and its ribose is a high-energy bond. The enzymes that consume NAD+ all break it, releasing nicotinamide and using the remaining ADP-ribose.
• The 2′ hydroxyl on the adenosine ribose. Phosphorylating this position gives NADP+, a separate coenzyme pool that cells keep largely reduced and use for biosynthesis.

The two forms can be told apart by light absorption. Both absorb in the ultraviolet near 260 nm because of the adenine. Only the reduced form, NADH, has a second absorption band at 340 nm, with a molar absorption coefficient of 6,220 M⁻¹cm⁻¹. That difference is the basis of a large share of classical enzyme assays, which follow a reaction by watching absorbance at 340 nm rise or fall.

How cells make NAD+

The review by Covarrubias and colleagues in Nature Reviews Molecular Cell Biology describes three routes to NAD+ in mammalian cells.

• The de novo pathway starts from the amino acid tryptophan and runs through the kynurenine pathway. The enzyme QPRT produces nicotinic acid mononucleotide, which is then converted to NAD+. The review describes this route as mainly active in the liver.
• The Preiss-Handler pathway starts from dietary nicotinic acid. The enzyme NAPRT converts it to nicotinic acid mononucleotide, and the NMNAT enzymes and NAD+ synthetase finish the conversion.
• The salvage pathway recycles nicotinamide, the fragment released every time an enzyme consumes NAD+. NAMPT converts nicotinamide to nicotinamide mononucleotide (NMN), and the NMNAT enzymes convert NMN to NAD+. Nicotinamide riboside (NR) joins the same route through the kinases NRK1 and NRK2. The review states that most NAD+ comes from this pathway.

Because NAD+ is consumed continuously and rebuilt from the nicotinamide that consumption releases, its concentration in a cell is a balance between two rates, not a fixed store. NAMPT is the rate-limiting step of the salvage pathway, which is why NAMPT inhibitors are a standard tool for depleting NAD+ in cell culture.

The enzymes that consume NAD+

In redox reactions NAD+ is converted to NADH and back, and nothing is lost. A second group of enzymes uses it up. The same review names four families.

These enzymes make NAD+ a signal as well as a cofactor. Sirtuin activity depends on NAD+ being available, and heavy PARP activation after DNA damage can draw the pool down.

Compartments and membranes

NAD+ is not spread evenly through the cell. The review describes three main pools, in the cytoplasm, the mitochondria and the nucleus, each regulated independently and each served by its own NMNAT enzyme (NMNAT2 in the cytosol, NMNAT1 in the nucleus, and NMNAT3 proposed for mitochondria). The mitochondrial transporter SLC25A51 is described as responsible for moving intact NAD+ into the organelle.

One point matters for anyone designing a cell-culture experiment: the same review describes NAD+ as not cell permeable. NAD+ added to a culture medium is not expected to cross the plasma membrane intact. It is exposed first to enzymes on the cell surface, such as CD38, and what enters the cell is largely its breakdown products and precursors. An experiment that adds NAD+ to medium and one that raises intracellular NAD+ are therefore not the same experiment, and the literature treats them separately.

NAD+ levels over a lifespan

The observation that brought NAD+ wide attention is that its tissue levels fall with age. Covarrubias and colleagues describe a gradual decline in tissue and cellular NAD+ levels across multiple model organisms, including rodents and humans, with reported declines ranging from roughly 10% to 65% depending on the organ and the age compared.

Why it falls is a separate question. In 2016, Camacho-Pereira and colleagues reported in Cell Metabolism that expression and activity of CD38 rise with age in mouse tissues, that mice lacking CD38 were protected from the age-related decline in NAD+, and that CD38 also degrades the precursor NMN in vivo. That work put increased consumption, and not only reduced synthesis, at the center of the explanation.

Two cautions apply when reading this literature. Measured NAD+ values depend heavily on how a sample was collected and extracted, because the molecule is consumed and interconverted within seconds in a fresh tissue sample. And most of the mechanistic work is in mice and cultured cells, so how far it carries over to people is still an open question.

What happens to NAD+ in plasma

There is little published work on NAD+ itself outside the cell in humans. One pilot study, by Grant and colleagues in Frontiers in Aging Neuroscience in 2019, measured plasma and urine during a six-hour intravenous infusion of 750 mg of NAD+ in saline. Eleven men aged 30 to 55 took part, eight receiving NAD+ and three receiving saline alone.

The authors reported no change in plasma NAD+ until after two hours, and a significant rise only at the six-hour point. Nicotinamide, methylnicotinamide and ADP-ribose rose in plasma, which the authors read as evidence of enzymes breaking NAD+ down. They list the small sample among the study's limitations, and the paper discloses that it was partly funded by a company one of the authors directs. It is a measurement study of where the molecule goes, not a study of effect, and it should be read as that.

Regulatory status

No drug product containing NAD+ is approved by the US Food and Drug Administration. The agency has published a notice reminding compounders to use ingredients suitable for sterile compounding, in which it says it is aware of compounders using food-grade NAD+ to make intravenous products and that food-grade ingredients are not suitable for that purpose without appropriate processing.

Research-grade NAD+ is a laboratory reagent. Anhydrolabs sells it for in-vitro laboratory research only, not for human or veterinary use.

Laboratory handling and storage

NAD+ is supplied as a white, lyophilized powder that takes up water readily. The practical points:

The opposite pH behavior of the two forms is used deliberately in sample preparation: an acid extract preserves NAD+ and destroys NADH, and an alkaline extract does the reverse, so the two can be measured separately from one sample.

Concentration is simple arithmetic from the molecular weight. A 500 mg vial holds about 0.754 millimoles of NAD+, so dissolving it in 10 mL gives 50 mg/mL, or about 75 mM. The peptide calculator does the same conversion for any mass and volume.

Anhydrolabs supplies NAD+ as a lyophilized powder in vacuum-sealed vials, and as 10-vial kits for groups that use it across many assays.