In a nutshell: summarizing our current knowledge of NAD and where we go next

An interview with Dr. Ross Grant

Dr. Ross Grant

Dr. Ross Grant is a biochemical pharmacologist and CEO of the Australasian Research Institute at Sydney Adventist Hospital. Dr. Grant has co-authored more than 100 academic articles in his research area, which focuses on understanding how a person’s lifestyle factors change their body’s biochemistry and immune activity, driving it toward either health or disease. In particular, he is interested in the influence of lifestyle factors on natural killer (NK) cell activity, redox balance (oxidative stress), and NAD+ metabolism, and how these influence cellular degeneration, particularly in the brain and central nervous system. He is a member of the Australian Neuroscience society (ANS), Nutrition Society of Australia (NSA), Australasian Society of Lifestyle Medicine, serves on the Advisory Board of NAD Research, Inc., and is one of our primary research partners.

NAD Research: I believe you’ve been investigating NAD longer than anyone. How long has it been, and what drew you to investigate this molecule?

Grant: I have been looking at it for quite a long time. It was popular back in the ‘90s to be investigating the kynurenine pathway, which tracked the metabolism of the essential amino acid tryptophan through a number of different compounds, upstream of NAD. Ironically, nobody was studying NAD at the time; we were looking at an intermediate metabolite, quinolinic acid, thought to be implicated in neurodegenerative disease. That was my focus: neuro-inflammatory disease. Researchers were interested in blocking the kynurenine pathway because quinolinic acid was a potentially neurotoxic intermediate that was elevated in people with neuro-inflammatory disease.

My concern was that, if you block the kynurenine pathway then you’re blocking what’s called de novo synthesis of NAD, which we knew was important in the production of cellular energy and as a redox couple for a lot of different enzymes. I published some early papers back in the ‘90s on what happens if you block the pathway (NAD decreases, particularly in some of the brain cells). I also looked at ways of being able to increase NAD. I also found that increasing a brain cell’s NAD+ would significantly reduce its susceptibility to oxidative damage. We knew that free radical activity was implicated in neurodegenerative diseases, so I looked to see whether NAD levels were decreased in an environment of high oxidative stress. (They are.) We published that study, as well, and then showed that nicotinic acid, as well as quinolinic acid, could both be used to increase NAD in some of the brain’s metabolic support cells. So yes, our group has been interested in NAD for quite some time—about 30 years.

Interest in NAD has really taken off more recently due to its role in cell degeneration and aging. In fact, our group was the first to actually publish, both in animal and human studies and in studies of the brain, that NAD decreases with age, as well as in relation to oxidative damage. A number of other people have replicated that now, so I guess NAD is a molecule that may be included in any “elixir of life.”

NAD Research: Elixir of life?

Grant: Any longevity formula. NAD won’t be the only ingredient, and nothing is going to be an absolute magic bullet. And, like everything else, there will probably be a sweet spot. Too little NAD is bad, but almost certainly too much is bad, as well. To think there would just be one magic ingredient would be a bit like saying, “Wheels work really well, so all we need for transport is wheels.” But actually axles, a chassis, and an engine are needed along with wheels. Wheels are essential for transport but they’re not enough.

NAD Research: Can you summarize the NAD findings—either made by your research group or by others—that are now accepted science?

Grant: Aside from our research, it is accepted science that NAD is essential for every living cell; it’s required for cellular energy production. We also know that NAD plays an essential role in DNA repair and maintenance of the genome. We know that it has a role in immune modulation, or making the immune system work better. It’s also involved in epigenetics: the switching on and off of genes.

The major contributions from our group have been that NAD levels decrease in an environment of oxidative stress. And that’s particularly in line with up-regulation of things like poly ADP-ribose polymerase (PARP) and NAD-dependent protein deacetylases, or CD38. In other words, inflammation, oxidative stress, and DNA damage all increase the turnover of NAD, which decreases its availability. If NAD gets too low, we get the apoptotic and possible necrotic pathways activated ultimately resulting in cell degeneration and death.

