NAD: Introduction to an Important Healthspan Molecule

NAD: Introduction to an Important Healthspan Molecule

Key Learning Objectives:

  • Learn about the different forms of NAD and what they do.
  • Understand why the NAD+ to NADH ratio is important.
  • Discover what happens to the NAD molecules in food during digestion.
  • Find out why “salvaging” niacinamide is critical to sustaining NAD+ levels.
  • Start to appreciate why redundancy in making NAD+ is important.

Introduction

Over the past few years there’s been a tremendous interest in boosting NAD+. In general, NAD+ decreases as we age, while boosting it supports several important healthspan processes and pro-longevity pathways. The attention NAD+ has received is well-deserved. Cells rely on NAD+ to carry out hundreds of metabolic functions, including a vast array of processes ranging from energy creation to maintaining healthy DNA.

In this article (the first in a series of more scientific articles) we’ll be covering the “big picture” when it comes to NAD. We’ll be doing a deeper dive on specific topics we introduce in this article in subsequent articles in this series. As you go through this series of articles please keep in mind that, like other molecules in the body, NAD+ is a means to an end. We don’t care about NAD+ on its own; we care about it because of what it allows cells to do. 

The human body is a complex system. The goal is to support its ability to self-regulate and respond to an ever changing environment. Rather than focusing only on NAD+ in isolation, it’s important to understand where it fits within the complex system and how it can be optimized in the context of improving performance of the whole system. Another thing to keep in mind is that one of our core values is “elevating the conversation.” Simplified information is easy to find … sometimes it’s still very useful, but it can also be misleading. By being better educated we believe it arms our community to ask better questions and see past simple stories. Understanding more about the biochemistry and studies might not be for everyone, but for those that do want to invest the time, we want to do our part to “elevate the conversation.” With these things in mind, let’s get started.

We don’t care about NAD+ on its own; we care about it because of what it allows cells to do.

What is NAD?

Nicotinamide adenine dinucleotide (NAD; also occasionally written diphosphopyridine nucleotide, DPN) (figure 1) is the coenzyme* form of vitamin B3 (niacin; nicotinic acid; niacinamide). It is found in all living cells, where it plays critical roles in cellular energy production (as adenosine triphosphate [ATP]) and several signaling pathways (e.g., sirtuins, PARPs) involved in healthy aging (i.e., healthspan).
*Some enzymes catalyze reactions by themselves, but many require helper substances such as vitamin coenzymes, metal ions, or ribonucleic acid (RNA) for activity.

NAD consists of two nucleotides (“di” being from the Greek and meaning two or twice). The molecule contains several units that are found in many molecules (e.g., ribose, adenine). But it’s the nicotinamide (NAM; niacinamide) unit that gives what is thought of as niacin activity (i.e., vitamin B3). Substances that contain NAM, or which can be converted into NAM in the body, are categorized as niacin equivalents. It’s the presence of niacin equivalents in the diet that is the key to building NAD and the intermediate molecules found in the NAD metabolome*.
*Metabolome is a scientific way of saying the metabolites made from and that make the NAD molecule.

Figure 1. NAD Diagram: Nicotinamide-containing Nucleotide (top) and Adenine-Containing Nucleotide (bottom)

NAD can have an additional phosphate group added to the ribose molecule of the adenine-containing nucleotide by an enzyme called NAD kinase* to create nicotinamide adenine dinucleotide phosphate (NADP).
*In addition to NAD kinase, there are several lesser-known mechanisms of generating NADP, all of which depend on mitochondrial enzymes (e.g., NADP-linked malic enzyme, NADP-linked isocitrate dehydrogenase, NADP-linked glutamate dehydrogenase, nicotinamide nucleotide transhydrogenase).

NAD refers to the what might be best thought of as the core molecule, while NAD+, NADH, NADP+, or NADPH are the forms that NAD exists in when it’s used in the body in redox reactions. There are many other intermediate molecules that are also part of the overall NAD metabolome. In biochemistry books, and some research articles you’ll often see the NAD molecule written as NAD(P). Putting the “P” in parenthesis following NAD is the accepted way to refer to both NAD and NADP rather than writing each.

Any dietary substance that contains or allows the body to build a nicotinamide molecule has some degree of niacin equivalent activity.

What Does the NAD Molecule Do?

NAD(P) as Redox Molecule

NAD(P) reactions play essential roles in many activities of cellular metabolism and energy production. One of these is the transfer of hydrogen (hydride transfer) and electrons (electron transfer) in oxidation or reduction (redox) metabolic reactions. In its redox role, NAD(P) exists in two forms: (1) NAD(P)+ (oxidized), and (2) NAD(P)H (reduced). These forms are converted back and forth as hydrogen (H) is “borrowed” and electrons (e-) are transferred. 

The NAD+/NADH redox reaction is an example. Oxidation is the loss of electrons. Reduction is the gain of electrons. The majority of the NAD(H) molecule in our cells exists in the oxidized NAD+ form. The NAD+ molecule can borrow hydrogen (H+) and carry 2 electrons (2e-). When it borrows the hydrogen (i.e. adds the H+) it also gains electrons, so becomes the reduced NADH form. 

In this NADH form it is an electron carrier, with the electrons serving as stored potential energy. This stored energy will be used to drive a process called oxidative phosphorylation (OXPHOS) that’s used to convert the energy in our food into cellular energy (we’ll talk more about this in the next section). 

During OXPHOS the NADH releases the hydrogen atom (i.e. returns the H+ it borrowed) and releases (i.e., loses) the electrons it carried. This shifts its form back to the oxidized NAD+. This redox reaction is shown in figure 2 for the NAD molecule, but NADP also shuttles between oxidized (NADP+) and reduced (NADPH) forms.

Figure 2. NAD+ ←→ NADH Redox Reaction

NAD+ and NADH are converted back and forth in cellular and mitochondrial reactions that break down food into energy (i.e., ATP).

