How is NAD+ Made? Preiss-Handler Pathway

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 the electron transport chain and the citric acid 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 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.

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