Peter Attia’s podcast, The Drive, is one of my favourites, but due to the complexity of conversation, if I’m not taking notes I might as well be listening to someone speak in a different language.

And I presume I’m not the only, so with that — here are my notes from episode #46 with Chris Masterjohn, PhD

Choline & Fatty Liver

• Choline is an essential component of the phospholipid phosphatidylcholine and this phospholipid is not just in our cell membranes it’s in the membranes of the lipoproteins that transport fat out of the liver so without enough choline you can’t make the LDL particle to get trigs out of the liver
• Doesn’t matter the etiology of fatty liver, as long as you have enough choline in the diet you can clear it from liver
• He recommends 1200 mg of choline a day in phosphatidylcholine form since this form is less likely to turn to TMAO in the gut but getting it from food is always the best way
• Excessive fructose will cause NAFLD (non-alcoholic fatty liver disease) not glucose
• He talks about a study where rats were fed a 60% fructose diet which they expected to cause fatty liver, but the rats never developed fatty liver. The diet they fed the animals was high in methionine (different to other fructose/fatty liver studies) and methionine is a precursor to choline, so this makes sense why they got the negative outcome for NAFLD
• If you add more protein to the diet, you naturally increase your intake of methionine, and this has been shown to reverse fatty liver in animal models

TMAO

• Found naturally in fish and produced in the gut in people with rare genetic disorders
• Enormous choline loading increases trimethylamine (the breakdown product of TMAO) which is responsible for the “fishy” scent
• In mice, TMAO is atherogenic so people made the assumption that the same held true for humans and eating choline or carnitine rich foods were “unsafe” for this reason since they had the potential to cause heart disease
• This idea does not make sense to Chris
• Fish is always associated with greater health, so if it was the TMAO that was the problem with meat and eggs too (high in choline and carnitine which can convert to TMAO), the same would apply to fish but it doesn’t
• 500 mg of choline bitartrate was shown to increase TMAO and blood clotting but you can not get that level of TMAO from getting your choline from food.. It is different to choline bitartrate
• Peter was eating 8 egg yolks a day on a 4:1 ketogenic diet and his TMAO levels did not budge
• TMAO response to eggs varies from person to person — some people have none some people have a lot and it depends on your gut microbiome

Diet & Choline

• How many eggs you eat in a day is different than how many eggs you eat in a sitting, we have a cap for absorbing choline
• Vegan diet would get choline from nuts and vegetables

Polyunsaturated Fatty Acids (PUFAs)

• In PUFAs, the carbon between the double bonds is very unstable and is vulnerable to lipid peroxidation (damage)
• The more omega 6 in cell membranes the more of a liability it becomes because oxidative stress and inflammation makes them vulnerable to damage
• Human study: followed over weeks, PUFAs lead to less liver fat so you could take this as a positive but if you have fatty liver of mostly PUFAs you are way more vulnerable for worse outcomes because of the increased targets for damage
• Oxidative stress does not just cause NAFLD or NASH (non-alcoholic steatohepatitis), it can cause steatosis because you get oxidative destruction of APOB particles (needed for transport of fat out of the liver)
• It isn’t just choline that allows export of fat from the liver, you need antioxidant protection in the liver because if you damage this particle before it exports the triglyceride, than you prevent it from doing so
• PUFAs are complicated because in short term with little going on, in the liver they will be better, but long term they are a disadvantage because it’s building the fire without the flame but when you light the fire it’s bad

APO Proteins

• APOB in liver: involved in VLDL export to get triglycerides out of liver but we also make denovo LDL in liver
• VLDL don’t pose atherogenic risk when you are insulin sensitive because they have low residence time (APOC3) (aka they don’t stick around for long)

NAD (Nicotinamide adenine dinucleotide)

• NAD is an extremely important molecule to various cellular reactions
• One role of NAD is as a substrate to sirtuins and PARPs (sirtuin and PARP activity has implications for longevity and healthy aging)
• NAD is an oxidizing agent, meaning it accepts electrons, reducing it to NADH (now an electron donor)
• Our cells are powered by these electron exchanges
• Mitochondria have complexes used to generate reducing agents to move electrons from one side of a double membrane building a gradient that allows for the synthesis of ATP (oxidative phosphorylation)