Shifting between the oxidized NAD(P)+ and reduced NAD(P)H forms as it borrows hydrogens is central to many metabolic processes. But the oxidoreductase enzymes that use NAD rarely use NADP (and vice versa). In general, metabolic processes that use NAD (i.e., shuttling between NAD+ and NADH) in a redox role tend to be catabolic (breaking molecules down into smaller units) and are used to produce a cellular energy molecule called ATP. These catabolic reactions include the suite of cellular processes that breakdown carbohydrates, fats, proteins, and alcohol, so they can be turned into cellular energy.

NADP is used in many anabolic metabolic reactions (constructing macromolecules from smaller units), including fatty acid synthesis and chain elongation, cholesterol synthesis and nucleic acid synthesis. NADPH is also used in several cell protective functions. As an example, it is the source of reducing equivalents for some cytochrome P450 enzymes that detoxify xenobiotics. NADPH also acts as a cofactor for glutathione reductase, the enzyme used to maintain reduced glutathione (GSH) levels by converting its oxidized form, glutathione disulfide (GSSG), back to GSH.[1] 

NADP and NADPH are converted back and forth in reactions that build molecules. NADPH also plays an important role in cellular protection.

NAD(H) and ATP Production

The NAD molecule, whether as NAD(H) or NADP(H) is involved in cellular and mitochondrial redox reactions. But it’s as NAD(H) (i.e., the NAD+ ←→ NADH redox reaction) where it plays a central role in four linked cellular energy pathways. 

One of these linked pathways is “Glycolysis,” which breaks down sugars. It starts inside cells and finishes in specialized organelles within cells called mitochondria. The other three linked pathways occur in mitochondria (i.e., cellular powerhouses). One is called “Beta-Oxidation.” It breaks down fats for energy. Another is called the “Krebs Cycle” (also called tricarboxylic acid cycle or the citric acid cycle). Both of these occur inside mitochondria. The third takes place in the folds (called cristae) of the inner membranes of mitochondria and is called oxidative phosphorylation (OXPHOS or electron transport). These four linked pathways need to perform at their best to optimize the NAD+ : NADH ratio and cellular energy production (in the form of ATP).

When these four linked processes turn food into energy, NAD is converted back and forth between its NAD+ and NADH forms as a way to carry stored energy to produce ATP. Glycolysis, beta-oxidation, and the Krebs Cycle build up NADH at the expense of NAD+ (i.e., NAD+ is sacrificed to produce NADH). These first three processes produce a small net gain of ATP, but an increase of NADH at the expense of NAD+. This NADH is then fed into electron transport (OXPHOS), where NAD+ is regenerated in the process of making the majority of ATP. When all of these four linked pathways are performing well, a high ratio of NAD+ to NADH is maintained. It’s this ratio, more than the absolute amount of NAD+, that appears to be critical for cellular and mitochondrial function.

Figure 3. Linked NAD(H) - ATP Pathways

The beginning processes of converting sugars and fats into energy build up NADH at the expense of NAD+. Mitochondrial OXPHOS turns the NADH back into NAD+ to make most of our ATP. Because of this, mitochondrial OXPHOS plays a big role in maintaining NAD+ levels.

Importance of the NAD+ to NADH Ratio

One of the insights arising from the scientific studies of calorie restriction is that the ratio of NAD+ to NADH (NAD+ : NADH ratio) might be important for the lifespan extension benefits. This ratio has been reported to decline with age, with NAD+ being decreased and NADH increased in older individuals.[2] 

While boosting the amount of NAD+ has been getting a lot of attention, improving the ratio between NAD+ and NADH might be more significant than the amount of cellular NAD+ in isolation.[3] In yeast experiments, calorie restriction decreases NADH much more dramatically than it affects NAD+.[4,5] This decrease in NADH is important for enhancing lifespan, because, on its own, it increases activity of the NAD+-consuming enzymes that boost longevity processes (e.g., Sirtuins) and DNA repair (e.g. PARPs) in yeast. This is thought to occur because NADH is an inhibitor of these enzymes, so lowering it releases the inhibition.[4]

A high ratio of NAD+ to NADH might be more important than the amount of NAD+ in isolation for promoting healthy aging.

Transferring electrons in NAD(P) redox reactions does not have a large effect on the total pool of NAD(P), since, rather than being broken down (catabolized), the NAD(P) molecule is interconverted between oxidized and reduced forms. As a result, redox reactions do not significantly alter total cellular levels of the NAD molecule, they simply shift its forms.[6]

While redox reactions don’t change the total amount of NAD, they can change the ratio of NAD+: NADH (and NADP to NADPH). Inducing reactions that oxidize NADH can shift the ratio in favor of NAD+. As an example, inducing the enzyme NADH: quinone oxidoreductase 1 (NQO1)—an enzyme that uses NADH as an electron donor (NADH → NAD+ + e-  + H+)—increases intracellular NAD+ levels because it shifts the NAD+ : NADH redox ratio in favor of oxidation (NAD+). A side effect of this reaction is that intracellular NAD+ levels increase.[7,8] Upregulation of the pathway that induces NQO1 occurs in calorie restriction and appears to be an important component of producing the benefits.[9,10] 

Cells maintain much higher amounts of the NAD molecule as NAD+, with an estimated 90% of NAD content in uncomplexed proteins being as oxidized NAD+.[11,12] The reason cellular NAD+ is maintained at such a high ratio is thought to be because of its role in several epigenetic transcriptional processes (i.e., processes that regulate how genes respond to the environment). In contrast, cellular NADP+: NADPH ratio favors maintenance of higher amounts of the reduced NADPH (which is correlated with upregulation of cell protective processes such as higher GSH).[1]  

For NAD(P), whether it’s NADand NADH, or NADP+ and NADPH, it’s the ratios, not the molecules in isolation that appear to be critical.

NAD+-Dependent Signaling

NAD(P) molecules are shifted back and forth between their oxidized and reduced forms during redox reactions. This means the NAD redox function alone wouldn’t create a big need to replenish niacins constantly in the diet. To put things in perspective, vitamin B2 (riboflavin) is also used extensively in redox reactions, but its daily value (DV) is 1.3 mg for men and 1.1 mg for women. These are less than 1/10th that of vitamin B3’s DV. Why the difference? Part of the answer is because NAD+ has non-redox uses.

