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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.
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.
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.
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. 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.
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.
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).
For NAD(P), whether it’s NAD+ and NADH, or NADP+ and NADPH, it’s the ratios, not the molecules in isolation that appear to be critical.
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 NAD+ levels 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). 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 NAD+ consuming 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.
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. 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 , shrimp and meat contain trace amounts of NMN , but niacin equivalents in animal foods occur mostly as the NAD+ and NADP molecules.
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. 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.
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) , 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.
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. 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. 
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.*
*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.
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, . There’s currently no evidence any NR gets past the liver intact—the existing evidence indicates it does not. 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. 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.
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. 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.
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.
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
NAD+ CONSUMPTION USES
Key Learning Objectives
- Learn why NAD+ is said to be “consumed” when used for signaling.
- Introduce the 3 main NAD+ “consumers.”
- Find out why overconsumption of NAD+ by one consumer leaves less for others.
- Discover how consumption uses change with aging.
WHAT DOES NAD+ DO?
The NAD(P) molecule was originally identified as a cofactor in cellular redox reactions (see section “NAD(P) as Redox Molecule” in NAD: Introduction to an Important Healthspan Molecule article).
In one of these redox roles, NAD(H) flips back and forth between NAD+ and NADH. This interconversion is used in the four linked processes—glycolysis, beta-oxidation, Krebs cycle, and electron transport—that allow cells and their mitochondrial networks to convert sugars and fats into cellular energy (i.e., ATP).
Figure 1. Cells and Mitochondria Use NAD(H) to Make ATP
The other major redox form is NADP, which shifts between NADP+ and NADPH to help cells make bigger molecules from smaller ones, and to enable a variety of cellular protective functions.
When the NAD(P) molecule is involved in redox, it doesn’t get “used up.” It’s changed between different forms, but the core molecule is conserved. If these were the only jobs cells needed the NAD(P) molecule to do, we’d require much less vitamin B3 activity in the diet.
But redox isn’t the only NAD(P) job. The NAD+ form of the molecule is required for certain cellular signaling reactions that change the way cells behave. Unlike redox, where the molecule is conserved, the NAD+ molecule is broken apart or “consumed” when used for signaling. It’s these NAD+ consumption uses that have been a main reason for the resurgence of scientific interest in NAD+ and strategies to boost it. And it’s this role that increases the need for niacin equivalents in the diet.
NAD+ consumption means that the molecule is being used almost like fuel to activate different signaling processes within cells.
Dietary compounds with vitamin B3 activity are niacin equivalents,(1) because they can be used to build the nicotinamide (niacinamide; NAM) unit of NAD+ (see upper pink box in figure 3). Niacin equivalent substrates include (1) NAM, (2) nicotinic acid (niacin, NA), (3) nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), and (5) L-tryptophan (Trp).
Science has been discovering that increasing the amount of niacin equivalents in the diet produces more NAD+, which leads to more being available for consumption uses. The end result is better cellular and mitochondrial performance.
Figure 2. Making and Using the NAD+ Molecule
WHAT IS NAD+ CONSUMPTION?
NAD+ consumption uses are called adenosine diphosphate (ADP)-ribosyl transfer reactions. This is because, when NAD+ is used in these reactions, enzymes transfer ADP-ribose (ADPR) and/or ADPR polymers (i.e., chains of ADPR units linked together), usually onto an acceptor molecule as part of the reaction.(2)
Figure 3 (NAD+ diagram) shows the entire NAD+ molecule. An ADP-ribosyl transfer reaction breaks the molecule apart, cleaving off the NAM unit (upper pink box). ADPR is all the remaining units of the NAD+ molecule (e.g., adenine, ribose sugars, and phosphates): it’s what gets transferred.
Each ADP-ribosyl transfer reaction is said to consume an NAD+ because of this breaking off of the NAM unit. The leftover NAM is a “salvageable” byproduct: it can be recycled to make a new NAD+ molecule. Regeneration of NAD+ from NAM occurs via the aptly named salvage pathway (see How is NAD+ Made? Part 3: Salvage). This recycling of NAM is essential for maintaining a pool of NAD+ molecules that can be used as needed for NAD+ signaling.
No matter how NAD+ might be made initially, consumption uses always increases niacinamide (NAM). To maintain the NAD+ pool NAM must be efficiently salvaged.
Major classes of ADP-ribosyltransferases include:
- Glycohydrolases (NADases that break nucleotides into nucleosides and phosphate)
- ADP-ribosylases (ADP-ribosylation that add one or more ADP-ribose moieties to a protein), and
- Deacetylases (Deacetylations that remove an acetyl group).
ADP-ribosyl transfer reactions mediate fundamental cellular processes, many of which are critical for healthy aging. They are involved in metabolic adjustments to the environment, influencing how genes respond to stress, diet, and lifestyle. They also impact cell division and growth, insulin secretion, DNA repair, elimination of damaged cells and proteins, cell signaling (such as AMPK, mTOR), and other pathways and processes needed to allow cells to function.
Figure 3. NAD+ Diagram
CONSUMPTION COMPETES FOR AVAILABLE NAD+
The main NAD+ consumption uses in humans are:
- Poly-ADP-ribose polymerases (PARPs)*
- Sirtuin deacetylases (Sirtuins)*
- Cluster of differentiation 38 (CD38)*
*These reactions will be discussed in more detail in subsequent articles in this series.
PARPs, Sirtuins, and CD38 compete for the same finite pool of NAD+. Excess demand by one, leaves less available for others. If utilization of NAD+ in one or more consumption uses is too great, it can lead to NAD+ depletion.(2–9)
Circumstances will dictate which consuming activities are the largest consumers of the available NAD+. In general, circumstances thought of as producing health (e.g. exercise, nutritious diet, sleep) favor use of NAD+ by Sirtuins, while stresses such as DNA damage and inflammation promote activity of PARPs and CD38, leaving less NAD+ available for Sirtuins. When less NAD+ is available for Sirtuins, it can cause a number of problems.
The 3 main NAD+ consumers are (1) Sirtuins, (2) PARPs, and (3) CD38. If one consumption use is too active, it can, in a sense, starve the others.
Sirtuins are products of our genes. They are important because they turn on and off many other genes, especially genes that determine how cells respond to stress. Sirtuins act a bit like a master regulator, dictating the way many genes express themselves. They play an especially important role in coordinating how cells adapt to nutritional status (including calorie restriction or fasting), exercise, stress, toxicity, and other environmental challenges.
The lifespan and healthspan extension effects of calorie restriction are, as an example, at least in part dependent on an increase in Sirtuin activation.(10–15) During calorie restriction, Sirtuins increase because 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+.
Figure 4. NAD+ Consumption Jobs
CONSUMPTION USES CHANGE WITH AGING
Increasing the expression and activity of Sirtuins promotes the cellular and mitochondrial processes needed for both healthier aging and longevity. So, in general, we want to have (1) greater amounts of NAD+ available for consumption use, and (2) more of it to flow to Sirtuins. Neither of these appears to occur with aging.
NAD+ levels decline in many tissues with age (9, 16, 17) and this decline is thought to contribute to the aging process.(7, 18–20) Increased activity of CD38, and to a lesser extent PARPs (presumably to counter accumulating DNA damage), with aging appear to be responsible for some of the age-related NAD+ decline.(21–24)
Stressing the NAD+ pool because of too much demand by PARPs can also have a side-effect of depleting cellular ATP levels, leading to cellular energy failure, which if left unchecked, can result in cellular dysfunction and eventually cell death.(25–28)
The big picture is that as we get older we don’t make enough NAD+. And what we do make gets used differently. Since the consumption uses are competing for a finite pool of NAD+, this also means less would be available for sirtuin activity. Conversely, when the amount of available NAD+ and sirtuin use of it are enhanced, tissues function in healthier ways.(29) The same is true when strategies are used to inhibit either CD38 (22, 30, 31) or PARPs,(21, 24): in both instances, the freeing up of NAD+ for use by sirtuins support aspects of cellular and mitochondrial function.
From a complex systems perspective, the strategy for healthier aging shouldn’t be to only increase NAD+; it should be to increase NAD+ while influencing how it’s being used.
1. 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 (National Academies Press (US), Washington (DC), 2012).
2. P. Belenky, K. L. Bogan, C. Brenner, Trends Biochem. Sci. 32, 12–19 (2007).
3. L. R. Stein, S.-I. Imai, Trends Endocrinol. Metab. 23, 420–428 (2012).
4. R. H. Houtkooper, C. Cantó, R. J. Wanders, J. Auwerx, Endocr. Rev. 31, 194–223 (2010).
5. A. A. Sauve, J. Pharmacol. Exp. Ther. 324, 883–893 (2008).
6. G. Magni et al., Cell. Mol. Life Sci. 61, 19–34 (2004).
7. S.-I. Imai, L. Guarente, NPJ Aging Mech Dis. 2, 16017 (2016).
8. C. Cantó, K. J. Menzies, J. Auwerx, Cell Metab. 22, 31–53 (2015).
9. S.-I. Imai, L. Guarente, Trends Cell Biol. 24, 464–471 (2014).
10. W. Qin et al., Neuronal SIRT1 Activation as a Novel Mechanism Underlying the Prevention of Alzheimer Disease Amyloid Neuropathology by Calorie Restriction. Journal of Biological Chemistry. 281 (2006), pp. 21745–21754.
11. W. Giblin, M. E. Skinner, D. B. Lombard, Trends Genet. 30, 271–286 (2014).
12. L. Guarente, Genes Dev. 27, 2072–2085 (2013).
13. J. Curtis, R. de Cabo, Utilizing Calorie Restriction to Evaluate the Role of Sirtuins in Healthspan and Lifespan of Mice. Sirtuins (2013), pp. 303–311.
14. M. V. Blagosklonny, Linking calorie restriction to longevity through sirtuins and autophagy: any role for TOR. Cell Death & Disease. 1 (2010), pp. e12–e12.
15. A. Zullo, E. Simone, M. Grimaldi, V. Musto, F. Mancini, Sirtuins as Mediator of the Anti-Ageing Effects of Calorie Restriction in Skeletal and Cardiac Muscle. International Journal of Molecular Sciences. 19 (2018), p. 928.
16. H. Massudi et al., Age-Associated Changes In Oxidative Stress and NAD Metabolism In Human Tissue. PLoS ONE. 7 (2012), p. e42357.
17. A. P. Gomes et al., Declining NAD Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging. Cell. 155 (2013), pp. 1624–1638.
18. C. C. S. Chini, M. G. Tarragó, E. N. Chini, Mol. Cell. Endocrinol. 455, 62–74 (2017).
19. S.-I. Imai, FEBS Lett. 585, 1657–1662 (2011).
20. S. Johnson, S.-I. Imai, F1000Res. 7, 132 (2018).
21. P. Bai et al., Cell Metab. 13, 461–468 (2011).
22. J. Camacho-Pereira et al., CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism. Cell Metabolism. 23 (2016), pp. 1127–1139.
23. S. Veith, A. Mangerich, RecQ helicases and PARP1 team up in maintaining genome integrity. Ageing Research Reviews. 23 (2015), pp. 12–28.
24. L. Mouchiroud et al., The NAD /Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling. Cell. 154 (2013), pp. 430–441.
25. N. A. Berger, J. L. Sims, D. M. Catino, S. J. Berger, Princess Takamatsu Symp. 13, 219–226 (1983).
26. N. A. Berger, Radiat. Res. 101, 4–15 (1985).
27. S. Chatterjee, S. J. Berger, N. A. Berger, Poly(ADP-ribose) polymerase: A guardian of the genome that facilitates DNA repair by protecting against DNA recombination. ADP-Ribosylation Reactions: From Bacterial Pathogenesis to Cancer (1999), pp. 23–30.