NAD Depletes as we Age

• Complex 1 (in mitochondria) turns NADH into NAD and as we age, the ratio of NAD to NADH goes down (less NAD), which is bad because this decreases the activity of sirtuins which do many things but are important for repairing DNA damage
• Also, NAD depletes as we age because sirtuins and PARPs, that use NAD for the purpose of protecting DNA and telomeres (and other things related to longevity), consume the NAD at a greater rate
• It’s hard to know why NAD is low
• Being in the overfed state is a problem
• If you look at NAD levels dropping, you’re looking at the PARPs and sirtuins consuming them for repair

Why you can’t just “eat” NAD

• Collectively we refer to it as niacin (B3) in the diet, usually consumed in the form of nicotinic acid from plant foods or nicotinamide from animal foods

NR → NMN → NAD

• If you take NR (nicotinamide riboside) you are taking it intact
• If you take NMN (nicotinamide mononucleotide) you are probably digesting it down to NR
• But you are definitely not absorbing NAD or NADH in the intestine
• When we eat food, nicotinic acid converts to nicotinamide which the intestine tries to convert to NAD but whatever it doesn’t, goes to liver and the liver is the main site for all the forms
• The liver will try to convert all these forms into NAD for the whole body
• Everything that comes out of liver and into circulation is nicotinamide (transport form) and then tissues will convert it to NAD
• You don’t see NAD or NADH transported in the blood
• If you take a supplement, you get a meaningless amount that gets transported as NAD or NADH

NR, NMN, NAD & Metabolism

• NR becomes NMN and then NAD
• if you take NR, the liver is basically taking it and making NAD, we know that the liver will convert it to nicotinamide
• All of the NAD in non-hepatic cells (non-liver) is derived from circulating nicotinamide that the liver released
• Nicotinamide (not charged) is the circulating form of niacin that all cells will use to make NAD (tiny bit made by the kidney)
• A tissue gets NAD because it took nicotinamide from the blood that the liver made with its store of NAD
• Once nicotinamide is in the cell it gets converted to NAD through two steps (ATP dependent)
• Gets converted to nicotinamide mononucleotide (NMN) and then gets converted into NAD
• A portion of the NMN becomes NR and then comes back to NMN kind of like a pressure release valve (converts to NR to kind of hold on to it)
• You have nicotinamide to NMN to NAD, you can go from NMN to NR but you have to go back over from NR to NMN to get to NAD

Sirtuins, PARPs, and the fate of intracellular nicotinamide

• Sirtuins and PARPs are NAD consuming enzymes for DNA repair and when they do this they release nicotinamide
• Nicotinamide is a negative inhibitor of the enzymes that use NAD (eg sirtuins and PARPs) through negative feedback loop
• We don’t want nicotinamide hanging around in our cells so we either do something with it fast or we methylate it and pee it out

Why it’s more effective to take NR over nicotinamide

• Nicotinamide is the circulating form that gets into the muscle but isn’t the substrate to take
• In a particular study, there was much less of an NAD response in muscle with oral nicotinamide than with NR
• Whatever supplement you take ends up in the liver
• Nicotinamide gets converted to NMN and NMN gets converted to NAD
• If sirtuins and PARPs are consuming NAD, they are releasing nicotinamide (already mentioned above)
• Having all this nicotinamide lying around is a liability because it acts as an inhibitor to these repair enzymes, so we try and get rid of it or try our best to convert it back to NAD
• If you take oral nicotinamide, it gets to the liver and is immediately a liability before it even becomes NAD
• The liver will either methylate it or convert it to NAD
• If you take oral NR, you convert it to NMN and in order to get back to nicotinamide you have to convert it to NAD first before it ever gets exposed to these detoxifying processes
• Chris believes that NR is a superior way to increase hepatic (liver) NAD, because when it gets to the liver, it can’t be immediately detoxified, it’s not an immediate threat to the sirtuins and PARPs and must be converted to NAD before anything else
• If you present with a threat, you can only make so much NAD at one time, you will have much more waste (via urine) than if you put NR in because it needs to make NAD before ever exposing to the detoxifying pathway
• The liver isn’t just making NAD for itself, it’s doing it to carry the reserves for the rest of the body as NAD
• NAD in liver isn’t just being immediately used for respiration, sirtuins, and PARPs, it has a reserve pool of NAD that it holds onto so it can slowly release nicotinamide to the rest of your tissues which then can make the immediate decisions whether to detoxify or make NAD
• If the liver can hold on to NAD safely, it has a better ability to release nicotinamide on a continuous as needed basis (somewhat parallel to the way we store glucose (nicotinamide) as glycogen (NAD) in the liver and titrate it out as glucose on an as need basis)

