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#46 - Chris Masterjohn, Ph.D

Summary

Methylazation = one carbon metabolism. Methr gene uses folate to recycle methyl donors back and forth to homocysteine. Some people have more of a particular enyzyme that helps this process than others.

If you aren't a good folate methylater, for whatever reason, choline is alternative. Best to get in diet (eggs, liver, meat, nuts, cruciferous vegetables).

Another way to go through methyl groups is by introducing superphysiological amounts of NR/NAD. To combat this you can take choline, TMG(?) or creatine.

50% of all methylization produces creatine. Creatine helps strength, hydration but also many other things.

If you are taking NR, take TMG one to one to make sure you are properly methylating.

Chris’s background, falling in love with biochemistry, and decision to pursue research over medicine [7:45]

  • Got his Ph.D. in Nutritional Sciences from the University of Connecticut
“I think one of the things that we often neglect in science is we get so caught up in the latest research that we forget to study the foundations of where things came from. My instinct is always to say, well, ‘where did this idea come from? What was the origin of this?’ And that inevitably leads to finding a fresh way to look at something because you realize the path that led you there, and that there were details that got left behind because no one knew what to do with those details at that time.”

Choline: what it is, why it is important, and how a deficiency can cause non-alcoholic fatty liver disease

  • And since choline contains TMAO, choline was now starting to be considered dangerous
  • Peter and Chris both had some problems with the science around this thesis
  • Choline is a methyl donor
  • Choline is an essential part of phosphatidylcholine (a phospholipid that is in our cell membranes)

What is a methyl group? And what is methylation?

  • All biological organisms are made of hydrocarbons
  • If you want to change one of those molecules, you’re going to have to put together carbons, or take them apart
  • To methylate = “to put on a methyl group”. Methyl group is a carbon group.
  • Alot in liver, egg yolks
  • Moderate amounts found in…Meats, Nuts, cruciferous veggies

Choline deficiency and fatty liver

  • Patients were being fed via total parenteral nutrition (TPN) and not given choline: developed fatty livers!
    • Choline is an essential part of the phospholipid called phosphatidylcholine
    • that phospholipid is not just in our cell membranes, but it’s also in the membranes of the lipoproteins that carry fat out of the liver
    • If you don’t have enough choline, you can’t make the LDL particle to get triglycerides out of the liver
    • There are human studies that put people in a controlled lab environment and when you take the choline out of their diet they will develop fatty liver disease
  • First, short term development of steatosis (the simple fat accumulation)
  • Steatosis does not necessarily lead to steatohepatitis ( fatty liver disease characterized by inflammation)
  • But all it takes is some hit of inflammation or oxidative stress that suddenly lights the fire
  • “The average person walking around out there who doesn’t have fatty liver, they don’t have fatty liver because of the choline in t their diet.” says Chris. Its both diet and choline
  • This experiment took people that didn’t have any fat in their liver and then they put them on this experimental diet where they took the choline out of what they were already eating
  • Those people didn’t have fatty liver ⇒ then they went on the TPN ⇒ they got fatty liver
  • You can cause fatty liver with sucrose, or alcohol, or excess fat
  • But no matter how you cause it, you just put in choline (or precursors to choline) at a high enough ratio and it will get rid of the fatty liver
“So if we know in humans that you get fatty liver when you take the choline down, and we know in animals that you get rid of fatty liver if you put the choline up, then I would think that if you took the humans with the fatty liver and you put the choline up, you’d get rid of it. But for some reason that hasn’t been tested the way you’d think it would be.”
  • “I can only give you a wild guess and my wild guess would be about 1200 milligrams of choline. And you could get that by eating food, but it would be very difficult.”
  • “I think the average choline intake is probably somewhere around 300 milligrams.”
  • Women = 425
  • Men = 550

NAFLD: increasing prevalence and potential causes [25:00]

Is NAFLD is more prevalent now than 20 or 30 years ago?

  • “Matched to obesity because that’s the overwhelmingly predisposing risk factor.”
  • “It’s hard to make an either or question about it, because It comes down to fat-in versus fat-out.”
  • Choline is such an overwhelming factor in the “fat out” part of the equation

Obesity and fatty liver disease

  • Chris says 67% of people who have obesity have fatty liver, and 76% of people who have fatty liver have obesity
  • The overwhelming reason (but not the only reason) for that is that the more obese you are, the more likely you are to have visceral adiposity
    • And the visceral fat pad directly empties into the portal vein (a blood vessel that carries blood from the gastrointestinal tract, gallbladder, pancreas and spleen to the liver)
    • “That’s probably a huge factor in why the metabolic health of someone who has relatively more visceral fat is so much worse than the person who has relatively more subcutaneous fat. It’s because you’re basically pounding the liver with fat all the time.”
    • You don’t even have to eat anything, it’s just you’re always engaging in lipolysis (freeing some free fatty acids)
    • “And if you have this gigantic tube going right into the liver, just feeding fatty acids in all the time, then I think that’s the major thing.”
  • Whenever people make one change in their diet. . .they’re actually changing like 30 or 40 things in their diet.
  • So the average person who takes fructose out (or goes on a low carb diet), they actually you probably increased protein, choline, riboflavin, zinc, etc.

Fatty liver, fructose, and choline

  • In grad school, Chris fed rats a 60% fructose diet (an enormous amount)
    • Why didnt rats get fatty liver? ⇒ Turns out the rat diet included amino acids that has 6 times more methionine (a precursor for choline)

In summary:

“When I look at the human data, I’m gonna say, ‘I do believe that reducing the fructose is going to reduce the amount of fat that the liver is producing from sugar,’ but I also believe that if you did that and you replaced it with protein, then that was part of the results. Or if you replaced it with vegetables, or you replaced it with egg yolks, those were part of the results.” —Chris

TMAO: Should we be worried about the TMAO content in choline and our foods? [39:15]

  • Being found naturally in fish
  • In humans, two different papers. . .
    • They were showing that TMAO levels in humans correlate with heart disease risk
    • And when humans eat either choline or carnitine, they will metabolize both of those to trimethylamine in the gut, and the liver will convert it to TMAO

⇒ After that, The argument was made that TMAO in those humans will cause heart disease the way that it causes heart disease in the genetically engineered mice

And how do humans get too much TMAO (according to the study authors)?