28. J. Krietsch, M. Rouleau, M. Lebel, G. Poirier, J.-Y. Masson, Poly(ADP) Ribose Polymerase at the Interface of DNA Damage Signaling and DNA Repair. Advances in DNA Repair in Cancer Therapy (2013), pp. 167–186.
29. G. Aragonès et al., Dietary proanthocyanidins boost hepatic NAD metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats. Scientific Reports. 6 (2016), , doi:10.1038/srep24977.
30. M. T. P. Barbosa et al., The enzyme CD38 (a NAD glycohydrolase, EC 188.8.131.52) is necessary for the development of diet-induced obesity. The FASEB Journal. 21 (2007), pp. 3629–3639.
31. C. Escande et al., Flavonoid Apigenin Is an Inhibitor of the NAD ase CD38: Implications for Cellular NAD Metabolism, Protein Acetylation, and Treatment of Metabolic Syndrome. Diabetes 2013;62:1084-1093. Diabetes. 63 (2014), pp. 1428–1428.
HOW IS NAD+ MADE? PREISS-HANDLER PATHWAY
HOW IS NAD+ MADE?
Key Learning Objectives
- Learn how NAD+ is made from niacin (nicotinic acid).
- Find out which form of vitamin B3 boosts liver NAD+ levels the fastest.
- Discover which type of vitamin B3 might be preferred by the gut microbiome.
- Understand why boosting ATP is critical for making NAD+ from niacin.
THE PREISS–HANDLER PATHWAY: INTRODUCTION
The common name for nicotinic acid (NA) is niacin (NIcotinic ACid + vitamIN). It was the 3rd of the B-complex family of vitamins discovered, hence its designation as vitamin B3.
In 1958, Jack Preiss and Philip Handler published a scientific paper describing how NAD+ was made from NA in three steps.(1) This pathway was later named the Preiss-Handler pathway after the co-discoverers. It describes the enzyme steps needed to convert NA into the NAD+ molecule.
Until the recent introduction of newer NAD+ precursors, NA and nicotinamide (NAM; niacinamide) had been grouped together as vitamin B3. They are thought to have complementary roles for NAD+ biosynthesis.(2)
Figure 1. Nicotinic Acid Diagram
THE PREISS–HANDLER PATHWAY: NAPRT ENZYME
The Preiss-Handler pathway begins with NA, whether the NA originates in food, from a dietary supplement, or is produced by the bacterial microflora in the intestines or saliva.
The first enzymatic reaction catalyzes the conversion of NA to its mononucleotide form, nicotinic acid mononucleotide (NaMN), by the enzyme nicotinic acid phosphoribosyltransferase (NaPRT). The reaction uses 5-phosphoribosyl-1-pyrophosphate (PRPP) as a cosubstrate.
NaMN is the same molecule produced as an end-product from L-tryptophan in de novo synthesis, so is common to both pathways (see How is NAD+ Made? De Novo Synthesis).
The most important factor for driving the pathway from niacin to NAD+ forward might be having more niacin available.
While phosphate activates the enzyme, the critical factor for inducing the enzyme appears to be the availability of NA.(3, 4) NaPRT is insensitive to the physiological concentration of NAD+ (i.e., having higher amounts of NAD+ doesn’t shut the enzyme off or slow it down). And NaPRT does not appear to be subject to feedback inhibition by NaMN. So NA availability appears to drive the overall pathway towards NAD+ synthesis.(2)
NaPRT is concentrated in liver, kidney, and small intestine, but is also expressed in other organs, including the brain and skeletal muscle, suggesting that many tissues can use NA to produce NAD+.(5–8) Tissues containing significant NaPRT activity are thought to use NA as the preferred source for NAD+ biosynthesis.(2, 9–11)
L-tryptophan can also be used to make the NaMN intermediate molecule formed in the Preiss-Handler pathway.
THE PREISS–HANDLER PATHWAY: NMNAT ENZYME
NaMN (whether produced by NA or L-tryptophan) is transformed into its dinucleotide form, nicotinic acid-adenine dinucleotide (NaAD), by a group of ATP-dependent isoenzymes collectively called nicotinate mononucleotide adenylyltransferase (NMNAT).
The NMNAT enzymes are common to both the Preiss-Handler and Salvage pathways. NMNAT is thought to be the rate-limiting step, but its activity might be functionally less important than having NA available in our cells. As an example, NaMN is rapidly converted to NaAD by NMNAT when red blood cells are exposed to physiological amounts of NA.(12) However, during the aging process, it appears that NMNAT might become functionally limiting, with its substrate (NaMN) being at the same levels in both young and older adults, but its product (NaAD) being reduced in older individuals.(13)
The NMNAT enzyme is considered rate-limiting, with its activity appearing to be one of the issues causing NAD+ levels to be decreased in older age.
There are three forms of NMNAT (NMNAT1, -2, and -3) in humans. These forms have distinct tissue and subcellular localizations. Human tissues including brain, heart, kidney, liver, lung, and skeletal muscle express one or more of the three NMNAT enzymes. Similar to the first step in the pathways which uses the NAPRT enzyme, the presence of this second enzyme in these tissues is evidence that they use NA to make NAD+.
In biochemical terms, NaAD formation consists of the adenylation of the pyridine mononucleotide, in this case NaMN. Adenylation is also known as AMPylation, because it attaches adenosine monophosphate (AMP) to NaNM to form the NaAD dinucleoside. ATP is the source of the AMP, so is catabolized in this reaction.(14–16)
Scientists think some tissues prefer using niacin to make NAD+.
THE PREISS–HANDLER PATHWAY: NADS ENZYME
In the final step in the pathway, NaAD is amidated to NAD+ by glutamine-dependent NAD+ synthase (NADS). Humans have two types of NADS: one is strongly expressed in the kidney, liver, and small intestine; the other predominates in the brain.
Similar to what occurs in the NMNAT enzyme step, ATP is consumed in this reaction, producing AMP and diphosphate, along with NAD+.(17)
We need ATP, cellular energy, to convert niacin into NAD+. It’s needed for two of the three enzyme steps.
Figure 2. Preiss-Handler Pathway of NAD+ Biosynthesis
USING NIACIN TO MAKE NAD+
The core ingredient in this stack is niacin (nicotinic acid; NA). It is a normal cellular metabolite in humans and a substrate for NAD+ generation in many tissues.(2)
Pharmacological doses of NA have been used to support healthy cholesterol levels in humans since the 1950s,(18) and have been extensively studied for prolonged periods of time. One of the main side effects at these doses is flushing.
NA is often thought of as the “flushing” vitamin B3, because, at the high doses used for cholesterol support, a majority of persons experience a flush of red on the skin about 30 to 60 minutes after taking NA. This is often accompanied by a tingling, itching or burning sensation.
Flushing tends to be dose-dependent: it occurs more frequently with higher doses. As an example, in an early study of niacin (from 1938), 5 percent of people taking 50 mg experienced flushing, but this increased to 50 percent at a dose of 100 mg and at 500 mg of niacin all individuals experienced flushing.(19) In a more recent study almost 25 percent of people taking 50 mg of NA twice daily reported flushing.(20) The lowest dose where mild flushing has been reported is 30 mg.(21)
It’s important to use a low dose of niacin (30 mg or lower) if the goal is to minimize the chances of experiencing flushing.
In addition to flushing, long-term supplementation with high doses of NA decreased insulin sensitivity (an unwanted metabolic effect),(22–24) so NA should not be considered a more is better supplement.
We used a dose below the threshold where flushing has been reported to occur. This is well below the dose required to produce unwanted metabolic effects. This dose would be expected to have modest effects on NAD+ if given alone, but additive effects with other NAD+ precursors and supportive ingredients in the overall formulation.
While it does not have the cache value of the newer niacins, NA markedly increases NAD+ levels in tissues, including liver, kidney, heart, skin, bone marrow, and white blood cells following oral dosing.(25–37)
Oral niacin has increased NAD+ in tissues including liver, kidney, heart and white blood cells in studies.
NA is very active in the gastrointestinal tract (presumably because of gut microflora).(8) It’s thought that NA might be the preferred vitamin B3 form to produce NAD+ in the small intestines.(2, 38)
While humans lack the nicotinamidase enzyme needed to convert NAM directly to NA, bacteria contain nicotinamidase and can deaminate NAM to NA.(39–41) Gut microflora can also synthesize NAD+ from niacin and produce niacin equivalents from tryptophan.(42–45) Niacin biosynthesis is present in the majority of human gut microflora genomes: 63% of all investigated gut microflora genomes contained one or more NAD+ biosynthesis pathways.(45)
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+.(10)
Niacin might be the preferred way of boosting NAD+ in the gut.
In general, tissue-specific increases in NAD+ levels following NA supplementation seem to correlate well with the tissues that have significant NaPRT activity, including colon, heart, kidney, and liver.(46)
Classical feeding studies reported that NA is a better NAD+ precursor than NAM in the liver, intestine, and kidney, all of which have high NaPRT activity.(24, 33)
Increased NAD+ has been reported with low doses of NA and responses have been more pronounced in persons with lower initial NAD+ levels.(47) A dose of 100 mg/d of NA for 8 weeks resulted in a progressive rise in lymphocyte NAD+ to nearly 5 times baseline levels.(48)
NA has upregulated poly(ADP-ribosylation) (PARP)—an NAD+ consumption enzyme involved in DNA repair—and/or DNA repair efficiency.(49) NA supplementation upregulates several other cell signaling pathways, including expression of transcription factors peroxisome proliferator-activated receptor (PPAR) α and δ, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in skeletal muscle,(50) and sirtuins.(51, 52)
In addition to possibly being the preferred niacin form to produce NAD+ in the intestines,(2)—which would imply that less NA is available to leave the digestive system since more of the oral dose would be used there—NA has produced the quickest increase in liver NAD+ levels.
Niacin has resulted in the fastest increase in liver NAD+ of the different forms of vitamin B3 studied.
In a study done in mice that measured liver response to equal doses of NA, NAM and nicotinamide riboside (NR), NA produced the fastest increase in both NAD+ and NADP+, more than doubling liver NAD+ at 2 hours. NA also produced the quickest increase in ADP-ribose derivative (ADPR). The increase in ADPR is evidence that NA efficiently upregulated NAD+-consuming activities rapidly.(35)
The rapid increase of liver NAD+ with NA found in the recent study is consistent with research from the 1960’s—NA arriving at the liver was able to increase NAD+ within minutes (reaching a peak after about 10 minutes) and sustain higher NAD+ higher liver levels for more than 8 hours.(8) This quick response might be explained by a bilitranslocase protein present in epithelial cells of the gastric mucosa, which has high affinity for NA but not for other forms of niacin.(53) This protein would be predicted to allow NA to be rapidly absorbed, which matches experimental results.
NA and NAM additively support NAD+ maintenance. It’s thought that NA and NAM are complementary since they are metabolized through different enzyme pathways.(2) When administered together, high doses of NAM do not prevent NA from increasing liver NAD+ levels.(45)
Some tissues appear to use niacin and niacinamide (NAM) differently as circumstances change … so it makes sense to give some of both. This gives cells more options for doing what they need.
It makes sense to supplement both NA and NAM, because responses to each appear to change depending upon the circumstances. High doses of NA (500 and 1000 mg/kg diet) elevated NAD+ in the blood, liver, heart and kidney, while the same doses of NAM only elevated blood and liver NAD+ (rat study). NAM, but not NA, elevated liver poly(ADP-ribose) (PARP) in control conditions, but when rats were exposed to a liver toxin, NA, but not NAM supplementation caused a greater accumulation of PARP.(54) This suggests that circumstances might significantly influence tissue response to different niacin equivalents, and that putting all the eggs in one niacin equivalent basket, so to speak, might not be the most prudent approach.