IV NAD

• Free extracellular NAD (outside the cell) activates granulocytes (white blood cells involved in immunity) to cause vasodilation and massive mTOR response
• When you inject NAD intravenously, you’ve bypassed the liver and its sitting in the plasma (blood)
• The immediate response is an inflammatory response
• Not clear where the NAD is going
• There are a variety of mammalian cell types that have the ability to take it into the cell but what is the normal physiology?
• To understand that, you need to look at how does NAD get out of the cell in the first place?
• It appears to be that extracellular NAD gets there from dead cells and dying cells and maybe cells that are stressed and secreting NAD as a signal to reflect the energy status of the cell
• Extracellular NAD = signalling molecule which makes sense because there are enzymes that consume NAD extracellularly, these enzymes breakdown the NAD
• What the signal is, we don’t know
• NAD injections transport NAD in a way that the body doesn’t transport it, and it could send a signal of what’s happening that is definitely not taking place but maybe it’s a good signal and has good consequences like a hormetic response but also could have negative consequences
• Why people are reporting benefits are either a hormetic response (signalling effect that by virtue of luck is beneficial) or it’s a placebo effect

Oral NR vs NMN

• Oral NMN is probably not absorbed intake because of the charged phosphate on it and generally charged phosphates can’t cross cells
• So even if it were that transporters could take NMN intact, it probably still wouldn’t be absorbed intact because the phosphatase that cleaves phosphates off of all molecules we eat do so non-specifically so it will do it to the NMN
• The overwhelming rule is that it is very difficult to carry charged phosphates across the intestines and cell membranes in general
• If anything, maybe it gets cleaved to NR and the NR gets across
• Oral NR gets the liver intact as NR because it doesn’t have the charged phosphate on it

NAD Turnover — Skin & Gut

• The turnover of NAD (in mice) in small intestine is 40X greater than muscle
• In the brain, NAD is generating molecules that are directly involved in neurotransmitter production, so that is probably why we see the brain affected in people with pellagra (niacin deficiency)
• The two tissues you see the most effect (rapid turnover) are the two tissues outside of the body (intestinal barrier and skin)
• Once we cross the intestinal barrier, we have incredible control
• When you are in the sun you are experiencing DNA damage and always repairing it which means you have this incredibly high NAD turnover in the skin
• Even things you would think are benign like going outdoors is causing damage that you are always repairing
• The skin has a huge demand for NAD
• If you take away this supply you are at risk for damage
• In the gut, maintaining the cell turnover is very energy intensive but it’s also the case that the gut is exposed to so many insults
• Chris’ suspicion is that benefits from increasing tissue NAD over the short-term is going to be in tissues with the highest turnover (skin and small intestine — both of which are hard to find what to measure)
• Over the long-term you would expect increased genomic stability, decreased accumulation of DNA damage and increased telomere length, all of which have not been measured
• Don’t have much data because we only have week-long studies, and we need longer term studies