  • Too much eggs and red meat (high in choline)
  • But, the did NOT mention fish ⇒ Fish has ~100x more choline than eggs and red meat
    • How could they have not brought fish into this discussion??

Chris strongly disagrees with the hypothesis that TMAO causes heart disease

  • “On a scale of one to ten, I wouldn’t say that my opinion that TMAO has negative properties in humans is zero. I think it’s maybe one or two or something like that.”
“But…my view of the overall story that eating eggs and meat are bad for you because they alter the microbiome in a way that makes you take the choline and the carnitine in those foods and convert it into TMAO and make you uniquely vulnerable to heart disease, I’m very close to zero on the probability of that story being true.”

Choline supplements and foods

phosphatidylcholine is better than choline bitartrate

  • “When I’m looking at choline supplements, I overwhelmingly prefer that someone would get phosphatidylcholine, which is the overwhelming form of choline that’s found in food.”
    • 1) Phosphatidylcholine is the form found in real food
    • 2) Phosphatidylcholine least likely to generate TMAO in the gut (because it’s absorption is better)

How to dose a choline supplement?

  • Chris uses lecithin, which is a phosphatidylcholine. But better to get from food
  • About 15% of phosphatidylcholine turns into choline
  • Example, if someone needs 1200 milligrams of choline…
    • When taking a lecithin supplement (a phosphatidylcholine), you would need to take ~8 or 10 grams in order to get between 1000-1500 milligrams of choline

Eggs and choline/TMAO

Choline and blood clotting

  • If you eat enough eggs you get a plasma TMAO response, but it’s three or four times lower than when you eat the same amount of choline as choline bitartrate.

The absorption of choline

IMPORTANT POINT: It’s not about how many eggs you eat in a day, it’s about how many eggs you eat at a sitting

  • The only reason eggs generate TMAO is because there’s some poorly characterized absorption cap to phosphatidylcholine and even though it’s better absorbed than the other choline that’s sold on the market, there’s some cap to how much choline you can absorb

Choline: Good guy or bad guy?

  • Peter: “The choline story is interesting and I think if you look at it over simplistically you could say, “Well, it’s a good guy and a bad guy,” but it seems to be more of a good guy than a bad guy.”
  • Chris: “Certainly from the perspective of fatty liver that’s clear [that it is a good guy].”

Types of fatty acids: How they may predispose us to different types of illnesses [53:30]

Defining the types of fats: 1) Saturated 2) monounsaturated and 3) polyunsaturated (We can further divide the polyunsaturated into a number of constituents)

  • A saturated fatty acid:
    • C8 has 8 carbons, versus a C12 would 12 carbons
    • Being saturated just means there are  ⇒ Every carbon is fully saturated with a hydrogen: no double bonds
  • Monounsaturated fat has one double bond
  • Polyunsaturated fat has two or more double bonds
  • One thing is that whenever you introduce a double bond, you create a kink in the molecule
    • So saturated fats ⇒ you can pack them together very well, so they tend to be solid
    • Less saturated fats (fats that are higher in monounsaturated and polyunsaturated fat) tend to be more liquid
      • I.e. Olive oil is more liquid than butter because it’s mostly monounsaturated fat

⇒ Also

  • In a fatty acid with two or more double bonds (polyunsaturated fat), the carbon that’s between two of those double bonds is very unstable and it is uniquely vulnerable to being damaged.
  • So that fatty acid is  (and in fact there are polyunsaturated fatty acids that are absolutely essential for us) ⇒ Omega-6 and omega-3 (both polyunsaturated) that are both essential to human physiology
  • But it’s also the case that the more of these that you have in your cell membranes the more of a liability it becomes
  • Because if you have oxidative stress and inflammation, those fats are more vulnerable to damage

So are PUFAs in liver worse than mono or saturated? Complicted

  • short term, PUFAs dont build up as much
  • longer term, if there is fatty buildup in liver, PUFAs much more likely to go from steatosis to steatohepatitis (ignite the fire you have been buiilding up)
    • this is because PUFA may more oxidative targets
  • In the alcoholic model Studies show that you get (opposite of the non-alcoholics) less steatosis with saturated fats than with polyunsaturated fats
    • I think the reason for this is that oxidative stress does not just cause you to go from NAFLD to NASH.”
    • It also can cause steatosis because you get oxidative destruction of the ApoB particle

ApoB particle

  • It’s not just choline that allows you to export triglycerides from the liver
  • You also need sufficient antioxidant protection in the liver because if you damage that particle before it ever exports to triglycerides, then you prevent it from doing so.
  • “Well, in the alcoholic model there’s so much oxidative stress already that’s provided by the alcohol, because the alcohol isn’t just a way to get fat in the liver, the alcohol’s metabolism generates oxidative stress.
    • “You’re taking a liver that doesn’t yet have fat accumulation, you’re putting a bunch of fat into it, and you’re adding on top of that an oxidative flame.”
    • “You actually wind up causing steatosis by causing oxidative damage to the ApoB particle so that they can’t leave the liver in that model.”

Why don’t we see low VLDL in patients with NAFLD? [59:45]

ApoB is used in two ways in the liver

  • First is VLDL export (which is our main way to get triglycerides out of the liver)
  • Second is for making de novo LDL in the liver

Shouldn’t that mean we see a reduction in VLDL in patients with NAFLD? ⇒ Peter has seen many patients with NAFLD but has not noticed an association with an alteration in VLDL

⇒ Why? Here is Peter’s possible explanation..

  • If you have NAFLD, you are very likely insulin resistant
  • If you are insulin resistant, you are upregulating APOC3
  • If you are upregulating APOC3, your VLDL are going to stick around longer than if you do not
  • That’s why we see these pathologic remnant VLDL particles that become atherogenic
  • In an insulin sensitive person, the VLDL really don’t pose much of an atherogenic risk because they stick around for such a short period of time (APOC3 is quite down regulated in that scenario)
  • If APOC3 is upregulated, you have VLDL starting to act like LDL ⇒ They stick around long enough in the plasma that they become atherogenic
  • So maybe the less VLDL that’s being exported in that patient is offset by a longer residence time due to more APOC3.
  • Chris points out: “Wouldn’t you also expect that from a lower rate of uptake of triglycerides from peripheral tissues?
  • Peter: “That’s a great point. You have a lower peripheral disposal of triglycerides so the whole thing can be confounded.”