It’s been suggested that supplementing both NA and NAM together may be better than the administration of NA or NAM alone.(55) We agree with this opinion and expect NA to have additive effects with other NAD+-generating substrates (both L-tryptophan and NAM). In other words, rather than supporting NAD+ through just one pathway (or one form of niacin equivalent), better self-regulation might occur—at lower individual doses of each molecule—when more options are given.
Making the NAD+ molecule is an example of a special type of biological redundancy called degeneracy. Degeneracy means that structurally dissimilar components/modules/pathways can perform similar functions (i.e. are effectively interchangeable) under certain conditions, but perform distinct functions in other conditions. Supporting several nutritionally unique ways to make the NAD+ molecule better supports the complex self-regulatory mechanisms involved in NAD+ maintenance. In other words, it gives more choices for cells to do what they need to do as circumstances change. So, rather than stressing one pathway by giving high doses of one type of niacin equivalent and hoping for the best, we believe it’s better to support all of them, but in moderation. This takes advantage of the functional redundancy for NAD+ generation in many tissues.
Giving a low amount of niacin plus other NAD+ precursors supports a type of redundancy called degeneracy. We think it’s important to help cells help themselves in the different ways they have of getting jobs done.
SUPPORTING THE PREISS-HANDLER PATHWAY: BOOSTING ATP
In addition to supplying NA, similar to other pathways, it’s important to offer full pathway support. This means that Preiss-Handler enzymes should be supported. Since NaMN is generated from both L-tryptophan and NA, supporting the NMNAT enzymes that convert the mononucleotide (NaMN) to its dinucleotide (NaAD) affects both the L-tryptophan de novo synthesis and the niacin Preiss-Handler pathway. If the activity of the NMNAT enzyme is slowed both pathways will be affected. ATP is catabolized in this reaction, so is an essential part of enzyme function.(12, 14, 16) ATP is also used in the next (and last) NADS enzyme reaction.
A study that measured levels of different metabolites of NAD+ pathways with aging found that NaMN levels did not change with older age, but levels of both NaAD and NAD+ decreased with age.(13) These findings, as well as other changes in substrates and products of enzymes that were found in aging, suggests that ATP status might be limiting the ability to make NAD+ from niacin and L-tryptophan, and to have healthy flow through the NAD+ metabolome as we get older. This suggests that ATP performance might be a bigger issue in the age-related decline in NAD+ levels than availability of the nutrient precursors.
Enzymes that use ATP in the different NAD+ pathways all seem to underperform in older age. In other words, it is ATP that appears to be the issue.
There are a number of ingredients in the formulation that support ATP production, including cofactors and supportive nutrients for electron transport and the Krebs cycle, as well as polyphenol and other support for mitochondrial optimization. Mitochondrial nutrients include CoQ10, PQQ, lipoic acid, and L-carnitine, as examples. We’ll go into more detail of full support for ATP in a subsequent article; however, we want to take a moment to highlight several ingredients that play substantials roles in ATP function.
ElevATP® has increased whole blood and intramuscular ATP concentrations in humans,(56, 57) which would be supportive of both NMNAT and NADS, allowing better flow from niacin to NAD+.
Creatine acts as an intracellular buffer for ATP, so is an important part of ATP homeostatic support. Creatine is used in the phosphocreatine (phosphagen) system to regenerate ATP from ADP in tissues. This system is especially important in circumstances where there’s high energy demand. In skeletal muscle, as an example, creatine phosphate (phosphocreatine) acts as a reservoir of high-energy phosphoryl groups that can be readily transferred to ADP to regenerate ATP through the action of the enzyme creatine kinase during higher intensity exercise.(58, 59)
ATP must be bound to a magnesium ion (Mg2+) in order to be biologically active: What is called ATP in cells usually occurs as a magnesium-ATP complex.(60, 61) Because of this magnesium status is essential for ATP performance.
Creatine and magnesium are examples of ingredients needed for ATP to perform well … to do its cellular energy job.
SUPPORTING THE PREISS-HANDLER PATHWAY: UPREGULATING NADS
NADS converts NaAD to NAD+. High doses of niacin equivalents (including NA, NAM, and NR) have produced huge relative increases in NaAD. NAM and NR produced greater increases than NA. This was a surprise since neither NAM nor NR can be directly converted into NaAD through any known pathway. NA was the only precursor expected to proceed to NAD+ through an NaAD intermediate.(35) The cellular mechanism for this substantial increase in NaAD from NAM and NR hasn’t been determined, but it’s at least possible that some of the increase might occur because the NADS enzyme reaction, similar to many reactions, is reversible at high NAD+ concentrations. The known NA pathway and these findings collectively suggest that supporting the function of the NaAD enzyme, especially when higher amounts of niacin equivalents are supplemented, might be a good idea (i.e., if we are building up higher amounts of its substrate we want to help the enzyme make its product).
BioVin® French Red Grapes Extract is included in this stack because consumption of grape seed proanthocyanidins dose-dependently and robustly increased liver NAD+ levels and sirtuin activity in healthy rats. Supplementation also dose-dependently upregulated NADS, with effects appearing at the lowest dose (comparable to about 56 mg for a 70 kg human adult).(62)
Polyphenol compounds called proanthocyanidins found in grapes help support the production of NAD+ from niacin.
SUPPORTING NIACIN METABOLISM AND ELIMINATION
The last part of the Preiss-Handler stack deals with improving capacity to handle elimination of NA. Niacin equivalents are constantly being eliminated in the urine as part of normal metabolism. As the amounts of niacin equivalents are increased in the diet (or with supplementation), the amount of metabolites found in the urine also increase.
Part of the reason for the relatively low daily values (DV) for niacin equivalents is because, at amounts much greater than the DV, metabolites found in the urine increase substantially.(63) At very high doses, such as the several grams/day that’s used to manage cholesterol, as much as 75% of NA is eliminated through the urine after 96 hours either as unmodified NA or one of its metabolites.(64) In a sense, this means much of the very high dose was wasted, but still required metabolic work to eliminate.
While the dose we selected for niacin in isolation would be considered low, the combination of niacin, niacinamide and L-tryptophan is an additive stack for augmenting niacin equivalents. So we think it’s prudent to support the processes involved in biotransformation and elimination, since they are being used under normal circumstances and might be more taxed as niacin equivalents in the diet increase.
NA is metabolized by the liver in one of two general ways: It is conjugated with glycine (these metabolites contribute to flushing) or methylated. In general, glycine conjugation is more important for NA, while methylation is more important for NAM; however, excess NA does use both biotransformation pathways.(35, 55, 65, 66)
Glycine support is provided using the glycine salt of magnesium (Magnesium Glycinate).
Methylation requires proper functioning of the folate cycle. Folate (as a combination of folic acid, calcium L-5′-methyltetrahydrofolate, and calcium folinate), vitamin B12 (as a mix of methylcobalamin and adenosylcobalamin) and riboflavin (vitamin B2) are used for the folate cycle as it interacts with the methionine cycle in methylation. Magnesium, vitamin B6 (as pyridoxal 5′-phosphate) and cysteine (as N-acetyl-L-cysteine) play roles in either the methionine cycle or the breakdown of one of its intermediates called homocysteine.(67)
Excess niacin equivalents are eliminated in the urine. We think it’s important to provide nutrient support so these elimination pathways don’t get over-stressed.
Figure 3. Preiss-Handler Support Stack
1. J. Preiss, P. Handler, J. Biol. Chem. 233, 493–500 (1958).
2. N. Hara et al., J. Biol. Chem. 282, 24574–24582 (2007).
3. J. W. Gross, M. Rajavel, C. Grubmeyer, Biochemistry. 37, 4189–4199 (1998).
4. L. Galassi et al., Biochimie. 94, 300–309 (2012).
5. J. Preiss, P. Handler, J. Am. Chem. Soc. 79, 1514–1515 (1957).
6. J. Imsande, P. Handler, Nicotinic acid mononucleotide pyrophosphorylase. J Biol Chem. 236, 525–530 (1961).
7. J. Imsande, J. Preiss, P. Handler, in Methods in Enzymology (Academic Press, 1963;vol. 6, pp. 345–352.
8. H. Ijichi, A. Ichiyama, O. Hayaishi, J. Biol. Chem. 241, 3701–3707 (1966).
9. K. Shibata, T. Hayakawa, H. Taguchi, K. Iwai, in Kynurenine and Serotonin Pathways: Progress in Tryptophan Research, R. Schwarcz, S. N. Young, R. R. Brown, Eds. (Springer New York, Boston, MA, 1991; pp. 207–218.
10. K. L. Bogan, C. Brenner, Annu. Rev. Nutr. 28, 115–130 (2008).
11. F. Zamporlini et al., FEBS J. 281, 5104–5119 (2014).
12. V. Micheli, H. A. Simmonds, S. Sestini, C. Ricci, Arch. Biochem. Biophys. 283, 40–45 (1990).
13. J. Clement, M. Wong, A. Poljak, P. Sachdev, N. Braidy, Rejuvenation Res. (2018), doi:10.1089/rej.2018.2077.
14. M. Schweiger et al., FEBS Lett. 492, 95–100 (2001).
15. F. Berger, C. Lau, M. Ziegler, Proc. Natl. Acad. Sci. U. S. A. 104, 3765–3770 (2007).
16. A. A.-B. Badawy, Int. J. Tryptophan Res. 10, 1178646917691938 (2017).
17. N. Hara et al., J. Biol. Chem. 278, 10914–10921 (2003).
18. J. R. Crouse III, Coron. Artery Dis. 7, 321–326 (1996).
19. T. D. Spies, W. B. Bean, R. E. Stone, J. Am. Med. Assoc. 111, 584–592 (1938).
20. J. Wink, G. Giacoppe, J. King, Am. Heart J. 143, 514–518 (2002).
21. W. H. Sebrell, R. E. Butler, JAMA. 111, 2286–2287 (1938).
22. S. Westphal, K. Borucki, E. Taneva, R. Makarova, C. Luley, Atherosclerosis. 193, 361–365 (2007).
23. E. Fabbrini et al., J. Clin. Endocrinol. Metab. 95, 2727–2735 (2010).
24. G. Fraterrigo et al., Cardiorenal Med. 2, 211–217 (2012).
25. B. Petrack, P. Greengard, H. Kalinsky, J. Biol. Chem. 241, 2367–2372 (1966).
26. O. Hayaishi, H. Ijichi, A. Ichiyama, Adv. Enzyme Regul. 5, 9–22 (1967).
27. P. B. Collins, S. Chaykin, Biochem. J. 125, 117P–117P (1971).
28. L. F. Lin, L. M. Henderson, J. Biol. Chem. 247, 8023–8030 (1972).
29. J. T. MacGregor, A. Burkhalter, Biochem. Pharmacol. 22, 2645–2658 (1973).
30. G. M. McCreanor, D. A. Bender, Br. J. Nutr. 56, 577–586 (1986).
31. D. A. Bender, R. Olufunwa, Br. J. Nutr. 59, 279–287 (1988).
32. G. J. Hageman, R. H. Stierum, M. H. van Herwijnen, M. S. van der Veer, J. C. Kleinjans, Nutr. Cancer. 32, 113–120 (1998).