Potential risk of taking taking NAD/precursor supplements

• A potential risk is that you are reducing your methyl group supply
• The reason is that when you do generate nicotinamide, you face the possibility that you may have to methylate it and get rid of it (in urine)
• If you are taking a huge dose of NR a day you are losing a lot of methyl groups and we know this in humans
• You see in studies, in urine and blood cells, there are significant increases in methylated nicotinamide

Methylation Cycle

• A methyl group is a 1-carbon unit
• The methyl cycle uses the amino acid methionine to donate that 1 carbon to dozens of different things
• We activate methionine using ATP by converting it to SAMe which is the universal methyl donor
• SAMe becomes homocysteine which is the inevitable by-product of using it
• Once we have homocysteine we have two choices, get rid of it (if we have an abundance of methyl groups) or we recycle it back to methionine

Two ways to recycle homocysteine

1. Using folate to take a methyl group from amino acid metabolism and can pass it on to B12 which passes it on to homocysteine to regenerate methionine
2. Or choline is oxidized to betaine, which can be the methyl donor to recycle homocysteine to methionine

Both of these are used equally (50/50) but will depend on dietary intake

MTHFR (methylenetetrahydrofolate reductase) Activity

• MTHFR enzyme is one of the enzymes that contributes to constructing the methyl group from amino acid metabolism that went onto the folate molecule and that’s how folate passes it on to B12 and eventually homocysteine
• Everyone has MTHFR
• There is a gradient of MTHFR activity it’s not black and white (polymorphisms)
• If you have a limited ability to use folate to regenerate methionine, you rely more on the choline pathway as a compensatory response
• Nutritionally, you would need to have an increase in choline intake
• The worse your MTHFR activity, the more choline you need
• When you compare people with the best vs the worst methyl folate status, the people with the worst methyl folate are the people that are doubling up the amount of choline used to take care of excess homocysteine

MTHFR & Riboflavin

• Studies showing a rise in homocysteine is limited to the people who are in the worst case scenario
• No one is talking about the fact that if you subdivide these people by their riboflavin status all the people that have the MTHFR genotype that create high homocysteine, have bad riboflavin status, and the thing that lowers MTHFR activity in people with these polymorphisms is that MTHFR is a riboflavin(B2)-dependent enzyme and it has a lower affinity to riboflavin as a cofactor
• You need better riboflavin status to optimize your MTHFR activity
• If you want to optimize the MTHFR activity you give B2 but if you approach it from getting rid of homocysteine you would give B6 (pyridoxine)

What happens when you eat a steak (ie. protein)

• You get a bunch of methionine, and you can use this methionine for methylation
• One of the negative aspects of this is that if you have a boatload of methionine that comes in, you are going to get a boatload of homocysteine
• But you don’t have the problem of needing to recycle that homocysteine because you have enough methionine available
• When lots of methionine is available, MTHFR is shut down and the enzyme that uses choline is shut down, so you aren’t recycling it anymore (these are the two pathways)
• You flip on another enzymes that isn’t usually active, that’s used to get rid of the homocysteine by just breaking it down
• On top of this, you want to get rid of the extra methyl groups
• Turning on this enzyme is going to use glycine as a buffer and then this glycine (if it can) will hold on to the methyl group for later use but if you methylate too much glycine, you end up peeing it out
• In this context, the B6 is activating the enzyme that kicked on when you ate the steak that helped you breakdown the homocysteine

MTHFR during a fast

• MTHFR shuts down in the fed state (if you are eating protein, because of the methionine)
• Methionine is the amino acid that falls the quickest when we fast, we don’t preserve it
• During a fast, you should have high MTHFR activity, because you would be needing the recycle homocysteine to methionine

Treating high homocysteine levels

• It’s proof of concept that if the B6 worked that the problem you addressed was not MTHFR, part of their high homocysteine was a result of fed-state homocysteine that should have been broken down using B6 but wasn’t because they didn’t have enough B6
• You can’t fix MTHFR activity with methylfolate
• High homocysteine levels contribute to oxidative stress and maybe CVD but mostly it’s a marker that things aren’t working well in the methylation cycle
• You have other alterations that could not be working in the cycle though
• For example, some people need 900–1200 mg of choline that’s because people with low MTHFR activity are doubling the amount of choline that they blow through
• They have problems that are the result of not having enough choline, which has nothing to do with homocysteine all that needs to be fixed is adding more choline to the system
• If homocysteine levels drop by adding choline, these people clearly have a higher choline requirement