It’s also confounded by different ethnicities…

  • African American patients even with type 2 diabetes will still have very low VLDL and triglycerides
  • A Hispanic patient with diabetes will generally have a high triglyceride and a high VLDL
  • Hispanics are actually far more susceptible to NAFLD than African American
  • Caucasians are in the middle

Understanding flux, and how machine learning may affect medicine in the near future [1:03:15]

“Mistaking a concentration for flux is one of the overwhelming interpretive problems in science.”

How can science overcome this problem?

Use our understanding of biochemical pathways

  1. We have mapped out many biochemical pathways, says Chris
  2. “If you know all the possible sequences and you can measure all the metabolites, you can often reconstruct what a video would have shown with fairly good precision.”
  3. Measure an abundance of things to see a fuller picture
    1. “If you measure 20 things in someone’s blood, you can have a much better idea of what’s going on, what probably happened than if you measure one thing in that blood.”
  4. ⇒ Serial measurements (even if it’s just a few markers)
  • Example, oral glucose tolerance test…
    • A person with a fasting glucose is 70 mg/dL and their fasting insulin is one, the probability that they’re going to have postprandial hyperinsulinemia is low
    • Similarly, you look at somebody whose fasting glucose is 130 and their fasting insulin is 30, the that person is likely headed towards a train wreck
  • So by sampling at 30, 60, 90, and 120 minutes after a glucose challenge, you can develop a very interesting kinetic pathway using just two or three measurements

Where will machine learning be most likely to displace human ability in medicine?

  • 1) Radiology: My intuition is most likely radiology, says Peter
  • 2) In the ICU: I’ve often thought the ICU would be the second most valuable place just based on the reams of data that are coming out
  • 3) Flux Peter’s new epiphany during this conversation is that there may be a very interesting opportunity here for machine learning to also start to differentiate this question of flux

NAD: How it works, supplements, sirtuins, and the central role of the liver [1:09:30]

NAD precursors are “the most sought after, discussed, asked about supplement on the market today

  • Many have heard of the precursor called nicotinamide riboside (NR)
  • Many think NAD is the “fountain of youth” because NAD is a substrate to sirtuins

Very popular NAD supplements

How NAD works

  • One process is called oxidative phosphorylation
    • In our cells we have mitochondria: have a ton of complexes which are basically used to generate reducing agents that move electrons to one side of a double membrane
    • This process builds up a gradient and then that gradient allows us to make a bunch of ATP

Figure 1. Oxidative phosphorylation. Image credit: wikipedia.org

image
  • The first of these complexes turns NADH to NAD
    • It has been postulated that as we age our ratio of NAD to NADH goes down: less NAD
    • And NAD is an important substrate for sirtuins (which do many things but primarily repair DNA damage)
      • So it would seem that we would want more NAD

Another reason for NAD depletion

  • Sirtuins and the PARPs are consuming a lot of NAD
  • These two enzymes that are using NAD for the purpose of protecting DNA, telomeres, and all the things that are postulated to be important to aging and longevity
  • Niacin in the diet, it’s a vitamin B3

Why can’t we just eat more NAD?

  • Collectively, we call all this stuff, Niacin
  • In plant foods it comes in the form of nicotinic acid
  • In animal foods, it comes in the form of nicotinamide
  • Much of the niacin that’s in the steak is going to be in the form of NAD or NADPH
  • What we’re absorbing in the intestines is either nicotinic acid or nicotinamide

When using supplements…

  • If you take a nicotinamide riboside (NR) supplement, you are absorbing the NR intact
  • If you take a nicotinamide mononucleotide (NMN) supplement, you are probably digesting that down to NR or nicotinamide and then absorbing them
  • But NAD and NADH can’t be digested in the intestines

So when we’re eating food,

  • Once we go into the enterocyte, we have nicotinic acid primarily being converted over to nicotinamide, and the intestinal cell tries to turn that into NAD
  • But whatever the intestinal cell doesn’t turn into NAD itself, it passes on to the liver
  • The Liver is the main site that’s really metabolizing all these forms for the entire body

In the liver…

  • It’s going to try to convert as much of those many forms into NAD as possible
  • Not even just for itself, but because it’s going to hold a reserve of NAD for the rest of the body
  • And then almost everything that comes out of the liver and goes into the circulation is (the transport form of NAD) nicotinamide
    • And then those tissues will convert the nicotinamide into NAD

NR

  • When taking an NR supplement, it becomes NMN which then becomes NAD
  • The liver is basically taking all of that NR and making NAD
  • All of the NAD that is in any of your cells that are not the liver (aside from 5% in the kidney made from protein) is ultimately derived from circulating nicotinamide that the liver put out

Is nicotinamide charged? ⇒ No

Does nicotinamide makes its way into a cell through an active or passive transporter? ⇒ Active

How does nicotinamide get converted into NAD once it gets to the cell?

Two steps that our ATP dependent

  • 1) It gets converted to nicotinamide mononucleotide (NMN)
  • 2) Then it gets converted into NAD
    • NOTE: A portion to the NMN becomes NR, and then comes back to NMN (Why? ⇒ If you’re trying to hold on to the nicotinamide and get it to NAD more than the rate at which you can make the NAD, you might convert some to NR to hold onto it)
  • In summary:
    • Nicotinamide (NAM) ⇒ NMN ⇒ NAD

What happens when sirtuins and PARPs consume NAD?

  • Enzymes like sirtuins and PARPs are using NAD for DNA repair
  • What they do when they consume the NAD is they release nicotinamide

⇒ Example: Imagine you are a muscle cell…

  • To get every molecule of NAD you have,  you took some nicotinamide from the blood originally
  • But then you did things with it
    • oxidative phosphorylation ⇒ you don’t “consume” NAD in that process, you just cycle it back and forth (You use it over and over and over and over and over again)
    • Sirtuins and PARPs ⇒ They are engaging in protection and those enzymes will consume the NAD to generate nicotinamide
  • The big problem here is that nicotinamide is an inhibitor of all those enzymes through negative feedback loop so you
  • MUST do something with the nicotinamide immediately, or you need to methylate it and pee it out

  • So you either use the nicotinamide to make NAD, or you get rid of it

What happens when you take a gram of NR?