Based upon this background it seems clear that optimal NAD levels will be beneficial to the body. The key question is, what are those optimal levels? In order to explore that question, I think we do need to establish a sound knowledge platform, which is what our current work is focused on: Number one, how can we make sure that the levels of NAD we measure reflect the levels of circulating NAD in the body? Importantly, when blood or plasma or tissue is removed from the body, the NAD in it can change very rapidly. We need to be able to stabilize it, so by the time we analyze it we get an accurate reading. The other thing we’ve been working on is ensuring that the levels we’re reading haven’t been affected by other factors, such as what our subjects have been eating or drinking, for example. We’re pretty happy with where we’re at there, in terms of the assays we’re using. We’re also able to test what’s called the NAD/NADH ratio or the redox ratio, which is NAD in its reduced form (NADH) and its oxidized form (NAD+). That’s a really important ratio when it comes to NAD’s role in energy production. A too low, fasting NAD+ to NADH ratio suggests inefficient mitochondrial function resulting in poor energy production and increased oxidative damage. However, we know that this ratio does tend to shift as people begin to age, if they experience infection, or even if their diet is unhealthy (i.e., if you eat a high calorie meal, the NAD+ to NADH ratio shifts in favor of NADH. Too much NADH can itself stimulate oxidative stress. So, to summarize: we need to be able to make sure the level of NAD we’re measuring in the samples we’re testing is an accurate reflection of NAD levels in the body; and we need to be able to preserve the NAD/NADH ratio, as well. Both are quite difficult, but we’ve developed a methodology for doing both. Now the next thing is to build from that base and measure what the NAD levels are in various populations. This is our next set of experiments, which will begin in early 2023.

So a key question is; “what are those optimal levels of NAD+?” While a lot of people have promoted NAD supplementation and treatments, sometimes for good clinical reasons, I do think we need to identify what optimal levels really are. So, our current research is focused on two things:

1. How can we make sure that the levels of NAD we measure reflect the levels of circulating NAD in the body (i.e., snap preserve the NAD+/NADH levels once the blood is taken form the body) and 2. Make sure the method we use to analyze the NAD levels is not affected by common interfering substances. Unfortunately, one popular method people are using is the microcycling assay. This is an enzyme-based assay that can be affected by things people eat or even some drugs. So, while it can give reasonable results, it can also produce erroneous results in the presence of Interfering substances. So, the method we developed is not affected by these substances.  So now that we have all the methodology in place for both preserving NAD+:NADH and avoiding interferences we know that the results we get are true NAD+:NADH levels. Now we want to have a look at NAD across various populations and see just what affects these levels from age to gender to lifestyle factors to disease etc.

In the past we have measured NAD levels from the samples drawn by various clinics who send them to our lab. But we don’t have a lot of detail on the lifestyles and other factors associated with those samples. We can get a general overview of what NAD levels are, but now we want to get accurate NAD levels for various populations: healthy or ill; old or young; male or female; pregnant or not; various disease conditions, etc. Once we’ve got that information we can deduce what optimal NAD levels are and start to treat suboptimal levels. After that, we’ll investigate the best means of raising NAD levels and what are the clinical benefits that come from that. Is it through the de novo pathway I mentioned before, i.e., from tryptophan through the kynurenine pathway? Or, is it better to come through nicotinic acid (i.e., the Priess-Handler pathway), or from nicotinamide or nicotinamide riboside or nicotinamide mononucleotide, or some combination of those? These are the things we’ll be attempting to determine. I guess there’s plenty of work for us to do in the future!

NAD Research: Can you share how you’re able to stabilize the NAD in your samples?

Grant: We use a two-step process, because first we’ve got to inactivate the enzymes that use up NAD, and we have a patent pending for that process. Then the second step is to prevent the interconversion of NAD with NADH, the redox couple. A standard microcycling assay forces the conversion of the NAD through NADH. That’s OK for some purposes, but not for ours.