MTHFR and Fatty Liver Disease

• Low MTHFR activity means choline is being used in greater amounts, so there is less choline to do its job in the liver (no data on this but it makes sense)
• 900–1200 mg/day of choline will do 2 things

1. Minimize markers of DNA damage
2. Bring choline utilization markers back down to what you would see in someone who didn’t have the MTHFR polymorphism

• If someone is using more choline to compensate for low MTHFR activity, there is hypothetically a greater chance of developing fatty liver disease
• He would prefer people increase choline with food but if they’re going to supplement do it with phosphatidylcholine over a choline salt

COMT (catechol-O-methyltransferase) enzyme

• Enzyme involved in the catabolism (breakdown) of catecholamines
• Moving beyond homocysteine, one whole side of the equation is what you are methylating (homocysteine is just a byproduct of the whole methylation cycle)
• 90% of what you are methylating is to synthesize creatine and phosphatidylcholine
• The other 10% is a gradient of a handful of things
• The next most sensitive thing to creatine synthesis is dopamine
• COMT is the enzyme that methylates dopamine

COMT, dopamine, & mental state

• If you methylate more dopamine, you are more mentally flexible and if you methylate less dopamine you are more mentally stable
• If you are in the middle, it could be a variation of your personality
• When you start to get to the ends of the spectrums you start to get into the possibility of psychiatric disorders as a result of being in these extremes
• You could be a warrior phenotype or a worrier phenotype
• The warrior is the person that has a higher rate of methylating dopamine, is more mentally flexible and is the person that faces battles and defeats and moves from one thing from the next
• The worrier gets stuck on things and these people have a low rate of methylating dopamine
• COMT activity is a gradient, there’s no one over the other
• The “worriers” low rate of methylation could mean that these people are better at doing things that require sustained focus (academics, competitive athletics, one study showed elderly are more likely to do exercise if you tell them to)
• Things getting stuck in your mind is good if its the right thing, so it’s not always worrying and being stuck on worries
• OCD is associated with low methylation
• Substance abuse and ADHD is associated with high methylation phenotype
• COMT is not a gene for a mental state, it just has a partial influence on the “stickiness” of your mind
• COMT genotype will not tell you the rate at which you are methylating dopamine it’s telling you the rate at which you COULD methylate dopamine given a certain supply of methyl donors

Low MTHFR activity & dietary recommendations & creatine synthesis

• 900–1200 mg of choline/day
• Preference is from food (eg. eggs)
• Supplement with creatine monohydrate because 45% of methyl demand is to synthesize creatine — creatine synthesis requires methylation
• This whole cycle, regardless of if you are taking methyl groups from choline or folate, you synthesize creatine by starting with guanidinoacetate which you take from the protein in your diet and you methylate it to make creatine
• Creatine is the only thing that is super sensitive to the methyl group supply
• When you eat that steak, you synthesize creatine, 5 hours later you synthesize way less
• Some things are so essential to the body that you don’t want them to ever change with the methyl supply
• Example: DNA methylation for gene expression, you don’t want to regulate 1000s of genes because you ate a steak and now you have a bunch more methyl groups, you want to control that for totally different reasons. DNA methylation is designed to stay constant. There are other things that have to be fairly stable like dopamine and other neurotransmitters, you don’t want these to go up and down a lot just because you ate, but creatine, is different
• The whole point of creatine synthesis is to keep it balanced, so if you synthesize no creatine in a day, your creatine goes from 120g to 118g (you pee out 2g/day) barely a dent
• Creatine is the ideal thing to vary with the methyl group supply because you can eat protein, do all the creatine synthesis you need and then 5 hours later in the fasted state you don’t synthesize creatine anymore
• You just need to make sure in say 60 days that the amount of creatine in your body isn’t zero
• Modifying your diet around MTHFR status depends on the problems you are facing and what goals you have
• For someone who has high homocysteine or psychiatric considerations, what you can expect from the low methylation state is for someone to be overly ruminating on things if you think this is a problem for you and feel like that you need to be more psychiatrically healthy you would stick more to a dietary protocol like increasing choline and taking creatine because if you cut your methyl demand in half it will matter half as much that you aren’t that great at methylating
• You increase choline because that is the alternative methyl donor
• You add creatine in the system to decrease the demand