Research on the lifespan extension effects of calorie restriction (CR) have led to the identification of non-redox transcriptional functions of NAD+ (i.e., NAD+-dependent signaling). As we’ve mentioned, CR decreases NADH (in yeast), which increases (i.e, improves) the NAD+ : NADH ratio. A side effect of this is activation of silent information regulator proteins (sirtuins). CR has also increased NADlevels in the brain and liver of mice, where it also activated sirtuin 1 (Sirt1) in both tissues.[13,14] In these studies, sirtuins increase because either more NAD+ is available, less NADH is inhibiting the reaction, or both. In either case, the “fuel,” so to speak, for the increase in sirtuin activity is NAD+. This use of NAD+ is an example of NAD+-dependent signaling.

NAD+-dependent signaling reactions, unlike NAD(P)+ : NAD(P)H redox reactions, where the dinucleotide is not catabolized, do break apart the NAD+ molecule, producing nicotinamide (NAM) as a byproduct (figure 4).[15] Because of this, they are often described as NAD+-consuming reactions (i.e., they consume the NAD+ molecule). The NAM produced in these signaling reactions can be salvaged and used to regenerate NAD+ via a salvage pathway. This salvage of NAM plays a key role in optimizing cellular and mitochondrial function. 

In the redox reactions NAD(P) is shifted back and forth between oxidized and reduced forms, but the core NAD molecule stays intact. In NAD+-dependent signaling the molecule is broken apart, with nicotinamide leftover.

Figure 4. Nicotinamide (NAM; Niacinamide) Diagram

NAD+-consuming reactions can collectively be thought of as adenosine diphosphate (ADP)-ribosyl transfer reactions. Major classes of ADP-ribosyltransferases include: (1) glycohydrolases (NADases that break nucleotides into nucleosides and phosphate) (2) ADP-ribosylases (ADP-ribosylation that add one or more ADP-ribose moieties to a protein), and (3) deacetylases (deacetylations that remove an acetyl group). The ADP-ribosyl transfer reactions that are of the most interest are shown in figure 5 and include: (1) cluster of differentiation 38 (CD38), (2) poly-ADP-ribose polymerases (PARPs), and (3) Sirtuin deacetylases (Sirtuins).*
*These reactions will be discussed in more detail in subsequent articles in this series. 

Maintaining a high ratio of NAD+ to NADH is important, because the NAD+-consuming enzymes mediate many fundamental cellular processes important for healthy aging. They are involved in adjustments to the environmental and in the control of many cellular events. The post-translational protein modifications induced by these enzymes impact gene expression, cell cycle progression, insulin secretion, DNA repair, apoptosis, and aging-associated pathways and processes. 

Figure 5. NAD+-Consuming Reactions

Increasing NAD+ and/or decreasing NADH increase flow through the pathways that use NAD+ to activate sirtuins and PARPS. This enhances many healthy aging and repair processes.

How Do We Increase NAD+?

Its roles in redox and signaling mean that the NAD molecule sits at the crossroads of cellular energy production (as NAD(H)) in OXPHOS reactions and ATP generation), anabolic reactions and cellular protection (as NADP(H)), and epigenetic response (as NAD+-dependent signaling reactions). NAD links cellular metabolism to signaling and transcriptional events needed for a healthy response to biobehavioral circumstances that affect healthspan and lifespan, including exercise and caloric restriction. These combined uses mean that the NAD molecule is an essential currency of cellular metabolism, and suggest that adequate levels, especially of its oxidized NAD+ form used in signaling reactions, is crucial for optimizing cellular functions. But how do we increase it?

To optimize cellular and mitochondrial performance, (1) niacins (vitamin B3) must be consistently supplied in the diet (hence the designation as a vitamin) and (2) nicotinamide (NAM) generated by NADconsuming reactions must be constantly salvaged and recycled to NAD+. Taking supplements containing some type of niacin equivalent has been getting a lot of attention in the anti-aging communities, but supporting salvage is also essential, especially if we want to optimize NAD+ without having to resort to large doses of vitamin B3.

Dietary Niacin

One way to increase NAD is by getting more vitamin B3 (niacins) in the diet. Niacin equivalents* are found in all dietary plant, animal, and fungal foods (like humans these organisms require NAD for life). Meat, eggs, fish, dairy, some vegetables, and whole grains are considered good sources of vitamin B3, but the form and bioavailability of niacin equivalents can differ substantially.
*Niacin equivalents is used to describe all dietary molecules that can contribute to niacin status in the body.

In general, plant foods contain niacin equivalents mostly as nicotinic acid (NA) and nicotinamide (NAM). Small amounts of NMN have also been found in some fruits and vegetables.[16] The niacin equivalents in cereal grains are bound up in a complex with hemicellulose. This is called niacytin and is nutritionally unavailable, because humans do not have the intestinal enzymes needed to liberate the bound niacin. As an example, corn contains abundant NA and NAM, but about 90% is not bioavailable, unless the corn undergoes nixtamalization—soaking and cooking corn in an alkaline solution, usually limewater.[17,18] Baking cereal grains can also improve bioavailability. Niacytin is concentrated in the outer layers of whole grains, which are removed by milling, so whole grains are a better source of niacin equivalents. 

The niacin equivalents in animal products are bioavailable. Milk contains trace amounts of nicotinamide riboside (NR) [19,20] and milk [20], shrimp and meat contain trace amounts of NMN [16], but niacin equivalents in animal foods occur mostly as the NAD+ and NADP molecules.[21]

Niacin equivalents are found in many foods, but bioavailability differs significantly depending on the type of food.