NAD Research: Thank you. And can you be more specific as to the lifestyle data that you’re hoping to collect along with your NAD samples?

Grant: Yes, it will be quite a comprehensive assessment. We’ll be looking at age, gender, types of nutritional intake down to a very granular level, including amounts of carbohydrate, micronutrients, and all that sort of thing. We’ll also be looking at subjects’ physical activity, how much exercise and sleep they get. We have a number of other markers that will tell us a bit about whether and which parts of the immune system are activated. We’ll also get a comprehensive list of medications or other supplements that they might be taking, and we will be asking them not to be taking NAD supplements. We want to get an indication as to whether any of those other medications and supplements might be having an effect on NAD. We’ll also assess the level of psychological stress, anxiety, and depression subjects are experiencing. So we’ll be collecting a fairly comprehensive set of background information. We’ve done this in a few other research projects—looking at lifestyle and its association with the biochemical changes contributing to degenerative disease. Our team is pretty used to working with these kind of big, complex data sets.

This study will be funded by NAD Research Inc. We’ve obtained the ethics approval for it, and we’re very excited to get started in the new year, 2023.

Interestingly, I’ve got some anecdotal evidence on this topic. A number of years ago I drew blood from a number of university students. I took the first sample on Friday, and then another sample the following Monday. It was fascinating to see the dramatic drop in NAD in some of the students. When I talked to them about what they did over the weekend, well, you can imagine what students will do over the weekend.

NAD Research: Study.

Grant: [Laughs] Yes. That’s right. But then after the study, they engaged in a little bit of alcohol. Actually, quite a bit of alcohol. Which did seem to drop the NAD quite dramatically in these young people. It’s rather fascinating that alcohol requires lots of NAD to metabolize. In fact, it’s the NAD availability that becomes the limiting factor for alcohol metabolism. So, someone with a higher NAD level would certainly do less damage from the alcohol they consume.

NAD Research: Very interesting. I might have to rethink my nightly cocktail. Do you have an idea of the number of people you’re hoping to study?

Grant: There will be 250. The group actually includes a pregnant cohort, which will be more of a challenge to get.

NAD Research: Well, that’s wonderful to hear. Women, pregnant women, are often left out.

Grant: Yeah. And yet you can imagine what additional metabolic demands there are on these women.

NAD Research: Can you summarize for us again what makes NAD levels in blood so difficult to measure?

Grant: Sure. Number one, NAD levels are very low in plasma. They’re also very labile due to the many enzymes in the blood that can metabolize the circulating NAD. It can get metabolized when you don’t want it to. There’s also the potential for significant interconversion of the reduced NAD to its oxidized form, in other words, NAD to NADH and vice versa.

NAD Research: The last researcher we interviewed, Dr. Jin-Xiong She, believes strongly that NAD infusions don’t raise cellular levels of NAD. Do you agree? Why or why not?

Grant: It’s not a simple question, so let me start by giving a little bit of background here. In the paper we published with Dr. Mestayer, we found that intravenous NAD, administered at a rate of 3 micromoles per minute, doesn’t result in an increase in blood or plasma levels of NAD for at least two hours. What that means is that the infused NAD is metabolized and/or put to use so quickly that blood and plasma levels don’t rise. We’ve demonstrated that in vitro studies, as well. We’ve added the NAD to a vial of blood and then checked NAD levels afterwards. It’s pretty clear that the blood itself is metabolizing the NAD very quickly. Additionally, during infusions, some NAD may be taken up by the tissues, possibly the liver. There are various enzymes that can help to take NAD up. It may also cross the blood brain barrier. We know that there is exchange between the central and peripheral nervous systems so NAD levels seem to be linked between the brain and periphery.