33. Q. Li et al., Alcohol. Clin. Exp. Res. 38, 1982–1992 (2014).
34. T. M. Jackson, J. M. Rawling, B. D. Roebuck, J. B. Kirkland, J. Nutr. 125, 1455–1461 (1995).
35. S. A. J. Trammell et al., Nat. Commun. 7, 12948 (2016).
36. H. L. Gensler, T. Williams, A. C. Huang, E. L. Jacobson, Nutr. Cancer. 34, 36–41 (1999).
37. A. C. Boyonoski et al., J. Nutr. 132, 115–120 (2002).
38. K. Shibata, T. Hayakawa, K. Iwai, Agric. Biol. Chem. 50, 3037–3041 (1986).
39. Y. Tanigawa et al., Biochimica et Biophysica Acta (BBA) – General Subjects. 201, 394–397 (1970).
40. M. Shimoyama et al., J. Bacteriol. 108, 191–195 (1971).
41. C. Bernofsky, Mol. Cell. Biochem. 33, 135–143 (1980).
42. P. Ellinger, M. M. Kader, Biochem. J. 44, 285–294 (1949).
43. P. Ellinger, Experientia. 6, 144–145 (1950).
44. Nutr. Rev. 4, 76–78 (1946).
45. S. Magnúsdóttir, D. Ravcheev, V. de Crécy-Lagard, I. Thiele, Front. Genet. 6, 148 (2015).
46. J. T. Eppig et al., Nucleic Acids Res. 33, D471–5 (2005).
47. T. M. Jackson, J. M. Rawling, B. D. Roebuck, J. B. Kirkland, J. Nutr. 125, 1455–1461 (1995).
48. A. B. Weitberg, Mutation Research/Environmental Mutagenesis and Related Subjects. 216, 197–201 (1989).
49. K. Weidele, S. Beneke, A. Bürkle, DNA Repair . 52, 12–23 (2017).
50. M. J. Watt, R. J. Southgate, A. G. Holmes, M. A. Febbraio, J. Mol. Endocrinol. 33, 533–544 (2004).
51. Y. Li et al., J. Nutr. Biochem. 26, 1338–1347 (2015).
52. Y. Li et al., Int. Immunopharmacol. 40, 211–218 (2016).
53. S. Passamonti, L. Battiston, G. L. Sottocasa, FEBS Lett. 482, 167–168 (2000).
54. H. Ijichi, A. Ichiyama, O. Hayaishi, J. Biol. Chem. (1966).
55. K. Shibata, T. Fukuwatari, C. Suzuki, J. Nutr. Sci. Vitaminol. . 60, 86–93 (2014).
56. T. Reyes-Izquierdo et al., J Aging Res Clin Practice. 2, 178–184 (2013).
57. T. Reyes-Izquierdo, C. Shu, R. Argumedo, B. Nemzer, Z. Pietrzkowski, J Aging Res Clin Pract. 3, 56–60 (2014).
58. P. L. Greenhaff, J. Physiol. 537, 657–657 (2001).
59. L. Guimarães-Ferreira, Einstein . 12, 126–131 (2014).
60. R. M. Touyz, Front. Biosci. 9, 1278–1293 (2004).
61. K. Pasternak, J. Kocot, A. Horecka, Journal of Elementology. 15, 601–616 (2010).
62. G. Aragonès et al., Sci. Rep. 6, 24977 (2016).
63. 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 (National Academies Press (US), Washington (DC), 2012.
64. R. M. Menon et al., J. Clin. Pharmacol. 47, 681–688 (2007).
65. K. Shibata, J. Nutr. 119, 892–895 (1989).
66. D. Li et al., Pharm. Biol. 51, 8–12 (2013).
67. A. L. Miller, G. S. Kelly, Altern. Med. Rev. 1, 220–235 (1996).
Neurohacker = source
HOW IS NAD+ MADE? DE NOVO SYNTHESIS
WHAT IS NAD+ AND HOW IS IT MADE?
Key Learning Objectives
- Start to appreciate why redundancy is important in living systems.
- Introduce the three pathways used to make NAD+.
- Discover how L-tryptophan at breakfast influences what happens at night.
- Learn about how L-tryptophan is used to make NAD+.
- Find out which dietary nutrients support making NAD+ from L-tryptophan
- Understand why preventing a build-up of quinolinic acid (QA) is important.
COMPLEX SYSTEMS SCIENCE AND REDUNDANCY
Complexity science is not about one thing in isolation; it’s about interacting networks of things (including our cellular network with mitochondrial networks and both with the gut microbiome), redundancy, self-regulation, and whole system responses. In this article (the second in our scientific series on NAD+) we’ll be introducing the redundancy in making the NAD+ molecule. Redundancy implies having more than one way to do something. In biological systems it means that, if plan A fails, there’s a backup plan … and probably a backup to the backup. This is exactly what we see with making the NAD+ molecule.
Biological complex adaptive systems often have more than one way to accomplish the same outcome. This allows for robustness, flexibility, and adaptability. These attributes are needed for successfully surviving in the face of real world challenges. Given the importance of NAD+ in cellular energy production, nutrient sensing, and many other functions, it should be no surprise that there’s more than one way we make it.
NAD+ can be made via the “De Novo Pathway,” starting from the essential amino acid L-tryptophan. It can be made by the “Preiss-Handler pathway,” using nicotinic acid, a form of vitamin B3 usually called niacin, which is known for producing flushing when taken in high amounts. And, it can be produced in the “Salvage Pathway” from niacinamide (nicotinamide), the non-flushing form of vitamin B3. Different tissues use these three pathways to greater or lesser extents to meet changing NAD+ needs. Because, in general, giving more choices allows for better adaptation, it makes sense to support all three pathways.
The type of redundancy that can be used in making NAD+ is called degeneracy. It means that structurally dissimilar components/modules/pathways can perform similar functions (i.e. are effectively interchangeable) under certain conditions, but perform distinct functions in other conditions.
THREE PATHWAYS, ONE MOLECULE
Because of its overall importance, most organisms have several alternatives for producing the NAD+ molecule. In humans, there are three major NAD+ biosynthesis pathways. These are:
NAD+ can be synthesized de novo through multiple enzymatic steps in the kynurenine pathway (KP), ultimately producing nicotinic acid mononucleotide (NaMN) after the last enzyme step. In biochemistry “de novo” means one biomolecule—NAD+ in this case—is synthesized anew from a different molecule. In this pathway, the niacin molecule is essentially built from scratch starting from the essential amino acid L-tryptophan (Trp). This is the only non-vitamin B3 pathway for producing NAD+. The de novo synthesis pathway is complete with the formation of NaMN.
Niacins (i.e., nicotinic acid- or niacinamide-containing compounds) supplied in the diet and/or supplements can be used to synthesize NAD+, with each form entering NAD+ biosynthesis pathways at different points and often relying on one or more unique enzymes.
Nicotinic acid (NA) and nicotinic acid riboside (NAR) produce NAD+ by the Preiss-Handler pathway. The Preiss–Handler pathway starts from a niacin (either NA or NAR). It proceeds to NAD+ in several enzymatic steps. NaNM is an intermediate in the pathway, so de novo synthesis from Trp shares several steps in this pathway to complete NAD+ synthesis.
A “salvage” pathway converts nicotinamide (NAM; Niacinamide) to NAD+ with nicotinamide mononucleotide (NMN) as an intermediate. It is called a salvage pathway, because, no matter what precursor (Trp or any of the niacins) or pathway produces NAD+, after NAD+ is consumed, NAM is generated and is a “salvageable precursor” to re-produce NAD+. So, the salvage pathway is the mechanism used to recycle NAM into NAD+. Nicotinamide riboside [NR] also uses the salvage pathway. Enzymes exist that can convert NR into NAM or NMN. In either instance, NAD+ formed by NR can be used in NAD+-consuming reactions and ends up as NAM. NMN would also be a salvage pathway niacin equivalent.
The NAD+ molecule can be made from the (1) amino acid L-tryptophan, (2) compounds containing a nicotinic acid (i.e., niacin), or (3) compounds containing a nicotinamide (i.e., niacinamide).
Figure 1. NAD+ Biosynthesis – Major Molecules and Pathway
NEUROHACKER COLLECTIVE’S COMPLEXITY SCIENCE APPROACH
Different NAD+ precursor molecules and pathways appear to be more (and less) active in certain tissues. But overall, the three NAD+ biosynthetic routes allow for some degree of functional redundancy. This is important because all humans are not alike, and our capacity for generating NAD+ via each pathway might change in certain tissues because of factors such as genetics, age, and health status.
Aging results in decreased NAD+ and changes to the NAD metabolome—metabolome is a scientific way of saying the metabolites made from and that make the NAD molecule. Alterations in both biosynthesis and consumption appear to stress NAD+ pools in ways that result in insufficient supply to meet the demands of healthy aging. To best support these demands, we believe it’s better to support the functional redundancy created by the three NAD+ biosynthesis pathways. This includes supplying at least one precursor for each pathway and supporting the different pathways with the cofactors needed for enzyme function. It means putting an emphasis on upregulating rate-limiting enzymes. And it might mean, depending on the pathway, nudging flows gently in directions that support higher levels of NAD+ and/or the type of responses seen in younger, healthy persons.
Our complex systems science approach supports all the biosynthesis pathways. We chose this approach because the body is capable of self-regulating NAD+ biosynthesis to meet tissue-specific and subcellular compartment NAD+ demands in ways that science does not completely understand. We believe it can do this more efficiently when it’s being supported comprehensively to create healthy flux through the three different pathways.
We think it’s important to nutritionally support the interconnected NAD+ pathways in ways that create healthy flows of all the molecules in the NAD+ metabolome.
Figure 2. NAD+ Biosynthesis Support Stack
DE NOVO SYNTHESIS PATHWAY SUPPORT: INTRODUCTION
Dietary L-tryptophan (Trp) (figure 3) is classified as a niacin equivalent in humans, because it can be used to generate NAD+ through a de novo synthesis pathway. This pathway forms the pyridine ring common to all niacins, so essentially builds a niacin molecule anew. De novo synthesis starts with Trp and proceeds as this essential amino acid is catabolized through the kynurenine pathway (KP). The KP is a subset of the entire de novo synthesis pathway, with the end-product produced still being an intermediate of NAD+ synthesis.
Figure 3. L-Tryptophan Diagram
In addition to KP, Trp is a precursor molecule for three other pathways. These are (1) hydroxylation (products include serotonin, 5-hydroxy-tryptamine, and melatonin); (2) decarboxylation (products include tryptamine); and (3) transamination (products include indolepyruvic acid). Estimates suggest that ~95% of Trp is degraded through KP, so Trp flux through these other 3 pathways is comparatively minor.[4,5]
L-tryptophan is used to build NAD+ from scratch. This is why the pathway is called ‘de novo,’ which in Latin means anew or from the beginning.
NIACIN EQUIVALENT ACTIVITY OF L-TRYPTOPHAN
Trp appears to generally be independent from (so additive to) niacins/niacinamides for NAD+ generation. As an example, in young women, niacinamide (NAM) supplementation did not affect flux through the de novo pathway, with no observed changes in KP intermediates as NAM doses were increased. The additive nature of Trp with NAM suggests that better self-regulatory benefits might be achieved if this pathway is supported along with pathways that use niacin/niacinamide.
It’s been estimated that 60 mg of Trp is the NAD+ equivalent to 1 mg of niacin in men. In Japanese women a conversion ratio of 67 mg Trp to make 1 mg niacin has been reported. Other conversion ratios have also been reported.[10,11] There’s some degree of individual variability, with many factors affecting conversion, including nutrients, hormones (e.g., cortisol, estrogen), pregnancy, drugs, and diseases. Some of these factors enhance, and others suppress the conversion of Trp to NAD+. In niacin-deficient animals, as an example, Trp has about 1/10 (molar ratio) the activity compared to NAM, indicating it is much more efficient in producing NAD+ under these circumstances.[13,14] Supplying enough Trp can correct vitamin B3 deficiency in animals fed a diet devoid of niacins, so a key point is that, despite any individual variance in conversion, Trp is a useful substrate for NAD+ synthesis, and is an especially important substrate under circumstances where tissues need it to be.
While Trp is not thought to contribute a large amount to the overall NAD+ pool under normal circumstances, there are times where its contribution takes on much greater importance. Trp has been suggested to be the primary source of NAD+ during immune system activation. In general, the flux of Trp into the KP increases with age and inflammation.[16–18] During neuroinflammation (at least in a mouse model of Alzheimer’s disease), 95% of the cerebral pool of Trp is catabolized through the KP.