Digestion of Niacin Equivalents

Larger niacin/niacinamide-containing molecules are enzymatically broken down during digestion. Intestinal mucosal (membrane bound or intracellular) enzymes break down NAD(P) niacin equivalents in the diet into NAM.[22–25] This process occurs mostly in the small intestine, where NAD+ is first converted by pyrophosphatases to nicotinamide mononucleotide (NMN). The NMN appears to be rapidly hydrolyzed to NR, with the NR more slowly converted to NAM before absorption occurs. The reaction from NR to NAM appears to be saturable and rate-limiting.[25] This would be expected to theoretically result in a slower rate of absorption and incorporation into NAD+ when NR is taken as a supplement compared to NA or NAM, which was supported by a comparative study that measured liver NAD+ after NA, NAM, and NR.[26]

NAM can be used locally in the gut (for NAD biosynthesis) or absorbed into circulation. It can also be deaminated by nicotinamide deamidase (ND) produced by gut microflora to form NA. NA is very active in the gastrointestinal tract (presumably because of gut microflora) [27], suggesting that there might be significant NAM to NA conversion in the intestines.

While humans lack the nicotinamidase enzyme needed to convert NAM directly to NA, bacteria contain nicotinamidase and can deaminate NAM to NA.[28–30] Gut microflora can also synthesize NAD from niacin and produce niacin equivalents from tryptophan.[31–33] Niacin biosynthesis is present in the majority of the human gut microflora genomes: 63% of all investigated gut microflora genomes contained one or more NAD biosynthesis pathways.[33] 

Similar to other tissues, the gut also uses niacin equivalents for its needs. It’s thought the gut might preferentially use the NA form since intestinal tissue contains all needed enzymes to convert NA to NAD.[6] Recent research suggests that cells have specialized receptors for NMN. Cells in the small intestine express these receptors in much higher amounts than other tissues, which allows the small intestine to take up and use NMN. [34]

The digestive tract contains enzymes that breakdown niacin-containing molecules like NAD+, NADH, nicotinamide riboside and nicotinamide mononucleotide. Available evidence indicates little of these larger molecules survives digestion intact.

What’s the Niacin Daily Value? Is It Enough?

The daily value (DV) for vitamin B3 is 14 mg/d for women and 16 mg/d men. These doses were determined based on studies that measured the amounts of niacin metabolites excreted in the urine (e.g., N1­-methyl­nicotinamide). As greater amounts of niacin are consumed, the amounts of these methylated elimination metabolites increase. The DV was set based on what amount of intake causes a big jump in these metabolites. When given at very high doses nicotinic acid and niacinamide are found unchanged in the urine, suggesting that the capacity to methylate them has been saturated.*[35]
*We’ll discuss this metabolism and elimination in more detail in a subsequent article, focusing in part on what it might imply for dosing and the body’s elimination mechanisms.

It’s important to support NAD+, because levels decline in many tissues with age.[15,36–38] This decline is thought to contribute to the aging process.[39–42] NAD+ levels are decreased in age-related metabolic and neurodegenerative disorders, as well as many circumstances linked with poorer health outcomes. Conversely, restoration of NAD+ to aged animals, by either augmenting biosynthesis or inhibiting consumption, promotes health, improves performance (including mitochondrial function), and extends lifespan in animal experiments.[43–50] These types of studies suggest that the daily value amount might not be sufficient to optimize cellular performance (especially as we age). 

While the DV might not be enough to support healthspan goals, giving very large doses of one or more types of niacin, whether vitamin B3 or newer niacins like NR or NMN, might not be the answer either. Elimination goes up as dose increases (so we’ll tend to waste more as we ramp up dosing). And, in general, despite successfully increasing the amount of NAD+, high doses of NR, as an example, haven’t translated into the expected benefits in metabolic health in humans.[51,52] In keeping with the Goldilocks principle and complexity science tenets, a more moderate dose, while also optimizing how NAD is made and used in the body might be a more sound approach.

While the DV might be insufficient to optimize function, especially as we age, taking excessively high doses of niacins might not be the the answer either.

Importance of “Salvage” to NAD+ Levels

The half-life of NAD+ in mammals is short (up to 10 hours) and the NAD+ pool is used and replenished several times a day.[27,53–58] An estimated 6 to 9 g of NAD+ are required daily to match this constant turnover.[58,59] With this much NAD+ being used, why is the DV so low?

There’s a large mismatch when comparing the DV (14-16 mg) to the estimated daily turnover (6000-9000 mg). The NAD+-consuming reactions are thought to be the primary reason for high turnover, pointing to the importance of salvaging the NAM that’s leftover from these reactions as a means to maintain healthy NAD+ levels.[6,59] As shown in figure 6, while tryptophan (Trp) and the different niacins (NAM, NA, NR) can generate NAD+, it’s the NAM that’s split off during the consuming reactions that needs to be continuously salvaged and recycled to maintain NAD+ levels. Efficient salvage of NAM allows the body to keep pace with NAD+ turnover.

We use much more NAD+ daily than even mega-dosing of vitamin B3 could keep up with. Most of the NAD+ used by our cells isn’t built from the vitamin B3 we get from food or supplements, it’s rebuilt from recycled NAM.

Figure 6. NAD+ Inputs and Uses

What’s the Best Niacin to Boost NAD+?

Since the oxidized NAD+ molecule is at the crossroads for major healthspan (i.e., healthy aging) and lifespan (i.e., long life) signaling processes, boosting NAD+ biosynthesis has been drawing attention as a therapeutic intervention to support healthy aging. A question often asked is, “What’s the best niacin to boost NAD+?”

Any precursor containing a niacin/niacinamide molecule has niacin equivalent activity. The niacin/niacinamide-containing precursors range from lower to higher molecular complexity (i.e., ranging from further removed to structurally closer to a finished NAD molecule). This continuum starts with the original vitamin B3 forms nicotinic acid (NA) and niacinamide (NAM). NR is a nucleoside—the NAM added to a 5-sided ribose sugar. NMN is a nucleotide—a nucleoside with one or more phosphates. NAD+, NADH, and NADP are the complete molecule. These more complicated molecules provide a more direct pathway to the finished NAD molecule … if the digestive system is removed, which isn’t the case with oral dosing. 