Josh Rabinowitz 2018 papers:

  • Showed that there is a little bit of NR that gets into the blood
  • And some of the NR gets into some of the cells, and not others (it can’t get to the brain, for example)
  • The NR could get into the muscle cells directly ⇒ The problem is that there’s not a lot there: “It’s basically practically meaningless.”
  • Other studies Showed that you do increase NAD in the muscle a lot when you supplement with the NR
  • But it all comes from nicotinamide that the liver had made in circulated in the blood
  • They showed that they got a much less NAD response in the muscle with oral nicotinamide than they did with NR even though they also showed with very elegant tracers that it all got there as nicotinamide

Why might it be more effective to take NR instead of NMN?

  • Imagine that the NR, the nicotinamide, nicotinic acid, whatever it was you ate, gets into the liver…
  • We go from nicotinamide to NMN to NAD
  • If we consume the NAD with sirtuins and PARPs, we come back to nicotinamide
  • And having that nicotinamide around is a liability, because it’s going to inhibit all of the repair enzymes (sirtuins and PARPs)
  • So we either want to make NAD out of it, or we want to get rid of it

So if you take oral nicotinamide…

  • The oral nicotinamide is immediately a liability before it ever becomes NAD because it can inhibit the sirtuins and the PARPs
  • Liver must make NAD or methylate the nicotinamide to pee it out ASAP...

⇒ If you take oral NR…

  • It then converts to NMN and then you  and expose it to the detoxification process have to make NAD before you ever generate nicotinamide
  • Why is this happening? ⇒ NR is a superior way to increase hepatic NAD because when it gets to the liver…
    • It can’t be immediately detoxified
    • It’s not immediately a threat to the sirtuins and PARPs
    • and it can only make NAD before it does anything else
  • If you’re presenting [the liver] with a thing that’s a threat (nicotinamide) and it has to make that decision, and it can only make so much NAD at once at one time, then you’re going to have much more waste in the detoxification pathway [as compared to] if you put the thing in (NR) that has to make NAD, that is not a threat when it makes NAD, and that has to actually generate NAD to ever be exposed to the detoxification pathway.”

The central role of the liver

  • It’s controlling the flux throughout the entire body
  • The liver is not just making NAD for itself, it’s making NAD because it carries all of the reserves for the rest of the body as NAD
  • Itt has a reserve pool of NAD that it holds onto for the specific purpose of a slow release of nicotinamide to the rest of the tissues that they will take up
  • And then the  to either detoxify it or make NAD. cells will have the immediate decision

⇒ Peter’s glucose analogy:

  • It sort of parallels glucose ⇒ we have to maintain about 5 grams of glucose in our bloodstream at all times (10 g you have diabetes, 2 g means you die)
  • So in this analogy, NAD is to glycogen what glucose is to nicotinamide

Intravenous NAD [1:33:00]

  • Some believe NAD given intravenously is the superior way to get it’s benefits
  • Peter does not believe that to be the case, but he’s not sure why

What is happening when you inject NAD into someone?

  • You’ve bypassed the liver, and it’s sitting there in the plasma
  • Per anecdotal reports of the immediate response to that, is that they’re are causing an inflammatory response
  • What is not clear is the kinetics of where that NAD is going

But what is the normal physiology? For people who are not injecting it, how does NAD get out of this cell in the first place?

  • It appears to be that extracellular NAD is from dead cells, dying cells, and maybe from cells that are undergoing some sort of stress response and are secreting it to some degree to reflect their energy status

NAD as a signaling molecule

I think it’s very clear that if there is any role for extracellular NAD, it is as a signaling molecule.”
  • There’s a whole class of extracellular NAD consuming enzymes
  • So it has to be the case, that extracellular NAD is primarily a signaling function
  • It’s definitely not a normal way of transporting NAD from tissue to tissue

IV NAD

  • So when we inject NAD we’re thinking about transporting it to cells
  • But the body thinks that it’s a signal so we’re amplifying a potentially negative signal

Oral NR: Is it the optimal way to get more NAD? [1:38:30]

Oral NMN

  • Example is riboflavin 5′-phosphate
  • “I would bet money that MNM is not absorbed intact.”
    • And that’s because NMN has a charged phosphate group on it: cannot cross cells.
    • “And even if it were to be true that there were transporters in the intestines that could take NMN up intact, it probably still would not be absorbed intact because the phosphatase is in the small intestine that cleave the phosphates off of all the molecules in the food you eat do so nonspecifically to all of the molecules in the food you eat.”
  • I believe the NR gets intact because

Oral NR

  • So we’re taking super physiologic doses of NR (500 to a 1000 milligrams daily which are found in common NR supplements)…
  • Using our glycogen analogy, we’re now increasing our  of nicotinamide that we can slow drip out when it’s demanded hepatic reservoir

The possible short-term and long-term benefits of NAD precursors

  • Rabinowitz papers showed that the turnover of NAD in the small intestine is 40 times what it is in muscle

How is this possible?

  • Consider the disease called pellagra (niacin deficiency) and NAD is low:
    • the four D’s of dermatitis, diarrhea and dementia, death

⇒ Pellagra effects

  • NAD is actually generating molecules that are directly involved in immediate neurotransmitter production ⇒
  • “I think that’s why you see brain effects in pellagra.”

  • If you exclude the brain, the two tissues where you are seeing the most dramatic effect are the two tissues that are outside of the body: skin and gut

⇒ skin issues?

  • Well, when you’re out in the sun, you’re always experiencing DNA damage and you’re always repairing it
  • So in the skin you have this very incredibly high NAD turnover, because even things that you normally think are benign (like just going outdoors) is actually causing damage that you are repairing always

⇒ In the gut. . .