Under some circumstances, aging and inflammation as examples, L-tryptophan takes on much greater importance in making NAD+.
Trp is thought to be a principal NAD+ precursor utilized in the liver, since the liver contains all the enzymes needed for de novo synthesis.[12,20,21] An estimated 90% of Trp degradation by KP occurs in the liver under normal circumstances. In general, flux through the KP pathway (and NAD+ production) in the liver increases dose-dependently with increasing intake of Trp up to about 5 grams.[22–24]
The NAD+ produced from Trp in the liver appears to be used locally, with nicotinamide (NAM), not the NAD+ molecule, released into circulation to be used in peripheral tissues.[12,25] Increasing Trp intake allows more flux through the KP, which increases the amount of NAM released into the vasculature (potentially upregulating the salvage pathway of NAD+ generation).[20,26,27] Conversely, under conditions of low Trp consumption, enzymes that direct Trp to non-NAD+ biosynthetic routes are down-regulated, suggesting a shift of all possible Trp catabolism to NAD+ generation when lower amounts are available in the diet. These findings highlight the importance of supplying modest amounts of Trp for both liver and systemic NAD+ maintenance.
Extrahepatic KP activity accounts for 5-10% of Trp degradation, and becomes more important during circumstances characterized by inflammation and immune activation. Extrahepatic KP does not include all enzymes of the pathway, so some intermediates of the KP are not produced in all tissues.[21,28]
When L-tryptophan (Trp) in the diet is low, non-NAD+ uses of this amino acid are down-regulated, suggesting the body is prioritizing making NAD+ over other possible uses of Trp.
THE KYNURENINE PATHWAY (SIMPLIFIED)
The kynurenine pathway (KP) is not a linear pathway. Several intermediates (e.g., kynurenine, 3-hydroxykynurenine [3-HK], 2-amino-3-carboxymuconate-6-semialdehyde [ACMS]) can branch Trp degradation to produce other metabolic intermediates (e.g., anthranilic acid [AA], kynurenic acid [KA], picolinic acid [PA], acetyl CoA) rather than continuing towards NAD+.. The simplified pathway described below will focus on the Trp to NAD+ progression.
While this is “simplified,” it does focus on the biochemistry and uses enzyme names and other scientific terms. This section is written for persons who want to know that level of detail. If that’s not you, it’s okay to skip, but do take a look at anything that is in bold and the quotes in bigger font and italics: those will hit the high notes of this section.
Figure 4. Simplified Kynurenine Pathway
Trp is initially converted to N′-formylkynurenine (NFK) by oxidative degradation using either tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO) (both enzymes are heme dependent but are independent of each other). The flux of Trp down the KP is determined primarily by Trp availability, and secondarily by activities of TDO and IDO.
The flux of L-tryptophan (Trp) down the kynurenine pathway (KP) is strongly influenced by how much Trp is available: More available Trp results in greater flow towards NAD+ production.
TDO is expressed mainly in the liver, but also exists and has activity in the brain. It’s responsible for most KP metabolism of Trp and is upregulated by Trp availability and cortisol (as well as other glucocorticoids).[12,17,30] Because of cortisol’s robust circadian rhythm, dietary Trp would be expected to be largely shunted into the KP in the morning. This makes biological sense, since, as an example, we don’t need a big increase in Trp being made into melatonin until nighttime.
IDO is widely expressed in all tissues (including blood and immune cells). IDO is thought to have negligible activity under normal conditions, but is upregulated during immune system activation and inflammation. Its activity also tends to increase with aging.
IDO is regulated by both pro- and anti-inflammatory molecules.[31–34] The principle inducer is interferon-gamma (IFN-γ) in the periphery and interleukin-6 (IL-6) in the central nervous system.[35–37] Other proinflammatory molecules including IFN-α, interleukins (IL-1β, IL-2) and tumor necrosis factor α (TNFα) can induce IDO, but do so with less activity. In general, anti-inflammatory cytokines inhibit IDO induction.
Because of this counter-regulation, IDO status is assumed to be determined by the overall balance between pro- and anti-inflammatory cytokines. During circumstances characterized by increased systemic inflammation or brain neuroinflammation, IDO dramatically increases Trp flux through the KP.[30,39–43] It’s been suggested that the higher activity of IDO might be a self-regulatory adaptation intended to increase overall NAD+ synthesis, so more of this molecule would be available for its consuming enzymes (e.g., PARP, CD38, Sirtuins) to support healthier immune and inflammatory responses.
During inflammation or immune system activation, KP, specifically an enzyme abbreviated IDO, is upregulated. This occurs because tissues need more NAD+ during these circumstances, and use L-tryptophan to make it.
Whether produced using TDO or IDO, NFK is converted to kynurenine (Kyn) by NFK formamidase (FAM), which does not have any known cofactors and is not rate-limiting. Kyn is the first stable metabolite formed in the KP. Very high doses of resveratrol (5 g/d) have increased Kyn in humans (and decreased Trp presumably because more of it is entering the KP). This study did not measure enzyme activity, but since resveratrol down-regulates IFN-γ and pro-inflammatory activation of KP via IDO in vitro, it’s possible that resveratrol upregulated TDO and/or FAM.
The next two steps are dependent on specific B vitamins (which have been included in the stack). Hydroxylation by kynurenine hydroxylase (K monooxygenase [KMO]) forms 3-HK. KMO uses the reduced NADPH coenzyme form of niacin. NADPH is a flavin adenine dinucleotide–dependent enzyme. Because of the flavin dependence, activity of KMO is diminished several-fold during vitamin B2 (riboflavin) deficiency.[46,47]
3-HT is converted to 3-hydroxyanthranilic acid (3-HAA) by kynureninase B (KYNase)—a pyridoxal 5′-phosphate (P5P) (vitamin B6) dependent enzyme. KYNase activity is impaired significantly by vitamin B6 deficiency.[46,48–53] Drugs, especially estrogen-containing therapies (e.g., oral contraceptives, hormone replacement therapy) can stress vitamin B6 status and also decrease KYNase activity.[54–57]
While deficiency of either vitamin B2 or B6 will inhibit flux through the KP, large intakes are not required to support these enzymes. Supplying an amount in the range of daily values (DV) has ensured that these enzymes are not rate-limiting, while significantly higher vitamin B2 and B6 have not further increased formation of KP intermediates formed after these enzymes.[57–60] Because of this, we think it’s prudent to supply approximately the DV amount of these vitamins to ensure adequacy, but high dosing does not appear warranted for KP function.
Vitamin B2 and B6 are used as cofactors in the KP. Ensuring daily values of both are being supplied is important to keep good flow through this pathway.
The next step uses 3-hydroxyanthranilic acid 3,4-dioxygenase (3-HAAO) to convert 3-HAA to ACMS. 3-HAAO is a non-heme iron-dependent (Fe2+) dioxygenase enzyme; it is considered the most active among KP enzymes, rapidly converting 3-HAA into ACMS. So under most circumstances the activity of this enzyme is not an issue. The exception might be during iron deficiency. Iron deficiency has reduced Trp utilization for NAD+ synthesis in animals (presumably because of this enzyme), and relative availability of endogenous iron and proteins that regulate its bioavailability appear to impact activity of 3-HAAO. Our intention is that this stack will be taken long-term. We don’t think it’s prudent to supplement iron for long periods of time unless iron status is being monitored. Because of this, we did not include iron in the stack. In persons who are iron-deficient, correcting iron status might be an important part of optimizing de novo synthesis of NAD+.
ACMS is an unstable intermediate (i.e., it spontaneously gets converted into other molecules) and does not require enzyme activity to convert to quinolinic acid (QA). But, only about 6% of ACMS is spontaneously condensed and rearranged into QA. The majority of ACMS is believed to be shunted away from NAD+ production (towards picolinic acid and acetyl-CoA synthesis) by α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD).[64,65] ACMSD is zinc-dependent. Change in zinc availability has been proposed to influence its activity. ACMSD inhibition (i.e., slowing of activity) nudges more ACMS towards QA and hence NAD+ biosynthesis, while overactivity of ACMSD reduces QA. Increased activity of PPARα down-regulates ACMSD. While not major players in this stack, several of our ingredient choices in the overall formula have upregulated PPARα in pre-clinical studies, so might theoretically nudge pathway flux more towards QA production (and hence in the NAD+ direction). These ingredients include: (1) strawberry seed (standardized for trans-tiliroside), (2) Kaempferia parviflora (black ginger), (3) rosemary extract (standardized for ursolic acid),[73,74] (4) cinnamon, and (5) apigenin.[76,77]
One of our core formulation principles is to have more than one ingredient that might do something and to include ingredients that do more than one thing.
QA production ends the KP portion of de novo synthesis. The next enzymatic step converts QA to a niacin-containing molecule. This step uses quinolinate phosphoribosyl transferase (QPRT) to create Nicotinic Acid Mononucleotide (NaMN)—magnesium and 5-phosphoribosyl-1-pyrophosphate (PRPP) are cosubstrates. NaMN converges with the Preiss-Handler pathway (discussed in the next article in this series): It is a common intermediate in both pathways and both pathways share the same enzymes steps to make NAD+ from this point.
Trp that makes it to QA in the liver is thought to be converted to NAD+ via subsequent enzyme reactions of QPRT and the Preiss-Handler pathway, since all needed enzymes are highly expressed in liver tissue.[78,79] The brain is also a big producer of NAD+ from QA.
QA might be best thought of as a “Goldilocks” molecule—we need some of it, but it is an excitatory molecule, so too much is neurotoxic to nerve cells in the brain. QPRT is expressed in the brain; it plays a major role in protection against the neurotoxic effects of quinolinic acid.[78,80–82] QPRT activity can increases in response to increased levels of QA, suggesting a protective role (i.e., the QPRT enzyme is being upregulated in response to QA to make sure this molecule isn’t allowed to build-up).
The last step in the KP on the journey to NAD+ takes an intermediate called quinolinic acid (QA) and converts it into a niacin-containing compound using an enzyme abbreviated QPRT. This is an important step to support, because we don’t want QA to build-up (it can damage neurons if too much accumulates).
The activity of QPRT can act as a rate-limiting factor for NAD+ generation from Trp. In other words, the enzymes in the KP that make QA from Trp do their jobs, but QPRT can’t keep pace. The result is that QA accumulates. This commonly occurs in tissues, especially the brain and nervous system, during circumstances characterized by inflammation (like the chronic inflammation of aging called “inflammaging”) and immune activation. As an example, although QPRT activity is known to upregulate in response to increases in the levels of QA, its capacity is insufficient to keep up with the QA being produced in circumstances characterized by neuroinflammation, resulting in a build-up of QA.[19,83–96] A similar finding occurs in plasma, where chronic inflammatory circumstances are associated with increased QA.[15,97–99] The highest levels of QA are found in spleen, lymph nodes, thymus, and many specific immune cell types and are increased following stimulation by immune activators. Higher QA levels have also been reported in cardiovascular disease. The key point is that QPRT activity is essential for preventing a build-up of QA.
Activity level of the de novo synthesis pathway is impacted with age,[18,102–106] but in tissue-specific ways. Activity of TDO (the non-inflammation first enzyme in the pathway) decreases significantly with age progression in the brain, liver and kidney. Activity of the inflammation-inducible IDO increases with age in the brain, but decreases in the liver and kidney. Activity of QPRT declines in the brain and liver, but increases in kidneys. Because of the critical importance of QPRT in generating the end-product of the KP (NaMN), the overall pathway performance is downregulated during aging in two tissues that heavily rely on it (e.g., brain, liver). Not surprisingly, in humans, QA levels are positively correlated with age (i.e., the amount of QA increases as we get older).[18,106]
Some enzymes involved in making NAD+ from L-tryptophan decrease with age in tissues like the brain and liver. This affects our ability to maintain healthy pools of NAD+.