Because of the role of digestion, it’s important to focus on niacin equivalent research that has used oral dosing. Injected niacins (whether intraperitoneal [i.p.] or into tissues) and cell culture experiments can advance understanding of cellular response and might help predict what could occur following an intravenous (i.v.) dose. But these types of studies are less likely to be predictive of pharmacokinetics and response following an oral dose. 

NAM and NA are used in the intestines to generate NAD+ (NA might be preferred for this) and absorbed from the intestines to enter the bloodstream for distribution to the liver and peripheral tissues.[23,27] While NAM is the primary circulating form of niacin [30,60] both of these forms of vitamin B3 increase tissue levels of NAD+ with each having some degree of tissue-specificity and different pharmacokinetics (we’ll discuss more about this in future articles on the Preiss-Handler Pathway and Salvage Pathway).

While different niacin forms might have unique abilities to increase NAD+ in some tissues in cell culture studies or when injected in animals (especially when salvage pathway enzymes are knocked out or inhibited), these effects can’t be assumed to occur with oral dosing, because the intestines and liver have substantial ability to metabolize niacin equivalents. As an example, intact molecules of nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) were found in multiple tissues following i.v. dosing, but the same niacins given orally were metabolized to nicotinamide (NAM) in the liver.[61] 

While nicotinamide riboside and nicotinamide mononucleotide (NMN) were found in multiple tissues following i.v. dosing, the same niacins given orally were metabolized to nicotinamide before making it to peripheral tissues.

In another study that used double-labelling of oral NR, while 54% of the hepatic NAD+ contained one heavy atom following a high dose of NR (i.e., parts of NR helped make liver NAD+), only 5% incorporated both heavy atoms (i.e., very little of the whole NR molecule made it to the liver). Peripheral tissue measurements for double-labelled NR were not done, [26]. There’s currently no evidence any NR gets past the liver intact—the existing evidence indicates it does not.[61] This does not mean NR wouldn’t positively impact the NAD metabolome in peripheral tissues; it means that when it does, it might be unrelated to the NR getting to these tissues in one piece, so to speak, and instead be because it is acting as a precursor for NAM.

Existing data suggests that there’s a very limited ability for the complete NR molecule to survive digestion and make it to the liver intact to be incorporated into NAD even following a high dose. This is consistent with earlier research that reported NR in the intestines was slowly converted to NAM prior to absorption.[25] NR can increase levels of NAD+ [62,63], but in a comparative study done in animals, all the niacins given (NR, NAM, and NA) served as precursors for liver NAD+ production, each with differing speeds and effects on the liver NAD metabolome over 24 hours.[26]

NAD+ and NADH are also sold as dietary supplements. As previously mentioned, intestinal enzymes cleave most NAD(P)H to NAM (via NMN and NR) and NAM can be converted to NA in the intestines.[22–25,27–30] So there’s little reason to believe that supplementing with NAD+ would survive digestion intact to offer a significant advantage over NAM or NA. 

In a study that examined urinary excretion of NAM and niacin metabolites, both oral NAM and NAD+ produced similar excretion levels, suggesting the NAD+ had similar (but not better) niacin activity. However, oral NADH did not increase urinary excretion of NAM and its metabolites, suggesting it was decomposed during digestion to compounds that did not have niacin activity.[64] Lastly, NAD+ and NADH, as oxidizing and reducing agents respectively, are unstable. Their suitability for inclusion as part of a multi-ingredient stack would be suspect.

Despite the importance of generating NAD+, there’s a paucity of data comparing oral dosing of niacin forms (NA, NAM, NR, NMN) to each other.

Complexity Science Embraces Redundancy

There’s a great deal of functional redundancy in NAD+ generation (i.e., there are several biosynthetic pathways capable of producing NAD+). Different tissues express the necessary enzymes and are thought to have more and less capacity for biosynthesis. These capacities can also change because of circumstances such as age and health.[6]

It’s critical to fully support functional redundancy. It’s likely that there’s some benefits to each pathway (in complexity science redundancy is a feature and a benefit). Science has known this to be true for the older niacins (NA and NAM) for many decades, with, as an example, NA lowering cholesterol while NAM does not. NA might have more NAD+ activity in tissues with higher amounts of enzymes used in the Preiss-Handler pathway (e.g., small intestines, kidney).[65,66] NAM and the salvage pathway might be the preferred means for NAD+ replenishment in many peripheral tissues, including the brain and skeletal muscle, where its enzymes have higher expression.[38,67]. Tryptophan can also be made into niacin via a de novo synthesis pathway, which adds to the functional redundancy.

Giving high doses of a single type of niacin, whether NA, NAM, NR, etc., does not take advantage of this redundancy. We believe a better approach for long-term health, and an approach that’s consistent with complexity science principles, is to support the functional redundancy inherent in the human body for NAD maintenance. This entails providing several substrates for NAD biosynthesis, as well as supporting rate-limiting steps in the different pathways. 

While support of biosynthesis is one part of an approach focused on optimizing self-regulatory capacities, an overall solution should also influence consumption pathways in manners that more closely replicate activity levels of these pathways found in younger, healthier persons. Specifically, this means upregulating sirtuin activity, reducing NAD+ consumption by CD38 (and to a lesser extent PARPs), and supporting a higher ratio of NAD+ : NADH by upregulation of NQO1 (we’ll be doing a deeper dive about each of these in future articles).

Similar to a food dish, the recipe for assisting the body to self-regulate the NAD metabolome to meet demands, especially the increased metabolic demands that arise with aging, stress, and even healthy habits like exercise, might be better when it includes more than one ingredient. 