Methylfolate & Glycine

• Methylfolate is the thing that controls whether you pee out glycine as a methyl buffer
• When methylfolate level is low, your body thinks that you are in a state of methyl abundance and that you should methylate glycine and pee it out
• You probably need more glycine as well then — hasn’t been shown but makes sense in the understanding of the system
• You want to have some methylfolate there but probably need more glycine
• We can synthesize some (3–5g) glycine, but if you want to optimize collagen turnover in the skin, or get better sleep, improve blood sugar, etc. the average person could use some glycine (collagen, gelatin, bone broth, or just straight up glycine)
• Collagen (in bone broth) is better at increasing collagen synthesis
• When you eat collagen, you get amino acids and collagen peptides
• In one study, taking 15 g of gelatine prior to exercise, with a little vitamin C, resulted in an increase in collagen synthesis in the tendons
• Rationale, in muscle (metabolically active/very good at taking things up) the connective tissues in joints are more dependent on pushing blood supply to it, so when you exercise and have the amino acids coming in, the increased blood flow to these areas will bring it there
• We would expect the glycine levels to be low in people with low methylation
• Food products with collagen: animal skin and bones (bone broth or soft bones)

Circling back to NR

• An enormous amount of the NR will end up being methylated as nicotinamide
• To get a sense of the impact — for every 1000 mg of nicotinamide that you detoxify, you are in theory decreasing the synthesis of creatine by 500 mg
• So, if you are synthesizing 2g of creatine a day, taking 2000mg of NR and if all of it were detoxified, you would be cutting your creatine synthesis in half
• You don’t need creatine to go to zero before you have problems
• Although 90% is in muscles and the most famous reason to take it is to support muscle, if you look at the physiology of creatine you are using it for so many more processes
• Nicotinamide could be sapping the methyl supply — this could impact mental state
• In people reporting mental instability after taking NR, Chris would say to take a lower dose and match with a methyl donor — in theory you could do creatine but you would have to test it — his recommendation is to match mg for mg with TMG (trimethylglycine)

Peter’s patient with high homocysteine

• Methylfolate and methyl B12 did nothing
• Adding B6 nothing
• Add TMG — boom, homocysteine falls
• The fact that B6 didn’t work indicates two things, either his enzyme for getting rid of homocysteine didn’t work that well, or this was not a fed state homocysteine issue
• You’re trying to use MTHFR or choline to re-methylate homocysteine when you are in the fasted state and if you don’t have a lot of methionine coming in you stop doing that entirely and start trying to get rid of homocysteine when you are in the fed state
• This patient must have had a problem with recycling in the fasted state because his MTHFR activity was low, so TMG worked because it is involved in the choline pathway
• When you use choline for methylation you convert it into TMG and that becomes the alternative methyl donor, so you could probably get the same result with just using choline
• There are no hard clinical endpoints for which MTHFR is diagnostic
• There are no hard clinical endpoints for which people with MTHFR needs dietary or supplemental recommendations but if you are in this situation you would want to change homocysteine levels or improve your mental state and improve energy

No one knows the true effect of supplementing NR

• Chris is tinkering with it for himself, just to experiment
• Some people report improvements in rosacea which makes sense because the skin has high NAD turnover
• IV NAD in Chris’ opinion is not a great idea and he would take NR over others to build up his liver reservoir of nicotinamide to slow drip into system
• Lots of anecdotes reporting the benefits of NR but could be placebo, there is no direct data yet

Learn more about Chris Masterjohn here.