Reduced ability to convert QA to NaMN is an issue for several reasons. In certain tissues and cells (e.g., neurons, astrocytes), and under some circumstances (e.g., neuroinflammation, immune system activation), NAD+ production from QA (and hence Trp) appears to be at least, if not more important than that from niacinamide (NAM) (and the salvage pathway) or nicotinic acid (NA) (and the Preiss–Handler pathway).[39,93,107] It’s thought that KP activation is upregulated during neuroinflammation in an attempt to maintain NAD+ levels through de novo synthesis from Trp. If QPRT is unable to effectively convert QA to NaMN, then NAD+ levels will be inadequate to meet cellular needs and tissue demands: the goal of the upregulation will not be met.
In addition to inefficient generation of NAD+, QA itself can be health-damaging. QA is an NMDA agonist and exerts excitotoxic effects on neurons.[108–110] So elevated QA is not only associated with many neuroinflammatory conditions, it is believed to be contributing to worsening them.
Inhibiting the KP—specifically IDO and QPRT—during neuroinflammatory circumstances might solve one problem (the build-up of QA), but cause others. Competitive inhibition of IDO and QPRT activities causes a dose-dependent decrease in intracellular NAD+ levels and sirtuin deacetylase-1 (SIRT1) activity in astrocytes and neurons (neurons are more affected than astrocytes): this results in impaired cell viability.
Because of these reasons, supporting upregulation of QPRT activity and protecting against QA’s health-damaging effects are important. Grape seed proanthocyanidin dose-dependently and robustly increased liver NAD+ levels and sirtuin activity in healthy rats. This was in part a result of increased KP activity. Supplementation increased TDO and also dose-dependently upregulated QPRT, with effects appearing at the lowest dose (comparable to about 56 mg for a 70 kg human adult).
Magnesium acts as core support because it is both a cosubstrate for QPRT and protects neuronal cells against QA toxicity.[112–115] In experiments, higher brain QA leads to increased Mg uptake into the brain, suggesting that if Mg is available in circulation under circumstances characterized by higher brain QA levels, it will be put to good use.
Several other ingredients in the stack have neuroprotective benefits against QA. One of these is L-carnitine.[117,118] Certain polyphenol molecules, including catechin (found in cocoa extract) and apigenin have neuroprotective effects against QA and prevent QA-mediated NAD+ depletion in cultured human neurons.
The last part of the stack has to do with experimental findings indicating that insufficient mineral intake decreased the conversion rate of Trp to NAD+ in rats. As previously mentioned several enzymes in the KP are dependent on minerals (e.g., iron, magnesium, and zinc). The stack contains 70 trace minerals (found in the peat extract component of elevATP®), including these three minerals.
We believe a comprehensive NAD+ stack should offer full pathway support of the KP. This means (1) supplying a modest amount of Trp, (2) including vitamin coenzymes and other nutritional cosubstrates needed to support enzyme function, and (3) and supporting activity of the enzyme step that acts as a functional rate-limited enzyme (QPRT).
1. Rongvaux A, Andris F, Van Gool F, Leo O. Reconstructing eukaryotic NAD metabolism. Bioessays. 2003;25: 683–690. doi:10.1002/bies.10297
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. Johnson S, Imai S-I. NAD + biosynthesis, aging, and disease. F1000Res. 2018;7: 132. doi:10.12688/f1000research.12120.1
4. Bender DA. Biochemistry of tryptophan in health and disease. Mol Aspects Med. 1983;6: 101–197.
5. Badawy AA. Tryptophan metabolism in alcoholism. Adv Exp Med Biol. 1999;467: 265–274.
6. Shin M, Nakakita S, Hashimoto C, Sano K, Umezawa C. NAD+ biosynthesis from tryptophan in the presence of nicotinic acid or vice versa by rat hepatocytes–effect of clofibrate-feeding. Int J Vitam Nutr Res. 1998;68: 104–108.
7. Fukuwatari T, Shibata K. Effect of nicotinamide administration on the tryptophan-nicotinamide pathway in humans. Int J Vitam Nutr Res. 2007;77: 255–262. doi:10.1024/0300-98184.108.40.206
8. Horwitt MK, Harvey CC, Rothwell WS, Cutler JL, Haffron D. Tryptophan-Niacin Relationships in ManStudies with Diets Deficient in Riboflavin and Niacin, Together with Observations on the Excretion of Nitrogen and Niacin Metabolites. J Nutr. Oxford University Press; 1956;60: 1–43. doi:10.1093/jn/60.suppl_1.1
9. Fukuwatari T, Ohta M, Kimura N, Sasaki R, Shibata K. Conversion Ratio of Tryptophan to Niacin in Japanese Women Fed a Purified Diet Conforming to the Japanese Dietary Reference Intakes. J Nutr Sci Vitaminol . 2004;50: 385–391. doi:10.3177/jnsv.50.385
10. Goldsmith GA. Niacin-tryptophan relationships in man and niacin requirement. Am J Clin Nutr. 1958;6: 479–486. doi:10.1093/ajcn/6.5.479
11. Nakagawa I, Takahashi T, Suzuki T, Masana Y. Effect in man of the addition of tryptophan oniacin to the diet on the excretion of their metabolites. J Nutr. 1969;99: 325–330. doi:10.1093/jn/99.3.325
12. Fukuwatari T, Shibata K. Nutritional aspect of tryptophan metabolism. Int J Tryptophan Res. 2013;6: 3–8. doi:10.4137/IJTR.S11588
13. Krehl WA, Bonner D, Yanofsky C. Utilization of niacin precursors and derivatives by the rat and neurospora. J Nutr. 1950;41: 159–172. doi:10.1093/jn/41.1.159
14. Shibata K, Swabe M, Fukuwatari T, Sugimoto E. Efficiency of D-tryptophan as Niacin in rats. Biosci Biotechnol Biochem. 2000;64: 206–209. doi:10.1271/bbb.64.206
15. Moffett JR, Namboodiri MA. Tryptophan and the immune response. Immunol Cell Biol. 2003;81: 247–265. doi:10.1046/j.1440-1711.2003.t01-1-01177.x
16. Frick B, Schroecksnadel K, Neurauter G, Leblhuber F, Fuchs D. Increasing production of homocysteine and neopterin and degradation of tryptophan with older age. Clin Biochem. 2004;37: 684–687. doi:10.1016/j.clinbiochem.2004.02.007
17. Chen Y, Guillemin GJ. Kynurenine Pathway Metabolites in Humans: Disease and Healthy States. Int J. SAGE Publications Ltd STM; 2009;2: IJTR.S2097. doi:10.4137/IJTR.S2097
18. de Bie J, Guest J, Guillemin GJ, Grant R. Central kynurenine pathway shift with age in women. J Neurochem. 2016;136: 995–1003. doi:10.1111/jnc.13496
19. Wu W, Nicolazzo JA, Wen L, Chung R, Stankovic R, Bao SS, et al. Expression of tryptophan 2,3-dioxygenase and production of kynurenine pathway metabolites in triple transgenic mice and human Alzheimer’s disease brain. PLoS One. 2013;8: e59749. doi:10.1371/journal.pone.0059749
20. Bender DA, Magboul BI, Wynick D. Probable mechanisms of regulation of the utilization of dietary tryptophan, nicotinamide and nicotinic acid as precursors of nicotinamide nucleotides in the rat. Br J Nutr. Cambridge University Press; 1982;48: 119–127. doi:10.1079/BJN19820094
21. Fukuwatari T, Morikawa Y, Sugimoto E, Shibata K. Effects of fatty liver induced by niacin-free diet with orotic acid on the metabolism of tryptophan to niacin in rats. Biosci Biotechnol Biochem. 2002;66: 1196–1204. doi:10.1271/bbb.66.1196
22. Shibata K, Matsuo H. Effect of dietary tryptophan levels on the urinary excretion of nicotinamide and its metabolites in rats fed a niacin-free diet or a constant total protein level. J Nutr. 1990;120: 1191–1197. doi:10.1093/jn/120.10.1191
23. Hiratsuka C, Fukuwatari T, Sano M, Saito K, Sasaki S, Shibata K. Supplementing healthy women with up to 5.0 g/d of L-tryptophan has no adverse effects. J Nutr. 2013;143: 859–866. doi:10.3945/jn.112.173823
24. Badawy AA-B, Dougherty DM. Assessment of the Human Kynurenine Pathway: Comparisons and Clinical Implications of Ethnic and Gender Differences in Plasma Tryptophan, Kynurenine Metabolites, and Enzyme Expressions at Baseline and After Acute Tryptophan Loading and Depletion. Int J Tryptophan Res. 2016;9: 31–49. doi:10.4137/IJTR.S38189
25. 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
26. Beadle GW, Mitchell HK, Nyc JF. Kynurenine as an Intermediate in the Formation of Nicotinic Acid from Tryptophane by Neurospora. Proc Natl Acad Sci U S A. National Academy of Sciences; 1947;33: 155–158. doi:10.1073/pnas.33.6.155
27. Bender DA, Olufunwa R. Utilization of tryptophan, nicotinamide and nicotinic acid as precursors for nicotinamide nucleotide synthesis in isolated rat liver cells. Br J Nutr. Cambridge University Press; 1988;59: 279–287. doi:10.1079/BJN19880035
28. Shibata K, Motooka K, Kurata K. The Differences in Growth and Activity of the Tryptophan-NAD Pathway between Wistar and Sprague Dawley Strains of Rats Fed on Tryptophan-Limited Diet. J Nutr Sci Vitaminol . 1982;28: 11–19. doi:10.3177/jnsv.28.11
29. Badawy AA-B. Tryptophan metabolism, disposition and utilization in pregnancy. Biosci Rep. 2015;35. doi:10.1042/BSR20150197
30. Magni G, Amici A, Emanuelli M, Raffaelli N, Ruggieri S. Enzymology of Nad + Synthesis : Mechanism of Enzyme Action, Part A. In: Purich DL, editor. Advances in Enzymology and Related Areas of Molecular Biology. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 1999. pp. 135–182. doi:10.1002/9780470123195.ch5
31. 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.