RELATED CONTENT:


REFERENCES: 

1. Pollak N, Dölle C, Ziegler M. The power to reduce: pyridine nucleotides--small molecules with a multitude of functions. Biochem J. 2007;402: 205–218. doi:10.1042/BJ20061638
2. Clement J, Wong M, Poljak A, Sachdev P, Braidy N. The Plasma NAD+ Metabolome Is Dysregulated in “Normal” Aging. Rejuvenation Res. 2018; doi:10.1089/rej.2018.2077
3. Ying W. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal. 2008;10: 179–206. doi:10.1089/ars.2007.1672
4. Lin S-J, Ford E, Haigis M, Liszt G, Guarente L. Calorie restriction extends yeast life span by lowering the level of NADH. Genes Dev. 2004;18: 12–16. doi:10.1101/gad.1164804
5. Evans C, Bogan KL, Song P, Burant CF, Kennedy RT, Brenner C. NAD+ metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity. BMC Chem Biol. 2010;10: 2. doi:10.1186/1472-6769-10-2
6. Bogan KL, Brenner C. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition. Annu Rev Nutr. 2008;28: 115–130. doi:10.1146/annurev.nutr.28.061807.155443
7. Ross D, Kepa JK, Winski SL, Beall HD, Anwar A, Siegel D. NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms. Chem Biol Interact. 2000;129: 77–97. doi:10.1016/S0009-2797(00)00199-X
8. Gaikwad A, Long DJ, Stringer JL, Jaiswal AK. In Vivo Role of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) in the Regulation of Intracellular Redox State and Accumulation of Abdominal Adipose Tissue. J Biol Chem. 2001;276: 22559–22564. doi:10.1074/jbc.M101053200
9. Pearson KJ, Lewis KN, Price NL, Chang JW, Perez E, Cascajo MV, et al. Nrf2 mediates cancer protection but not prolongevity induced by caloric restriction. Proc Natl Acad Sci U S A. 2008;105: 2325–2330. doi:10.1073/pnas.0712162105
10. Diaz-Ruiz A, Lanasa M, Garcia J, Mora H, Fan F, Martin-Montalvo A, et al. Overexpression of CYB5R3 and NQO1, two NAD+ -producing enzymes, mimics aspects of caloric restriction. Aging Cell. 2018; e12767. doi:10.1111/acel.12767
11. Tischler ME, Friedrichs D, Coll K, Williamson JR. Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats. Arch Biochem Biophys. 1977;184: 222–236. doi:10.1016/0003-9861(77)90346-0
12. Williamson DH, Lund P, Krebs HA. The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J. 1967;103: 514–527.
13. Qin W, Yang T, Ho L, Zhao Z, Wang J, Chen L, et al. J Biol Chem. 2006;281: 21745–21754. doi:10.1074/jbc.M602909200
14. Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P. Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature. 2005;434: 113–118. doi:10.1038/nature03354
15. Gomes AP, Price NL, Ling AJY, Moslehi JJ, Montgomery MK, Rajman L, et al. Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging. Cell. 2013;155: 1624–1638. doi:10.1016/j.cell.2013.11.037
16. Mills KF, Yoshida S, Stein LR, Grozio A, Kubota S, Sasaki Y, et al. Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice. Cell Metab. 2016;24: 795–806. doi:10.1016/j.cmet.2016.09.013
17. Krehl WA, Teply LJ, Elvehjem CA. CORN AS AN ETIOLOGICAL FACTOR IN THE PRODUCTION OF A NICOTINIC ACID DEFICIENCY IN THE RAT. Science. 1945;101: 283. doi:10.1126/science.101.2620.283
18. Krehl WA, Teply LJ, Sarma PS, Elvehjem CA. GROWTH-RETARDING EFFECT OF CORN IN NICOTINIC ACID-LOW RATIONS AND ITS COUNTERACTION BY TRYPTOPHANE. Science. 1945;101: 489–490. doi:10.1126/science.101.2628.489
19. Bieganowski P, Brenner C. Discoveries of Nicotinamide Riboside as a Nutrient and Conserved NRK Genes Establish a Preiss-Handler Independent Route to NAD+ in Fungi and Humans. Cell. Elsevier; 2004;117: 495–502. doi:10.1016/S0092-8674(04)00416-7
20. Ummarino S, Mozzon M, Zamporlini F, Amici A, Mazzola F, Orsomando G, et al. Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay. Food Chem. 2017;221: 161–168. doi:10.1016/j.foodchem.2016.10.032
21. Henderson LM. Niacin. Annu Rev Nutr. 1983;3: 289–307. doi:10.1146/annurev.nu.03.070183.001445
22. Turner JB, Hughes DE. THE ABSORPTION OF SOME B-GROUP VITAMINS BY SURVIVING RAT INTESTINE PREPARATIONS. Exp Physiol. 1962;47: 107–123. doi:10.1113/expphysiol.1962.sp001582
23. Kaplan NO, Goldin A, Humphreys SR, Ciotti MM, Stolzenbach FE. Pyridine nucleotide synthesis in the mouse. J Biol Chem. 1956;219: 287–298.
24. Gross CJ, Henderson LM. Digestion and absorption of NAD by the small intestine of the rat. J Nutr. 1983;113: 412–420. doi:10.1093/jn/113.2.412
25. Baum CL, Selhub J, Rosenberg IH. The hydrolysis of nicotinamide adenine nucleotide by brush border membranes of rat intestine. Biochem J. Portland Press Limited; 1982;204: 203–207. doi:10.1042/bj2040203
26. Trammell SAJ, Schmidt MS, Weidemann BJ, Redpath P, Jaksch F, Dellinger RW, et al. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans. Nat Commun. 2016;7: 12948. doi:10.1038/ncomms12948
27. Ijichi H, Ichiyama A, Hayaishi O. Studies on the Biosynthesis of Nicotinamide Adenine Dinucleotide III. COMPARATIVE IN VIVO STUDIES ON NICOTINIC ACID, NICOTINAMIDE, AND QUINOLINIC ACID AS PRECURSORS OF NICOTINAMIDE ADENINE DINUCLEOTIDE. J Biol Chem. ASBMB; 1966;241: 3701–3707.
28. Tanigawa Y, Shimoyama M, Murashima R, Ito T, Yamaguchi K, Ueda I. The role of microorganisms as a function of nicotinamide deamidation in rat stomach. Biochimica et Biophysica Acta (BBA) - General Subjects. 1970;201: 394–397. doi:10.1016/0304-4165(70)90318-1
29. Bernofsky C. Physiology aspects of pyridine nucleotide regulation in mammals. Mol Cell Biochem. 1980;33: 135–143.
30. Shimoyama M, Tanigawa Y, Ito T, Murashima R, Ueda I, Tomoda T. Nicotinamide deamidation by microorganisms in rat stomach. J Bacteriol. 1971;108: 191–195.
31. Ellinger P, Kader MM. The nicotinamide-saving action of tryptophan and the biosynthesis of nicotinamide by the intestinal flora of the rat. Biochem J. 1949;44: 285–294
32. Ellinger P. The role of intestinal flora and body-tissue in the biosynthesis of nicotinamide in rat and man. Experientia. 1950;6: 144–145. doi:10.1007/BF02153093
33. Magnúsdóttir S, Ravcheev D, de Crécy-Lagard V, Thiele I. Systematic genome assessment of B-vitamin biosynthesis suggests co-operation among gut microbes. Front Genet. 2015;6: 148. doi:10.3389/fgene.2015.00148
34. Grozio A, Mills KF, Yoshino J, Bruzzone S, Sociali G, Tokizane K, et al. Slc12a8 is a nicotinamide mononucleotide transporter. Nature Metabolism. 2019;1: 47–57. doi:10.1038/s42255-018-0009-4