However, I think most of the NAD is getting metabolized by various ectoenzymes. These are enzymes that are on the cell surface and that function outside the cell, where they would be accessible to the circulating NAD. These ectoenzymes include CD38, CD73, and CD157. What they do is convert the infused NAD to a number of different types of nicotinamide plus ADP ribose, nicotinamide mononucleotide plus AMP or adenosine monophosphate, and nicotinamide riboside plus ADP or adenosine diphosphate. The end result of all of this is the potential for a number of health benefits, in spite of what seems to be relatively limited uptake of NAD into most tissues.

We might find some tissues are very good at NAD uptake. In our study with Dr. Mestayer, we didn’t actually look at NAD levels in the red blood cells. We should have, so we’ll do that in our next study. But we also think NAD may be taken up in the liver, which needs huge amounts of NAD.

Nevertheless, the point is, even when NAD is metabolized by those ectoenzymes, you actually end up with production of a number of very useful metabolites for every molecule of NAD. As mentioned previously the infused NAD can be broken down to nicotinamide, nicotinamide mononucleotide, or nicotinamide riboside. And if you know the NAD production pathways, each of them—the nicotinamide, nicotinamide mononucleotide, and nicotinamide riboside—are actually precursors for NAD production. All of them can be taken up into the cell. The mononucleotide probably has to be converted to the riboside to be taken up, just because of its ionic restrictions. Once taken up into cells they can then participate in what’s called the salvage pathway for NAD production. So it is highly likely that the infused NAD is metabolized very rapidly into these NAD precursors, which then might be taken up into the tissue and converted to NAD in that tissue. And when you’re infusing NAD at three micromoles per minute, that’s a large amount of precursors. So in the end, I suspect that you actually will get an increase in tissue NAD simply because you’ve now got lots of precursors that are available for the NAD salvage pathway. We still need research to confirm that, and I’m really am looking forward to doing it.

The second point I want to make regarding NAD infusions examines the question of what else do you get as a result of infused NAD? I’ll mention just two of the very important results. One is a significant increase in AMP, or adenosine monophosphate, which activates enzymes that are associated with health-promoting benefits. For example, AMP mimics the health benefits of exercise, such as reducing blood sugar, improving fatty acid oxidation, and reducing some of what’s called endoplasmic reticulum stress. In other words, AMP (that is produced from the breakdown of the infused NAD) actually helps to reduce some of the stress in the cell and reduces inflammation. So there are a number of benefits from just the AMP.

Second, when NAD is infused and is metabolized rapidly, you actually get an increase in adenosine, which does a lot of very important things—in fact, too many to list in an interview. To begin, adenosine suppresses neuronal excitations. This may be why infused NAD is so effective at helping patients who are withdrawing from substances like alcohol. Alcohol withdrawal results in an increase in neuronal excitation in the brain, and adenosine suppresses it. This means you could detox a lot more efficiently and with a lot fewer side effects. Another interesting thing is that adenosine has its own anti-inflammatory activity. It reduces some of the inflammatory pathways that are switched on in patients who are detoxing from substance use. This might be part of the explanation for why infused NAD is so effective at easing patient detox. I’m really excited about future research to better understand the clinical results that have been observed.

So, to summarize briefly: we don’t see an immediate increase in plasma NAD during an infusion due to its rapid metabolism. That metabolism produces precursors, which themselves will most likely increase intracellular NAD along with all of the other potential clinical benefits from adenosine and the AMP.

NAD Research: Great. Thank you. This is a side topic, but could the fact that intravenous NAD doesn’t seem to raise intracellular NAD levels (although we don’t know if that’s a fact) be a possible benefit when treating cancer patients? This might require a backstory, too.