  • I think part of it is that it’s so energy intensive to maintain the cell turnover in the gut
  • But also the gut is exposed to so many insults of just total lack of quality control over the things that come inside it
  • NAD Most benefit short term in those tissues with the highest turnover (the skin and the small intestine)
  • The challenge: It’s hard to design the ideal study to measure those things (as compared to measuring glucose and insulin and triglycerides and lipoproteins which they’ve already done)

What might be the long-term benefit of NAD?

  • Increased genomic stability
  • Decreased accumulation of DNA damage
  • Increased telomere length
  • None of which have been measured

The need for longer studies

  • To understand what it’s doing inside all of the other cells in the body, you’re going to need a much longer time horizon

What is the possible harm of taking an NAD precursor? [1:47:15]

If you’re cost-insensitive, what’s the downside in spending $100 a month to take these compounds?

“The potential risk is that you’re sapping your methyl group supply.”
  • Part of this cycle is when you do generate nicotinamide, you face the possibility that you have to methylate it and you have to pee out the methylated metabolite
  • If you’re taking 1,000 milligrams or 2,000 milligrams of NR a day (the RDA for niacin is around 15 milligrams), you are losing a lot of methyl groups

The MTHFR gene [1:49:45]

Methyl groups

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Figure 2. Methyl groups are used in the synthesis and regulation of many compounds. Image credit: Chris Masterjohn

What we do there is…

  • Using ATP, we activate methionine to S-Adenosyl methionine (the universal methyl donor)
    • So no matter what we’re synthesizing or regulating, it is always S-Adenosyl-methionine that donates that one carbon, that methyl group
  • Through two steps it becomes homocysteine (the inevitable byproduct of using it)
image

Figure 3. The methylation system produces S-adenosylmethionine as the universal methyl donor. Image credit: Chris Masterjohn

  • Once we have homocysteine, we have two choices
    • 1) We can either get rid of the homocysteine (if we have an abundance of methyl groups) or…
    • 2) We recycle the homocysteine back to methionine (if we don’t have enough methyl groups)
  • How to get from homocysteine back to methionine? 2 ways…
    • 1) Folate (vitamin B9) can take a methyl group from amino acid metabolism and can pass it on to B12 ⇒ B12 then passes it onto homocysteine ⇒ Homocysteine regenerates methionine. Or…
    • 2) Choline can be oxidized to betaine ⇒ which can be then the methyl donor to recycle homocysteine to methionine
    • These two pathways are equivalent meaning in the avg. person they’re probably 50/50, but it’s going to depend on their dietary intake
image

Figure 4. Half of methylation is supported by folate and B12, half by choline or betaine. Image credit: Chris Masterjohn

What is the MTHFR enzyme?

  • One of the enzymes that is involved in constructing the methyl group that came from the amino acid metabolism that became the folate molecule
  • That’s how folate passed the methyl group on to B12 and then on to homocysteine to recycle methionine

Everyone has MTHFR

  • We all have MTHFR, but there are variants of the gene
  • The two common polymorphisms (gene variants in the MTHFR gene) are so spread out in the population that what you actually see as a gradient of MTHFR activity that’s fairly even spread across the population
image

Figure 5. MTHFR polymorphisms cause an almost continuous gradient of activity equally distributed across the population. Image credit: Chris Masterjohn

⇒ If you have limited ability to use folate for methylation, there are several things that happen…

  • The first is that you use up more choline
    • You do that because you suck at using folate and you have no problem using choline.
    • These people with the worst MTHFR activity are the ones who require 1,200 milligrams of choline

Which SNPs would be the “worst” to have?

It’s more like a spectrum

  • Varies by population It’s more like a spectrum than wild type vs mutation
  • There’s not a category that even has a large minority of the people in it

Comparing the two extreme ends of methylation: The people with the “best” and the “worst” “methyl folate status”

  • The people who have bad methyl folate status are the people who are doubling up the amount of choline that they’re blowing through for the cycle.
  • Peter points out that there’s an undeniable correlation when looking at your MTHFR status, the lower you are in your capacity to efficiently do so, the higher your homocysteine is, all things equal.

⇒ Therefore, wouldn’t we expect that if they’re truly as efficient at methylation even using choline, shouldn’t they be able to compensate for that?

  • Chris:
    • “What you see in the literature is that studies that can show a statistically significant rise in homocysteine, it is generally limited to the people who are in the worst-case scenario.”
    • What’s interesting is that if you subdivide those people by their riboflavin status, all the people who have the MTHFR genotype that look bad, that have high homocysteine, have bad riboflavin status.
    • “The thing that lowers MTHFR activity with those polymorphisms is that MTHFR is a riboflavin-dependent enzyme (vitamin B2).”
    • “It has a lower affinity for the riboflavin as a cofactor, so you need better riboflavin status to optimize your MTHFR.”

What about using vitamin B6?

  • Peter: “This is so counterintuitive because the thing that we find, clinically, that has the best hammer is B6.”
  • Chris: “Because you’re not thinking about optimizing MTHFR activity, you’re thinking about getting the homocysteine.”
  • Peter: “Do you think that’s the wrong way to think about it, clinically?”
  • Chris: “Yeah, I think it’s the wrong way to think about it. I think it’s too simplistic . . . But if someone has high homocysteine that goes away with B6 supplementation, that person probably needed high B6.”

The methylation pathway [1:58:15]

Great resource from ChrisStart Here for MTHFR and Methylation

Example, you eat a steak…

  • Good thing: There’s a bunch of methionine in there and you can use that methionine for methylation
  • The bad thing: If you had this boatload of methionine that comes in, you’re going to generate a boatload of homocysteine
    • You also don’t have the problem of needing to recycle that homocysteine because you got a boatload of methionine coming in (it’s probably dripping into the liver and you can probably predict that if you have a lot right now, you’re going to have even more coming in 10 minutes)

⇒ What do you do?

  • You totally shut down MTHFR which is the enzyme that uses choline
  • And then you flip on this other enzyme that’s not usually active that gets rid of the homocysteine
  • On top of that, you want to get rid of the extra methyl groups
  • You turn on this other enzyme that is going to use the amino acid glycine as a buffer
  • That glycine is going to, if it can, hold on to the methyl group for later use, but if you methylate too much of that glycine, you end up peeing it out
image

Figure 6. When SAMe is present in excess, homocysteine is broken down instead of recycled and glycine soaks up the extra methyl groups. Image credit: Chris Masterjohn

⇒ So what is the B6 doing (which Peter uses clinically to reduce homocysteine)?