35. Institute of Medicine (US) Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and its Panel on Folate, Other B Vitamins, and Choline. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline [Internet]. Washington (DC): National Academies Press (US); 2012. doi:10.17226/6015
36. Massudi H, Grant R, Braidy N, Guest J, Farnsworth B, Guillemin GJ. Age-associated changes in oxidative stress and NAD+ metabolism in human tissue. PLoS One. 2012;7: e42357. doi:10.1371/journal.pone.0042357
37. Imai S-I, Guarente L. Trends Cell Biol. 2014;24: 464–471. doi:10.1016/j.tcb.2014.04.002
38. Imai S-I, Guarente L. It takes two to tango: NAD+ and sirtuins in aging/longevity control. NPJ Aging Mech Dis. 2016;2: 16017. doi:10.1038/npjamd.2016.17
39. Imai S-I. Dissecting systemic control of metabolism and aging in the NAD World: the importance of SIRT1 and NAMPT-mediated NAD biosynthesis. FEBS Lett. 2011;585: 1657–1662. doi:10.1016/j.febslet.2011.04.060
40. Yoshino J, Mills KF, Yoon MJ, Imai S-I. Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Cell Metab. 2011;14: 528–536. doi:10.1016/j.cmet.2011.08.014
41. Chini CCS, Tarragó MG, Chini EN. NAD and the aging process: Role in life, death and everything in between. Mol Cell Endocrinol. 2017;455: 62–74. doi:10.1016/j.mce.2016.11.003
42. Johnson S, Imai S-I. F1000Res. 2018;7: 132. doi:10.12688/f1000research.12120.1
43. Ramsey KM, Mills KF, Satoh A, Imai S-I. Age-associated loss of Sirt1-mediated enhancement of glucose-stimulated insulin secretion in beta cell-specific Sirt1-overexpressing (BESTO) mice. Aging Cell. 2008;7: 78–88. doi:10.1111/j.1474-9726.2007.00355.x
44. Haigis MC, Sinclair DA. Annu Rev Pathol. 2010;5: 253–295. doi:10.1146/annurev.pathol.4.110807.092250
45. Viscomi C, Bottani E, Civiletto G, Cerutti R, Moggio M, Fagiolari G, et al. In vivo correction of COX deficiency by activation of the AMPK/PGC-1α axis. Cell Metab. 2011;14: 80–90. doi:10.1016/j.cmet.2011.04.011
46. Mouchiroud L, Houtkooper RH, Moullan N, Katsyuba E, Ryu D, Cantó C, et al. The NAD(+)/Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling. Cell. 2013;154: 430–441. doi:10.1016/j.cell.2013.06.016
47. Cerutti R, Pirinen E, Lamperti C, Marchet S, Sauve AA, Li W, et al. Cell Metab. 2014;19: 1042–1049. doi:10.1016/j.cmet.2014.04.001
48. Khan NA, Auranen M, Paetau I, Pirinen E, Euro L, Forsström S, et al. Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3. EMBO Mol Med. 2014;6: 721–731. doi:10.1002/emmm.201403943
49. Frederick DW, Loro E, Liu L, Davila A Jr, Chellappa K, Silverman IM, et al. Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle. Cell Metab. 2016;24: 269–282. doi:10.1016/j.cmet.2016.07.005
50. Yoshino J, Baur JA, Imai S-I. NAD+ Intermediates: The Biology and Therapeutic Potential of NMN and NR. Cell Metab. 2018;27: 513–528. doi:10.1016/j.cmet.2017.11.002
51. Martens CR, Denman BA, Mazzo MR, Armstrong ML, Reisdorph N, McQueen MB, et al. Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults. Nat Commun. 2018;9: 1286. doi:10.1038/s41467-018-03421-7
52. Dollerup OL, Christensen B, Svart M, Schmidt MS, Sulek K, Ringgaard S, et al. A randomized placebo-controlled clinical trial of nicotinamide riboside in obese men: safety, insulin-sensitivity, and lipid-mobilizing effects. Am J Clin Nutr. 2018;108: 343–353. doi:10.1093/ajcn/nqy132
53. Nishizuka Y, Hayaishi O. Studies on the Biosynthesis of Nicotinamide Adenine Dinucleotide I. ENZYMIC SYNTHESIS OF NIACIN RIBONUCLEOTIDES FROM 3-HYDROXYANTHRANILIC ACID IN MAMMALIAN TISSUES. J Biol Chem. American Society for Biochemistry and Molecular Biology; 1963;238: 3369–3377.
54. Elliott G, Rechsteiner M. Pyridine nucleotide metabolism in mitotic cells. J Cell Physiol. 1975;86: 641–651. doi:10.1002/jcp.1040860509
55. Rechsteiner M, Hillyard D, Olivera BM. Turnover at nicotinamide adenine dinucleotide in cultures of human cells. J Cell Physiol. 1976;88: 207–217. doi:10.1002/jcp.1040880210
56. Rechsteiner M, Hillyard D, Olivera BM. Magnitude and significance of NAD turnover in human cell line D98/AH2. Nature. Nature Publishing Group; 1976;259: 695. doi:10.1038/259695a0
57. Williams GT, Lau KM, Coote JM, Johnstone AP. NAD metabolism and mitogen stimulation of human lymphocytes. Exp Cell Res. 1985;160: 419–426.
58. Yang Y, Sauve AA. NAD+ metabolism: Bioenergetics, signaling and manipulation for therapy. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 2016;1864: 1787–1800. doi:10.1016/j.bbapap.2016.06.014
59. Chiarugi A, Dölle C, Felici R, Ziegler M.  Nat Rev Cancer. 2012;12: 741–752. doi:10.1038/nrc3340
60. Chaykin S, Dagani M, Johnson L, Samli M, Battaile J. Biochimica et Biophysica Acta (BBA) - General Subjects. 1965;100: 351–365. doi:10.1016/0304-4165(65)90004-8
61. Liu L, Su X, Quinn WJ 3rd, Hui S, Krukenberg K, Frederick DW, et al. Quantitative Analysis of NAD Synthesis-Breakdown Fluxes. Cell Metab. 2018;27: 1067–1080.e5. doi:10.1016/j.cmet.2018.03.018
62. Nikiforov A, Dölle C, Niere M, Ziegler M. Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells: FROM ENTRY OF EXTRACELLULAR PRECURSORS TO MITOCHONDRIAL NAD GENERATION. J Biol Chem. 2011;286: 21767–21778. doi:10.1074/jbc.M110.213298
63. Airhart SE, Shireman LM, Risler LJ, Anderson GD, Nagana Gowda GA, Raftery D, et al. An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers. PLoS One. 2017;12: e0186459. doi:10.1371/journal.pone.0186459
64. Kimura N, Fukuwatari T, Sasaki R, Shibata K. Comparison of Metabolic Fates of Nicotinamide, NAD+ and NADH Administered Orally and Intraperitoneally; Characterization of Oral NADH. J Nutr Sci Vitaminol . 2006;52: 142–148. doi:10.3177/jnsv.52.142
65. Shibata K, Hayakawa T, Taguchi H, Iwai K. Regulation of Pyridine Nucleotide Coenzyme Metabolism. In: Schwarcz R, Young SN, Brown RR, editors. Kynurenine and Serotonin Pathways: Progress in Tryptophan Research. Boston, MA: Springer New York; 1991. pp. 207–218. doi:10.1007/978-1-4684-5952-4_19
66. Hara N, Yamada K, Shibata T, Osago H, Hashimoto T, Tsuchiya M. Elevation of cellular NAD levels by nicotinic acid and involvement of nicotinic acid phosphoribosyltransferase in human cells. J Biol Chem. 2007;282: 24574–24582. doi:10.1074/jbc.M610357200
67. 
Revollo JR, Grimm AA, Imai S-I. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J Biol Chem. 2004;279: 50754–50763. doi:10.1074/jbc.M408388200