Grant: Yes. This might be a topic for another conversation because the relationship between increased NAD levels and cancer is not simple. We’ve done a bit of the work on NAD in cancer cells ourselves, measuring the NAD levels in a number of cancer cells in culture and showing that they were three to five times above what they would be in a normal, healthy cell. We didn’t publish that data at the time but others have since done that. So we know that cancer cells, in general, need high levels of intracellular NAD to grow and divide. That’s because cancer cells have an unstable genome, which they’re constantly trying to repair. They’re also trying to divide and multiply. They don’t have control in terms of their cell division, which is what makes them dangerous. Importantly cancer cells mostly generate the NAD they need via the salvage pathway, most of it coming from the recycling of nicotinamide. Potentially, it could also come from nicotinamide riboside or the nicotinamide mononucleotide. The interesting thing is that there’s no evidence that we know of that cancer cells get the NAD they need by taking up extracellular NAD. So taking NAD itself probably won’t promote increased NAD levels for the cancer. Now what about taking NAD precursors?

I’ve made an observation—again, I didn’t publish it because it was a number of years ago—that increasing NAD levels within cancer cells actually produces a potential apoptotic, inhibitory, effect. Part of the reason is because cancer cells are really damaged cells. You only get a cancer cell once you have damaged the DNA in ways that haven’t been repaired. This damage prevents the cell from going into its normal cell death, or apoptotic, pathway.

We’ve all had damaged cells. We get them all the time. We’ve been out in the sun too long and ended up with sunburn. Our body’s first option is to try and repair the damaged cells. If they can’t be repaired, the second option is to kill the cell. That’s what peeling after a sunburn is. If the cell doesn’t die, the body’s next option is to attach death receptors on the outside to try to alert the immune system that there’s a damaged cell that needs to be removed. The immune system hopefully recognizes the call and sends cytotoxic T lymphocytes, or natural killer cells, to destroy the damaged cell.

When you develop a cancer, the problem is that you’ve actually damaged some of your fail-safe machinery, so the body can’t kill the cell even though it wants to. The cell might even have damaged some of its death receptors, like P2X7, which is a very common one. In fact, P2X7 is a death receptor that has a relationship with NAD levels. If the NAD levels are high, you can activate P2X7. However, most cancers have a nonfunctional P2X7, so can’t be activated. At the same time, there is immune evasion, so for various reasons, the cytotoxic cells, lymphocytes, can’t see the death receptors.

So what needs to be understood is that most cancers aren’t little evolutionary deviants trying to take over the body. In fact, they’re little deaf, dumb, and blind cells that have been damaged, and all of their means for doing what they’re supposed to do—repair or die—have been damaged. The only machinery that they’ve got left working is their replication machinery…so they keep on blindly dividing. Cancer cells will die if they can get the signal.

Interestingly, very early on we did some cell culture experiments with cancer cells and found that in some cells if we increased the NAD to supernormal levels, we could actually get some of these cancer cells to kill themselves. I thought that was fascinating. We haven’t pursued that line of research, but it is an important one.

So the fact that the NAD is increased in various cancer cells is one thing. They do need it. However, they probably get most of it from circulating nicotinamide. Will taking more NAD make them grow faster? They need other things to grow faster, as well. NAD isn’t the only thing they need. So we’d need to do a series of experiments, probably in culture, to see whether or not NAD supplementation can change growth rates.

But since we’re on the topic, I need to mention one other thing. The immune system also needs high levels of NAD. So even though cancer cells might consume a lot of NAD, your body’s anti-tumor arsenal also needs a lot of NAD. If you’ve got good NAD levels, your immune system is likely to be more efficient at actively suppressing any tumor growth. So, yes it would be ideal if you could starve the cancer of its NAD, while still promoting NAD production within your T lymphocytes. There are some ways this could be done, which aren’t ready for publicizing at this point, but they would be good to study because there would be huge benefits. They won’t work for all cancer types; it depends on what the cancers themselves use for their NAD substrates. Some cancer cells lack some of the other enzymes necessary to generate NAD from various precursors. There might be ways to take advantage of this, which would be useful to investigate, but of course, it would need funding to move this line of research forward.

NAD Research: Well, cancer research does seem to be an arena that gets a lot of funding. That leads us to our next question: how much funding would you need to pursue all of the various research projects in your pipeline?