32. Chiarugi A, Carpenedo R, Molina MT, Mattoli L, Pellicciari R, Moroni F. Comparison of the Neurochemical and Behavioral Effects Resulting from the Inhibition of Kynurenine Hydroxylase and/or Kynureninase. J Neurochem. 2002;65: 1176–1183. doi:10.1046/j.1471-4159.1995.65031176.x
33. Badawy AA-B. Pellagra and alcoholism: a biochemical perspective. Alcohol Alcohol. 2014;49: 238–250. doi:10.1093/alcalc/agu010
34. Badawy AA-B. Tryptophan availability for kynurenine pathway metabolism across the life span: Control mechanisms and focus on aging, exercise, diet and nutritional supplements. Neuropharmacology. 2017;112: 248–263. doi:10.1016/j.neuropharm.2015.11.015
35. Pfefferkorn ER, Rebhun S, Eckel M. Characterization of an indoleamine 2,3-dioxygenase induced by gamma-interferon in cultured human fibroblasts. J Interferon Res. 1986;6: 267–279. doi:10.1089/jir.1986.6.267
36. Werner E, Werner-Felmayer G. Substrate and Cofactor Requirements of Indoleamine 2,3-Dioxygenase in Interferon-Gamma-Treated Cells: Utilization of Oxygen Rather Than Superoxide. CDM. 2007;8: 201–203. doi:10.2174/138920007780362482
37. Samikkannu T, Saiyed ZM, Rao KVK, Babu DK, Rodriguez JW, Papuashvili MN, et al. Differential regulation of indoleamine-2,3-dioxygenase (IDO) by HIV type 1 clade B and C Tat protein. AIDS Res Hum Retroviruses. 2009;25: 329–335. doi:10.1089/aid.2008.0225
38. Boasso A, Shearer G. How Does Indoleamine 2,3-Dioxygenase Contribute to HIV-Mediated Immune Dysregulation. CDM. 2007;8: 217–223. doi:10.2174/138920007780362527
39. Ozaki Y, Edelstein MP, Duch DS. The actions of interferon and antiinflammatory agents on induction of indoleamine 2,3-dioxygenase in human peripheral blood monocytes. Biochem Biophys Res Commun. 1987;144: 1147–1153. doi:10.1016/0006-291X(87)91431-8
40. Grant RS, Naif H, Thuruthyil SJ, Nasr N, Littlejohn T, Takikawa O, et al. Induction of Indolamine 2,3-Dioxygenase in Primary Human Macrophages by Human Immunodeficiency Virus Type 1 Is Strain Dependent. J Virol. American Society for Microbiology Journals; 2000;74: 4110–4115. doi:10.1128/JVI.74.9.4110-4115.2000
41. Boasso A, Herbeuval J-P, Hardy AW, Anderson SA, Dolan MJ, Fuchs D, et al. HIV inhibits CD4+ T-cell proliferation by inducing indoleamine 2,3-dioxygenase in plasmacytoid dendritic cells. Blood. 2007;109: 3351–3359. doi:10.1182/blood-2006-07-034785
42. Badawy AA-B. Kynurenine Pathway of Tryptophan Metabolism: Regulatory and Functional Aspects. Int J Tryptophan Res. 2017;10: 1178646917691938. doi:10.1177/1178646917691938
43. Kanai M, Funakoshi H, Takahashi H, Hayakawa T, Mizuno S, Matsumoto K, et al. Tryptophan 2,3-dioxygenase is a key modulator of physiological neurogenesis and anxiety-related behavior in mice. Mol Brain. 2009;2: 8. doi:10.1186/1756-6606-2-8
44. Gualdoni GA, Fuchs D, Zlabinger GJ, Gostner JM. Resveratrol intake enhances indoleamine-2,3-dioxygenase activity in humans. Pharmacol Rep. 2016;68: 1065–1068. doi:10.1016/j.pharep.2016.06.008
45. Wirleitner B, Schroecksnadel K, Winkler C, Schennach H, Fuchs D. Resveratrol suppresses interferon-gamma-induced biochemical pathways in human peripheral blood mononuclear cells in vitro. Immunol Lett. 2005;100: 159–163. doi:10.1016/j.imlet.2005.03.008
46. Verjee ZHM. Tryptopthan metabolism in baboons: effect of riboflavin and pyridoxine deficiency. Int J Biochem. 1971;2: 711–718. doi:10.1016/0020-711X(71)90065-6
47. Bender DA. Tryptophan And Niacin Nutrition—Is there a Problem? In: Filippini GA, Costa CVL, Bertazzo A, editors. Recent Advances in Tryptophan Research. Boston, MA: Springer US; 1996. pp. 565–569. doi:10.1007/978-1-4613-0381-7_92
48. Knox WE. The relation of liver kynureninase to tryptophan metabolism in pyridoxine deficiency. Biochem J. Portland Press Limited; 1953;53: 379–385. doi:10.1042/bj0530379
49. Ogasawara N, Hagino Y, Kotake Y. Kynurenine-transaminase, kynureninase and the increase of xanthurenic acid excretion. J Biochem. 1962;52: 162–166.
50. Takeuchi F, Shibata Y. Kynurenine metabolism in vitamin-B-6-deficient rat liver after tryptophan injection. Biochem J. Portland Press Limited; 1984;220: 693–699. doi:10.1042/bj2200693
51. Bender DA, Njagi ENM, Danielian PS. Tryptophan metabolism in vitamin B6-deficient mice. Br J Nutr. Cambridge University Press; 1990;63: 27–36. doi:10.1079/BJN19900089
52. Shibata K, Mushiage M, Kondo T, Hayakawa T, Tsuge H. Effects of vitamin B6 deficiency on the conversion ratio of tryptophan to niacin. Biosci Biotechnol Biochem. 1995;59: 2060–2063. doi:10.1271/bbb.59.2060
53. van de Kamp JL, Smolen A. Response of kynurenine pathway enzymes to pregnancy and dietary level of vitamin B-6. Pharmacol Biochem Behav. 1995;51: 753–758. doi:10.1016/0091-3057(95)00026-S
54. Bender DA. Inhibition in vitro of the enzymes of the oxidative pathway of tryptophan metabolism and of nicotinamide nucleotide synthesis by benserazide, carbidopa and isoniazid. Biochem Pharmacol. 1980;29: 707–712. doi:10.1016/0006-2952(80)90544-4
55. Bender DA, Laing AE, Vale JA, Papadaki L, Pugh M. The effects of oestrogen administration on tryptophan metabolism in rats and in menopausal women receiving hormone replacement therapy. Biochem Pharmacol. 1983;32: 843–848. doi:10.1016/0006-2952(83)90586-5
56. Bender DA, Totoe L. Inhibition of tryptophan metabolism by oestrogens in the rat: a factor in the aetiology of pellagra. Br J Nutr. Cambridge University Press; 1984;51: 219–224. doi:10.1079/BJN19840026
57. Rios-Avila L, Coats B, Chi Y-Y, Midttun Ø, Ueland PM, Stacpoole PW, et al. Metabolite profile analysis reveals association of vitamin B-6 with metabolites related to one-carbon metabolism and tryptophan catabolism but not with biomarkers of inflammation in oral contraceptive users and reveals the effects of oral contraceptives on these processes. J Nutr. 2015;145: 87–95. doi:10.3945/jn.114.201095
58. Patterson JI, Brown RR, Linkswiler H, Harper AE. Excretion of tryptophan-niacin metabolites by young men: effects of tryptophan, leucine, and vitamin B6 intakes. Am J Clin Nutr. 1980;33: 2157–2167. doi:10.1093/ajcn/33.10.2157
59. Hankes LV, Schmaeler M, Jansen CR, Brown RR. Vitamin Effects on Tryptophan-Niacin Metabolism in Primary Hepatoma Patients. In: Huether G, Kochen W, Simat TJ, Steinhart H, editors. Tryptophan, Serotonin, and Melatonin: Basic Aspects and Applications. Boston, MA: Springer US; 1999. pp. 283–287. doi:10.1007/978-1-4615-4709-9_36
60. Shibata K, Hirose J, Fukuwatari T. Method for Evaluation of the Requirements of B-group Vitamins Using Tryptophan Metabolites in Human Urine. Int J Tryptophan Res. 2015;8: 31–39. doi:10.4137/IJTR.S24412
61. Stachowski EK, Schwarcz R. Regulation of quinolinic acid neosynthesis in mouse, rat and human brain by iron and iron chelators in vitro. J Neural Transm. 2012;119: 123–131. doi:10.1007/s00702-011-0694-6
62. Oduho GW, Han Y, Baker DH. Iron deficiency reduces the efficacy of tryptophan as a niacin precursor. J Nutr. 1994;124: 444–450. doi:10.1093/jn/124.3.444
63. Shibata K. Organ Co-Relationship in Tryptophan Metabolism and Factors That Govern the Biosynthesis of Nicotinamide from Tryptophan. J Nutr Sci Vitaminol . 2018;64: 90–98. doi:10.3177/jnsv.64.90
64. Fukuoka S-I, Ishiguro K, Yanagihara K, Tanabe A, Egashira Y, Sanada H, et al. Identification and Expression of a cDNA Encoding Human α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase (ACMSD): A KEY ENZYME FOR THE TRYPTOPHAN-NIACINE PATHWAY AND “QUINOLINATE HYPOTHESIS.” J Biol Chem. 2002;277: 35162–35167. doi:10.1074/jbc.M200819200
65. Li T, Iwaki H, Fu R, Hasegawa Y, Zhang H, Liu A. Alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is a new member of the amidohydrolase superfamily. Biochemistry. 2006;45: 6628–6634. doi:10.1021/bi060108c
66. Martynowski D, Eyobo Y, Li T, Yang K, Liu A, Zhang H. Crystal structure of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase: insight into the active site and catalytic mechanism of a novel decarboxylation reaction. Biochemistry. 2006;45: 10412–10421. doi:10.1021/bi060903q
67. Shibata K, Fukuwatari T. Large amounts of picolinic acid are lethal but small amounts increase the conversion of tryptophan-nicotinamide in rats. J Nutr Sci Vitaminol . 2014;60: 334–339. doi:10.3177/jnsv.60.334
68. Pellicciari R, Liscio P, Giacchè N, De Franco F, Carotti A, Robertson J, et al. α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase (ACMSD) Inhibitors as Novel Modulators of De Novo Nicotinamide Adenine Dinucleotide (NAD+) Biosynthesis. J Med Chem. 2018;61: 745–759. doi:10.1021/acs.jmedchem.7b01254
69. Brundin L, Sellgren CM, Lim CK, Grit J, Pålsson E, Landén M, et al. An enzyme in the kynurenine pathway that governs vulnerability to suicidal behavior by regulating excitotoxicity and neuroinflammation. Transl Psychiatry. 2016;6: e865. doi:10.1038/tp.2016.133
70. Matsuda H, Gomi R-T, Hirai S, Egashira Y. Effect of dietary phytol on the expression of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase, a key enzyme of tryptophan-niacin metabolism, in rats. Biosci Biotechnol Biochem. 2013;77: 1416–1419. doi:10.1271/bbb.130029
71. Goto T, Teraminami A, Lee J-Y, Ohyama K, Funakoshi K, Kim Y-I, et al. Tiliroside, a glycosidic flavonoid, ameliorates obesity-induced metabolic disorders via activation of adiponectin signaling followed by enhancement of fatty acid oxidation in liver and skeletal muscle in obese-diabetic mice. J Nutr Biochem. 2012;23: 768–776. doi:10.1016/j.jnutbio.2011.04.001
72. Kobayashi H, Horiguchi-Babamoto E, Suzuki M, Makihara H, Tomozawa H, Tsubata M, et al. Effects of ethyl acetate extract of Kaempferia parviflora on brown adipose tissue. J Nat Med. 2016;70: 54–61. doi:10.1007/s11418-015-0936-2
73. Jia Y, Kim S, Kim J, Kim B, Wu C, Lee JH, et al. Ursolic acid improves lipid and glucose metabolism in high-fat-fed C57BL/6J mice by activating peroxisome proliferator-activated receptor alpha and hepatic autophagy. Mol Nutr Food Res. 2015;59: 344–354. doi:10.1002/mnfr.201400399
74. Zhang Y, Song C, Li H, Hou J, Li D. Ursolic acid prevents augmented peripheral inflammation and inflammatory hyperalgesia in high-fat diet-induced obese rats by restoring downregulated spinal PPARα. Mol Med Rep. 2016;13: 5309–5316. doi:10.3892/mmr.2016.5172
75. Sheng X, Zhang Y, Gong Z, Huang C, Zang YQ. Improved Insulin Resistance and Lipid Metabolism by Cinnamon Extract through Activation of Peroxisome Proliferator-Activated Receptors. PPAR Res. 2008;2008: 581348. doi:10.1155/2008/581348
76. Shi T, Zhuang R, Zhou H, Wang F, Shao Y, Cai Z. [Effect of apigenin on protein expressions of PPARs in liver tissues of rats with nonalcoholic steatohepatitis]. Zhonghua Gan Zang Bing Za Zhi. 2015;23: 124–129. doi:10.3760/cma.j.issn.1007-3418.2015.02.010
77. Wang F, Liu J-C, Zhou R-J, Zhao X, Liu M, Ye H, et al. Apigenin protects against alcohol-induced liver injury in mice by regulating hepatic CYP2E1-mediated oxidative stress and PPARα-mediated lipogenic gene expression. Chem Biol Interact. 2017;275: 171–177. doi:10.1016/j.cbi.2017.08.006
78. Okuno E, Schwarcz R. Purification of quinolinic acid phosphoribosyltransferase from rat liver and brain. Biochimica et Biophysica Acta (BBA) – General Subjects. 1985;841: 112–119. doi:10.1016/0304-4165(85)90280-6
79. Fernando FS, Conforti L, Tosi S, Smith AD, Coleman MP. Human homologue of a gene mutated in the slow Wallerian degeneration (C57BL/Wlds) mouse. Gene. 2002;284: 23–29. doi:10.1016/S0378-1119(02)00394-3
80. Wolfensberger M, Amsler U, Cuénod M, Foster AC, Whetsell WO Jr, Schwarcz R. Identification of quinolinic acid in rat and human brain tissue. Neurosci Lett. 1983;41: 247–252.