2 Comments

  • Jeremy
    What about studies showing that nicotinamide and its derivatives/precursors inhibit sirtuins and lead to hypomethylation? My understanding is that NR was originally targeted as a supplement because it could be converted directly to NAD+ without driving up levels of NAM which has anti-NAD+ effects. It seems like increasing dietary niacin will only lead to negative aging effects. The hunt is still on for an effective non-NAM route to increasing NAD+. I'm curious why Neurohacker Collective is promoting dietary niacin as a potential method of increasing NAD+ when its inhibition of protective pathways has long been established? Below are a few relevant sources but I can provide many more if needed. http://www.jbc.org/content/277/47/45099.short https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.0020040 https://www.cambridge.org/core/journals/british-journal-of-nutrition/article/nicotinamide-supplementation-induces-detrimental-metabolic-and-epigenetic-changes-in-developing-rats/2D0BC868E50D39D88EA4EB5A0B98817E https://www.tandfonline.com/doi/abs/10.3109/13880209.2012.697175
  • Greg Kelly, ND
    Lead Product Formulator
    Great questions, they show you understand the issues with just trying to boost NAD+ by supplying its precursors without taking a complex systems science approach. We have future articles in production covering your questions in greater detail. But for now we want to highlight a few things. (1) All precursors that build the NAD molecule increase niacinamide (NAM). In fact, in the only comparative study to date, NR increased liver NAM levels to a greater degree than NAM itself did, and niacin (nicotinic acid or NA) had the least effect (see figure 5D in reference 26 [Trammel 2009]). This is why Neurohacker believes it is so important to support NAM salvage, which is why we wrote about it above (IMPORTANCE OF “SALVAGE” TO NAD+ LEVELS) and provide nutritional support for salvage pathway enzymes in our new product Eternus. (2) All NAD+ precursors tax methylation to some degree (you can see this in fig 5E in reference 26 [Trammel 2009]). This is a dose-dependent issue (higher doses tax methylation much more than lower doses) and is why Neurohacker believes it's important to use more modest doses of NAD+ precursors and to also support methylation processes at the same time (which is why Eternus contains the methyl forms of folic acid and B12). (3) While NR is promoted as something that can be converted directly to NAD+ without driving up levels of NAM, in practice this doesn't occur following an oral dose (again see figure 5D in reference 26 [Trammel 2009]). (4) As you point out, in vitro, NAM is a non-competitive sirtuin inhibitor at high concentrations. But, because this inhibition occurs in cell cultures at high concentrations, does not mean it would occur in the body at cellular concentrations of NAM. In studies where NAM has been given to living animals, in contrast to the predicted in vitro results, NAM increases sirtuin activity. The reason for the different in vitro and in vivo result is likely because, in our cells and tissues, NAM does not build up to the concentrations needed to inhibit sirtuin activity. It’s fairly rapidly converted to NMN, which then produces NAD+, assuming an efficient flow in the salvage pathway is maintained, which is another reason we think it's essential to support salvage.
Sign in or Register to Comment