Grant: We need to establish the optimal NAD ranges for health and better understand the various disease and conditions that affect NAD levels. That study has funding from NAD Research. I’m really looking forward to seeing the end results so that we can comprehensively characterize the clinical benefits of IV NAD therapy that we observe at clinics like Springfield Wellness Center. In addition to confirming their results, we’ll be able to understand some of the mechanisms behind them.

The second group of studies (i.e. investigating what were the best protocols/precursors, etc., for raising NAD+ levels and then also quantifying the clinical benefits of raising NAD levels in various disease cohorts)  will probably require $100,000 – $150,000 US. We could do it cheaper, it would just limit the number of metabolites we were able to measure. Nevertheless, we could collect the samples and then wait until we’ve got more funding to analyze them.

In parallel with the IV NAD clinical therapies, it would be good to comprehensively characterize the fate of that infused NAD. In other words, track its kinetics and the concentration of the metabolites in both the aqueous and cellular fractions. We should be able to do most of that in conjunction with what we do in that more comprehensive clinical study. Then, once we understand how things work, we can modify our NAD treatments to get the best outcome, which will mean intervention studies to determine the most effective way of administering NAD tailored to various clinical cohorts. For example, when treating with NAD to address neurodegenerative diseases, there’s strong advocacy by some to utilize NMN (nicotinamide mononucleotide). That should be fine as long as the enzyme that converts NMN through to NAD is working well. But there are some conditions, particularly amongst some of the elderly, where those enzymes are not necessarily working that well. If NMN is going up and NAD is going down, which can happen within some of the aged groups, then it switches on an enzyme called SARM1. When SARM1 gets switched on, it actually starts to chew up axons, the long communication filaments between neurons. You really don’t want that to be happening. And, it doesn’t seem to happen when you supplement NAD through other means. Ideally you want to raise your NAD while keeping your NMN levels relatively low, thus protecting your neurons.

This is an example of why we need to be careful about overstating the benefits of NAD supplementation. It’s not as simple as, “Take this supplement and you’ll live forever.”

So anyway, that was a little aside. But establishing the most effective ways of increasing our NAD to concentrations that are relevant within the tissues that we’re looking at is a useful line of research. Some of the early studies I did showed that nicotinic acid was particularly good for some of the cells of the brain. Some of the other precursors might be better for some of the other tissues. And then, of course, further investigation would enable us to fine-tune all of the various tools that we’ve got and apply them to a range of conditions, all the way from mental health disorders, to neurodegenerative disease, to energy metabolism to tissue anomalies, whether that’s liver-associated damage or enhancing muscular performance, or even just treating some of the mitochondriopathies, which there are certainly case studies out there showing that NAD is of benefit.

NAD Research: Do we know how the body prioritizes what it does with the NAD available to it?

Grant: The body has a complex way of being able to deal with NAD because it’s used in so many different ways. NAD is a master switch. And there are different pools of NAD, so the way it’s managed within the mitochondria is a little bit different than the way it’s managed within the nucleus or within the cytoplasm or the way extracellular NAD is managed. But yes, in general the areas that are low in NAD+ will most likely be serviced first. The body works actively to maintain its NAD in each of the cell compartments. Once NAD drops below a certain level—consistently we’ve shown in experimentation–below about 50%–you start to get some significant cell death. And again, if you have higher levels of NAD, the cells are more able to resist damage, particularly to the DNA from causes such as oxidative stress.

We’ve done a series of studies looking at NAD in relation to ultraviolet damage for skin cells or oxidative damage for brain cells. The cells can resist a whole lot more damage when the NAD levels are even two to three times what they are at baseline. So this brings us back to where the optimal level is. If you go over that, do you get any benefit? I suspect not. So yes, the body does have priorities in the way it uses NAD, but it has some very complex interrelationships between different types of cells, tissues, and organs, and they all take different precursors. Some will have specific uptake mechanisms even for the NAD itself. We know that NAD is necessary even down in the renal filtrate. It was certainly a very intelligent mind that put it all together. I suspect we won’t get to the bottom of it all before the end of my lifetime.

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