81. Feldblum S, Rougier A, Loiseau H, Loiseau P, Cohadon F, Morselli PL, et al. Quinolinic-Phosphoribosyl Transferase Activity is Decreased in Epileptic Human Brain Tissue. Epilepsia. 1988;29: 523–529. doi:10.1111/j.1528-1157.1988.tb03756.x
82. Ko¨hler C, Eriksson LG, Okuno E, Schwarcz R. Localization of quinolinic acid metabolizing enzymes in the rat brain. immunohistochemical studies using antibodies to 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase. Neuroscience. 1988;27: 49–76. doi:10.1016/0306-4522(88)90219-9
83. Foster AC, Whetsell WO, Bird ED, Schwarcz R. Quinolinic acid phosphoribosyltransferase in human and rat brain: Activity in Huntington’s disease and in quinolinate-lesioned rat striatum. Brain Res. 1985;336: 207–214. doi:10.1016/0006-8993(85)90647-X
84. Heyes MP. The kynurenine pathway and neurologic disease. Therapeutic strategies. Adv Exp Med Biol. 1996;398: 125–129.
85. Guidetti P, Hemachandra Reddy P, Tagle DA, Schwarcz R. Early kynurenergic impairment in Huntington’s Disease and in a transgenic animal model. Neurosci Lett. 2000;283: 233–235. doi:10.1016/S0304-3940(00)00956-3
86. Stone TW. Kynurenines in the CNS: from endogenous obscurity to therapeutic importance. Prog Neurobiol. 2001;64: 185–218.
87. Guillemin GJ, Brew BJ. Implications of the kynurenine pathway and quinolinic acid in Alzheimer’s disease. Redox Rep. 2002;7: 199–206. doi:10.1179/135100002125000550
88. Guillemin GJ, Brew BJ, Noonan CE, Takikawa O, Cullen KM. Indoleamine 2,3 dioxygenase and quinolinic acid immunoreactivity in Alzheimer’s disease hippocampus. Neuropathol Appl Neurobiol. 2005;31: 395–404. doi:10.1111/j.1365-2990.2005.00655.x
89. Guidetti P, Schwarcz R. 3-Hydroxykynurenine and Quinolinate: Pathogenic Synergism in Early Grade Huntington’s Disease? In: Allegri G, Costa CVL, Ragazzi E, Steinhart H, Varesio L, editors. Developments in Tryptophan and Serotonin Metabolism. Boston, MA: Springer US; 2003. pp. 137–145. doi:10.1007/978-1-4615-0135-0_16
90. Guillemin GJ, Smythe G, Takikawa O, Brew BJ. Expression of indoleamine 2,3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons. Glia. 2005;49: 15–23. doi:10.1002/glia.20090
91. Guillemin GJ, Meininger V, Brew BJ. Implications for the kynurenine pathway and quinolinic acid in amyotrophic lateral sclerosis. Neurodegener Dis. 2005;2: 166–176. doi:10.1159/000089622
92. Wonodi I, Schwarcz R. Cortical kynurenine pathway metabolism: a novel target for cognitive enhancement in Schizophrenia. Schizophr Bull. 2010;36: 211–218. doi:10.1093/schbul/sbq002
93. Braidy N, Guillemin GJ, Grant R. Effects of Kynurenine Pathway Inhibition on NAD Metabolism and Cell Viability in Human Primary Astrocytes and Neurons. Int J Tryptophan Res. 2011;4: 29–37. doi:10.4137/IJTR.S7052
94. Campbell BM, Charych E, Lee AW, Möller T. Kynurenines in CNS disease: regulation by inflammatory cytokines. Front Neurosci. 2014;8: 12. doi:10.3389/fnins.2014.00012
95. Giil LM, Midttun Ø, Refsum H, Ulvik A, Advani R, Smith AD, et al. Kynurenine Pathway Metabolites in Alzheimer’s Disease. J Alzheimers Dis. 2017;60: 495–504. doi:10.3233/JAD-170485
96. Chang K-H, Cheng M-L, Tang H-Y, Huang C-Y, Wu Y-R, Chen C-M. Alternations of Metabolic Profile and Kynurenine Metabolism in the Plasma of Parkinson’s Disease. Mol Neurobiol. 2018;55: 6319–6328. doi:10.1007/s12035-017-0845-3
97. Moffett JR, Espey MG, Gaudet SJ, Namboodiri MAA. Antibodies to quinolinic acid reveal localization in select immune cells rather than neurons or astroglia. Brain Res. 1993;623: 337–340. doi:10.1016/0006-8993(93)91450-7
98. Murray MF. Tryptophan depletion and HIV infection: a metabolic link to pathogenesis. Lancet Infect Dis. 2003;3: 644–652.
99. Bipath P, Levay PF, Viljoen M. The kynurenine pathway activities in a sub-Saharan HIV/AIDS population. BMC Infect Dis. 2015;15: 346. doi:10.1186/s12879-015-1087-5
100. Valle M, Price RW, Nilsson A, Heyes M, Verotta D. CSF quinolinic acid levels are determined by local HIV infection: cross-sectional analysis and modelling of dynamics following antiretroviral therapy. Brain. 2004;127: 1047–1060. doi:10.1093/brain/awh130
101. Mangge H, Stelzer I, Reininghaus EZ, Weghuber D, Postolache TT, Fuchs D. Disturbed tryptophan metabolism in cardiovascular disease. Curr Med Chem. 2014;21: 1931–1937.
102. Yoshida R, Nukiwa T, Watanabe Y, Fujiwara M, Hirata F, Hayaishi O. Regulation of indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis of mice. Arch Biochem Biophys. 1980;203: 343–351. doi:10.1016/0003-9861(80)90185-X
103. Truscott RJW, Elderfield AJ. Relationship between Serum Tryptophan and Tryptophan Metabolite Levels after Tryptophan Ingestion in Normal Subjects and Age-Related Cataract Patients. Clin Sci. 1995;89: 591–599. doi:10.1042/cs0890591
104. Comai S, Costa CVL, Ragazzi E, Bertazzo A, Allegri G. The effect of age on the enzyme activities of tryptophan metabolism along the kynurenine pathway in rats. Clin Chim Acta. 2005;360: 67–80. doi:10.1016/j.cccn.2005.04.013
105. Braidy N, Guillemin GJ, Mansour H, Chan-Ling T, Grant R. Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats. FEBS J. 2011;278: 4425–4434. doi:10.1111/j.1742-4658.2011.08366.x
106. Capuron L, Schroecksnadel S, Féart C, Aubert A, Higueret D, Barberger-Gateau P, et al. Chronic low-grade inflammation in elderly persons is associated with altered tryptophan and tyrosine metabolism: role in neuropsychiatric symptoms. Biol Psychiatry. 2011;70: 175–182. doi:10.1016/j.biopsych.2010.12.006
107. Braidy N, Grant R, Adams S, Brew BJ, Guillemin GJ. Mechanism for quinolinic acid cytotoxicity in human astrocytes and neurons. Neurotox Res. 2009;16: 77–86. doi:10.1007/s12640-009-9051-z
108. Stone TW, Connick JH, Addae JI, Smith DAS, Brooks PA. The Neuropharmacology of Quinolinic Acid and the Kynurenines. In: Roberts PJ, Storm-Mathisen J, Bradford HF, editors. Excitatory Amino Acids. London: Palgrave Macmillan UK; 1986. pp. 367–380. doi:10.1007/978-1-349-08479-1_24
109. Grant RS, Kapoor V. Murine Glial Cells Regenerate NAD, After Peroxide-Induced Depletion, Using Either Nicotinic Acid, Nicotinamide, or Quinolinic Acid as Substrates. J Neurochem. 2002;70: 1759–1763. doi:10.1046/j.1471-4159.1998.70041759.x
110. Rahman A, Ting K, Cullen KM, Braidy N, Brew BJ, Guillemin GJ. The excitotoxin quinolinic acid induces tau phosphorylation in human neurons. PLoS One. 2009;4: e6344. doi:10.1371/journal.pone.0006344
111. Aragonès G, Suárez M, Ardid-Ruiz A, Vinaixa M, Rodríguez MA, Correig X, et al. Dietary proanthocyanidins boost hepatic NAD(+) metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats. Sci Rep. 2016;6: 24977. doi:10.1038/srep24977
112. El-Defrawy SR, Boegman RJ, Jhamandas K, Beninger RJ. The neurotoxic actions of quinolinic acid in the central nervous system. Can J Physiol Pharmacol. NRC Research Press; 1986;64: 369–375. doi:10.1139/y86-060
113. Wolf G, Keilhoff G, Fischer S, Hass P. Subcutaneously applied magnesium protects reliably against quinolinate-induced N-methyl-d-aspartate (NMDA)-mediated neurodegeneration and convulsions in rats: Are there therapeutical implications? Neurosci Lett. 1990;117: 207–211. doi:10.1016/0304-3940(90)90145-Y
114. Schurr A, West CA, Rigor BM. Neurotoxicity of quinolinic acid and its derivatives in hypoxic rat hippocampal slices. Brain Res. 1991;568: 199–204. doi:10.1016/0006-8993(91)91398-K
115. Xiao H, Yang C, He Y, Zheng N. Neurotoxicity of quinolinic acid to spiral ganglion cells in rats. J Huazhong Univ Sci Technolog Med Sci. 2010;30: 397–402. doi:10.1007/s11596-010-0364-1
116. Rothe F, Wolf G, Fischer S, Hass P, Keilhoff G, Abicht K. Quinolinate and kainate facilitate magnesium penetration into brain tissue. Neuroreport. 4: 205–207. doi:10.1097/00001756-199302000-00023
117. Silva-Adaya D, Pérez-De La Cruz V, Herrera-Mundo MN, Mendoza-Macedo K, Villeda-Hernández J, Binienda Z, et al. Excitotoxic damage, disrupted energy metabolism, and oxidative stress in the rat brain: antioxidant and neuroprotective effects of L-carnitine. J Neurochem. 2008;105: 677–689. doi:10.1111/j.1471-4159.2007.05174.x
118. Elinos-Calderón D, Robledo-Arratia Y, Pérez-De La Cruz V, Pedraza-Chaverrí J, Ali SF, Santamaría A. Early nerve ending rescue from oxidative damage and energy failure by L: -carnitine as post-treatment in two neurotoxic models in rat: recovery of antioxidant and reductive capacities. Exp Brain Res. 2009;197: 287–296. doi:10.1007/s00221-009-1913-3
119. Braidy N, Grant R, Adams S, Guillemin GJ. Neuroprotective effects of naturally occurring polyphenols on quinolinic acid-induced excitotoxicity in human neurons. FEBS J. 2010;277: 368–382. doi:10.1111/j.1742-4658.2009.07487.x