  • It’s activating the enzyme that kicked on when you ate the steak that helped you break down the homocysteine

⇒ In the fed state: Your MTHFR shuts down in the fed state if you ate protein

⇒ In the fasted state: The amino acid that falls most quickly when you fast is methionine

⇒ So three days into a fast, you should have very high MTHFR activity? ⇒ “Yes.”

image

Figure 7. When SAMe is present in excess, the lack of methylfolate turns on the glycine buffer system. Image credit: Chris Masterjohn

⇒ Tying it back to the patient taking B6 for high homocysteine

  • This is proof of concept that the B6 worked
  • But the problem you addressed was not MTHFR
  • “That means that part of their high homocysteine, it was a result of fed state homocysteine that should’ve been broken down using the vitamin B6 that wasn’t broken down because they didn’t have enough B6.”
  • The B6 is working because those people need more B6.

Is homocysteine a good indicator of whether you’re messing up your methylation?

  • Some data says that maybe the homocysteine is a problem in it of itself because:
    • it caused oxidative stress and might contribute to cardiovascular disease,
    • But it’s also just a marker that things aren’t working right in that cycle.

⇒ Not the only thing that goes wrong in the cycle. For example,

  • Some people need 900 to 1,200 milligrams of choline because people who have low MTHFR activity are doubling the amount of choline that they blow through
  • This issue has nothing to do with the homocysteine
  • “The data indicate that those people have a higher choline requirement and the way that you address that is by consuming enough choline.”

MTHFR and fatty liver

Given that a subset of patients are going to be burning through choline quicker and therefore have less choline to do its job in the liver?

  • “I would expect that that could be the case, but I don’t know of data on that.”
  • Hopefully, there’s a graduate student listening to this because that would be a really interesting and elegant experiment to demonstrate that!

For those in the worst case scenario of MTHFR

  • 900 or 1,200 milligrams of choline will do two things:
    • 1) it will minimize markers of oxidative DNA damage
    • 2) it brings choline utilization markers back down to what you would find in someone who didn’t have the MTHFR or polymorphism.
  • If someone is using up more of their choline, I wouldn’t necessarily expect them to have fatty liver, but I would expect them to have a higher probability of developing it if you put the other conditions in.

Recommendation for a choline supplement?

  • Chris would prefer food, first
  • But if you’re going to do a supplement, Chris would prefer phosphatidylcholine over a choline salt

The COMT gene [2:04:30]

What is getting synthesized in the methylation process?

  • 90% of the methylation process is done to synthesize creatine and to synthesize phosphatidylcholine
  • Of the remaining 10%, the next most sensitive to methylation is dopamine
image

Figure 8. Most of methylation is used for the synthesis of creatine and phosphatidylcholine, with other uses includingthe catabolism of neurotransmitters. Image credit: Chris Masterjohn

  • COMT is the enzyme that methylates dopamine
  • If you methylate more dopamine you are mentally more flexible
  • If you methylate less dopamine, you are mentally more stable
  • If you’re in the middle of that, it could just be a variation of your personality
  • But at the extreme ends of that spectrum, you start to get into the possibility of psychiatric disorders
  • This can apply to the people in the middle of that spectrum (i.e. not in the extremes)
  • The warrior = the person who has a higher rate of methylating dopamine, is more mentally flexible, and that’s the person that picks the battle, faces it, picks the battle, faces it, defeats, and just moves from one thing to the next
  • The worrier = low rate of methylation, gets stuck on things they’re worrying about
  • There aren’t as many different types ⇒ It’s basically 50% of people are in the middle, 25% are in one end, 25% are on the other end

Creatine: The uses and benefits and it’s important role in methylation [2:10:15]

MTHFR

  • You want to get between 900 and 1,200 milligrams of choline a day
  • Get choline from food first (eggs, meat, protein)
  • If you need to supplement, use a phosphatidylcholine
  • Take 5 grams of creatine monohydrate ⇒ “supplementation with creatine makes a lot of sense because 45% of your methyl demand is to synthesize creatine”

Creatine

  • Creatine monohydrate is something that most people take for exercise
  • Years ago people thought to load 20 grams a day for five days than five grams (cycle on and off)
  • Now the literature suggests 5 g/day is plenty, says Peter

Peter: We think of that as creatine as a phosphate donor (i.e. weight training, the burst is really a phosphate donation from creatine, it’s not an ATP driven process)

Choline’s role in synthesizing creatine

  • Whether you’re taking methyl groups from choline or you’re taking methyl groups from folate…
  • What you do when you synthesize creatine is you start with guanidinoacetate
  • which you make from the protein in your diet and then you methylate it and that makes creatine

Creatine’s sensitivity to methyl group supply. Your going to stop making creatine before you stop expressing the correct genes.

“Creatine is the only thing that is super sensitive to the methyl group supply”
image

Figure 9. Creatine synthesis is most sensitive to the supply of methyl groups, while phosphatidylcholine and gene expression are least sensitive and neurotransmitters are intermediate. Image credit: Chris Masterjohn

  • You eat a steak
  • You synthesize creatine right then
  • But five hours later, you’re synthesizing nowhere near as much creatine

Why is this the case?

  • Some things never change with the supply of methyl groups in your body
    • i.e. DNA methylation for gene expression ⇒ you don’t want to regulate thousands of genes because you ate a steak and you had a bunch extra methyl groups
  • Others are “fairly acutely stable
    • i.e. dopamine and other neurotransmitters you don’t want that to go up “log fold” because you ate a steak
  • Creatine is very sensitive to the amount of methyl group supply

Creatine in the average person

  • Avg. person has 120 grams of creatine in their body and they will lose 2 grams of creatine everyday that they pee out in their urine
  • “The whole point of creatine synthesis is to keep the accounts balanced.”
  • If you synthesized no creatine in an entire day, your creatine drops from 120 grams to 118 grams ⇒ “It’s like barely a dent.”
  • You just have to make sure that in 60 days from now, your creatine isn’t zero, it’s still 120 grams in your body

Like AMPK is a great surrogate for ATP, do we have similar enzymes that give us a sense of CP versus C, creatine phosphate versus creatine?

  • “I don’t think there’s an analogous enzyme, but there’s a completely analogous energy state.”
  • When you’re exercising, for example,
    • ATP levels go down and AMP levels go up ⇒ meanwhile your creatine phosphate levels go down and your creatine levels go up
    • When you recover, everything reverses.

Creatine’s impact on performance

  • Many athletes like to use creatine is for increasing that maximal performance, or the duration of short maximal performance
  • But would creatine affect your ability to run a marathon? ⇒ “Yes. I think it  have an impact on your ability to run a marathon.” say Chris
  • will

  • Because creatine does things in muscle that are independent of phosphate donation
  • It hydrates the muscle which…
    • Makes your muscles look bigger
    • And also directly increases your muscular strength

Dietary strategies for MTHFR: choline, creatine, folate and glycine [2:16:45]

Videos by Chris on this topic:

Who should consider modifying their diet for MTHFR reasons?

⇒ It depends on…

  1. what problems are you facing ?
  2. what goals do you have ?
  3. And to what degree do you want to be anal about doing things that have  payoff?
  4. theoretical

Strategy for the low methylators:

  • Such as someone who has either high homocysteine or they have psychiatric difficulties
  • Or they are overly ruminating on things or feeling like mental health is an uphill battle

What to do:

  • Most data backed thing is increasing your choline
  • Should also increase creatine intake
  • Methyl folate is also important (but overlooked in many cases)
  • Glycine is also important
image

Figure 10. Ways to get choline from foods. Image credit: Chris Masterjohn

Methyl folate and glycine

  • Methyl folate is the thing that controls whether you pee out glycine as a methyl buffer
  • “When your methyl folate level is low, your body thinks that you are in a state of methyl abundance and that you need to methylate glycine and pee out the methyl groups and the glycine into the urine.”
  • So you probably need more glycine

Glycine

image

Figure 11. Glycine is calming and has antipsychotic and sleep-promoting effects. Image credit: Chris Masterjohn

Peter isn’t impressed with the data that taking collagen or glycine will help collagen synthesis

  • “For the benefit of replacing their own collagen. I’ve always been like I just don’t see how that works.”
  • Chris can’t argue: “There is no good data showing that you will have less wrinkles if you take collagen. I’ve never heard of any data on bone health.”

Peter think that glycine is more likely to be beneficial for:

  • Blood sugar
  • Sleep

Can collagen improve the health of tendons?

  • Showed that before you exercise… if you take 15 grams of gelatin (but not 5 grams) with a little vitamin C, you will increase collagen synthesis in the tendons
  • The rationale for the study:
    • your muscles are very metabolically active and very good at taking things up when it wants to,
    • but the connective tissues in your joints are very dependent on you just pushing more blood supply there.
    • So, when you exercise, you have the amino acids coming in before you exercise, and then when you’re exercising, there’s increased blood flow that gets the collagen peptides into those tissues.

What foods would be high enough in glycine?

  • Bones are the highest, and skin is intermediate.
  • For bones…
    • It’s not the marrow, It’s the broth
    • It’s dependent on the protein content ⇒ “If you’re making it homemade, you don’t know exactly what the protein content is, I would use the metric of whether it’s well gelled.”
    • Several bone broth products on the market ⇒ Label says 10 grams of protein per cup which equal 3 grams of glycine
    • Ideally, you should take 3 to 5 grams
  • You’re gonna drink that before bed every night, and it might help you sleep? ⇒ In theory.”
image

Figure 12. Nourishing the low-MTHFR phenotype. Image credit: Chris Masterjohn

How to mitigate the negative effects of NAD supplements [2:23:45]

Losing your methyl group supply

From the Brenner study, we learned…

  • That an enormous amount of this NR is getting methylated as nicotinamide
  • Not possible to extract the data to say exactly how much because they’re in concentrations, and we don’t know what the up and down flux is over time

Roughly speaking…

  • But as a rough calculation to get a sense of how much impact you could have if the impact were maximal…
    • For every 1,000 milligrams of nicotinamide that you detoxify you are in theory decreasing your synthesis of creatine by 500 milligrams
    • You’re synthesizing two grams of creatine in a day
    • So if you’re taking 2,000 milligrams of NR and all of it were detoxified, you’d be cutting your creatine synthesis in half
    • And you don’t need your creatine to go to zero before you have problems

Benefits of creatine

Why do bodybuilders take creatine?

  • Because they’re hoping to maybe increase their body stores by about 30%.
  • Just leveraging a little bit marginal increased creatine makes big results in your strength
image

Figure 13. Creatine supplementation improves depression. Image credit: Chris Masterjohn

Just looking at the physiology of creatine…

  • you use it to make your sperm swim
  • And you’re using it to pump acid into your stomach
“Creatine is important in all kinds of areas that you would not expect to think about it in.”

Should someone take creatine daily even when not exercising?

  • On the whole, if you take 5 grams a day, and you just always do that, you’re eventually going to get to “hit that steady state”
  • Although you could argue that it might be better if…
    • You take it after exercise with a carbohydrate bolus
    • And if you split it up into two doses in a day

So are NR supplement dangerous?

“My position is not that this stuff is really dangerous.”

However…

  • Anecdotally, Chris sees people who are taking this stuff who experience
    • seesawing in their energy levels
    • seesawing in their mental and emotional states
  • These are things that could be explained by 1,000 other things
  • BUT…this is
  • “exactly what I would expect to happen in someone whose methyl group supply was being sapped.”

How to minimize the potential downside of taking NAD precursors

A case study of a person with high homocysteine [2:28:00]

Case study: A patient of Peter’s with high homocysteine (13-15 range)

Could NOT get his levels to go down, Peter tried…

  • Standard dose of methyl B12, Double dose of B12, Adding 50 milligrams of B6 three times a week, Then tried 50 milligrams of B6 daily
  • Finally tried TMG and his homocysteine falls by 50%

What was going on with this guy?

  • B6 doing nothing indicates one of two things
  • 1 Either that guy’s enzyme for getting rid of the homocysteine didn’t work that well
  • 2 This was not a fed-state homocysteine issue
  • “My guess is that unlike the guy whose homocysteine went down with the B6, this guy had a recycling issue. He did not have a problem of disposing of the homocysteine in the fed state. He had a problem of recycling in the fasted state.”
  • When you use choline for methylation, what you do is you convert it into trimethylglycine (TMG), and that becomes the methyl donor.

Does it really matter what your homocysteine levels are?

  • “I don’t think the argument is super strong that by fixing the homocysteine is reducing cardiovascular risk.”
  • “I look at homocysteine, and I say, ‘Well, look. Young, healthy people have a homocysteine on average between seven and nine, that’s probably a sweet spot.’”

What is the level of evidence that you need to take an action? [2:32:15]

The following is a transcript of their discussion…

Chris: This comes down to a philosophic question of what is the level of evidence that you need to take an action?

Peter: Versus the level of evidence to not take an action, which people always forget that they have to ask that question just as well.

Chris:

Well, also versus the level of evidence to state that you have a certain degree of certainty or confidence in something.

We can be fairly rational and within a fairly narrow window on what do we believe are the principles that we need to secure to say we have a certain degree of confidence that something is true.

But we can never create that window for saying what is the level of evidence that I need in order to take an action, because that comes back to your subjective values.

A lot of this is assumption as well. Even if you’re coming to the question of what is the probability that we should assess that something is true? For me, I would take as a background assumption that things that have a proven track record in human history over a long period of time should be assumed as a default.

Someone else may take the assumption that nothing is the default or that the status quo is the default.

If you’re basically saying that we have to have meta-analyses of large, randomized, controlled trials on clinical endpoints in order to have something, you are assuming that in the absence of that evidence we will follow the status quo.

Even in the case of us debating how confident we are that something is true, we have reasons like that to have a spectrum of agreement or disagreement.

But when it comes down to should I take this supplement, that comes to subjective value over what kind of risks do you wanna take. Right?

There are some people that want to optimize their metabolism to make their body run like a well oiled machine, the best way that they know how, and they’re willing to spend some money or design their diet around doing so. And I think most of what I said mostly applies to those people.

I think for people who are looking for hard clinical endpoints, in fact, this is why there’s Twitter wars over MTHFR sometime.

Peter: Oh, thank God I missed those.

Chris:

I’ve gotten into some tussles on the internet with some people who looked at this from the perspective of, “look, there’s no hard clinical endpoints for which MTHFR is diagnostic. There’s no hard clinical endpoints for which people with MTHFR, for whom that population has some specific dietary or supplemental regimen that alters that clinical endpoint.”

And I think that’s true.

Where I come from if I were in that situation, I would wanna bring my homocysteine down to what by all probability appears to be the healthy level.

If I were that person, I would want a reason, well, I feel like my mind is too sticky, and I wanna loosen it up a little bit. If I were that person, I would wanna have good energy, and I would wanna use a rational assessment of why my energy might be low and what I can do to bring it up to normal.

And I’m probably never gonna convince anyone that if I feel better, that there’s a hard, testable clinical end point to that.

Does Chris supplement with NAD precursors? And can it improve symptoms of rosacea? [2:35:45]

He takes Tru Niagen

  • He takes 75 milligrams with breakfast and 75 micrograms with lunch
  • Breaks the 150 mg pill in half
  • “I might go up. I’m just playing around with it. It’s just a tinkering thing.”

Do NR supplements help with rosacea?

  • In Peter’s patients who take NR, the one thing that seems to across the board improve is rosacea
  • Perhaps, rosacea as an endpoint in a clinical study would have good results to it, suggests Chris

Summarizing Chris’s thoughts

  • IV NAD is not a great idea
  • It’s much better to build up a hepatic reservoir of nicotinamide that’s converted through the NR that you can then slow trickle into circulation as needed

Decision making in the face of inconclusive data, and trying to disentangle the placebo effect [2:39:00]

Chris references a video he made about NR

“People are gonna make a decision to take this or not, and there is no clear data on what it does. So, you either take the position that you’re gonna wait 10 or 20 years until we know something better, or you take the position that you’re gonna tinker. And if you’re gonna tinker, you’re gonna tinker a lot more successfully if you have a working model of what’s going on than if you don’t.”
  • Peter: “Rapa would probably be the single most ‘out there’ thing that I do, but my model’s robust. Now, I also think I have much more data to point to. So, even though people can say, “Oh my god, rapamycin is so scary,” I get to point to what happens in the yeast, the flies, the worms, the mice, the rats, the dogs, the kangaroos, so I feel like I’m standing on the shoulder of much more evidence, even though I’m interfering with a much more important sensor.”

How to follow Chris’s work [2:48:45]

SELECTED LINKS / RELATED MATERIAL

Great resources from Chris:

Chris’s article criticizing the TMAO studiesDoes Carnitine From Red Meat Contribute to Heart Disease Through Intestinal Bacterial Metabolism to TMAO? | Chris Masterjohn (westonaprice.org) [42:30]

Video where Chris talks about matching your NR dose mg for mg with a methyl donorWhy You Should Be Careful With Niacin and Nicotinamide Riboside | Chris Masterjohn Lite #54 | Chris Masterjohn PhD (youtube.com)

What you need to know about your methylation systemStart Here for MTHFR and Methylation

MTHFR videos:

Nutrition in Neuroscience (Part 1 | Part 2 | Part 3 | Part 4)

Niacin series (Part 1 | Part 2)

Other Related Links:

Chris’s article criticizing the TMAO studiesDoes Carnitine From Red Meat Contribute to Heart Disease Through Intestinal Bacterial Metabolism to TMAO? | Chris Masterjohn (westonaprice.org) [42:30]

Observational data showing that people who eat more fish have more TMAO in their bloodTrimethylamine N-Oxide: The Good, the Bad and the Unknown (Velasquez et al., 2016) [43:15]

Chris’s podcast where he discusses the 2018 Rabinowitz paperNiacin Part 1: What It Is and Why You Need It | Chris Masterjohn (chrismasterjohnphd.com) [1:27:45]

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