More Than Methylation: the Crucial Role of Choline with Dr Jonathan Bortz

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More Than Methylation: the Crucial Role of Choline with Dr Jonathan Bortz

How often are you thinking about a patient's choline status?

In this episode, Dr Jonathan Bortz discusses his journey into developing pharmaceuticals and supplements, and how it brought him to look at choline in a different light. He talks about the story of choline as a “one carbon metabolism,” as well as the various uses of choline in the body, including its role in methylation, genetics, and non-alcoholic fatty liver disease (NAFLD).

Covered in this episode

[00:49] Welcoming Dr Jonathan Bortz
[01:32] Discussing Jonathan’s background
[09:25] Jonathan describes his journey to choline
[14:54] Choline and it’s uses in the body
[22:14] Choline, oestrogen and foetal development
[34:05] Discussion on choline and methylation 
[40:21] SNPs and NAFLD
[49:09] Dosing choline
[50:08] How choline deficiency can lead to NAFLD
[57:02] Treatment in early stages of NAFLD
[1:03:16] Betaine supplementation in NAFLD


Mark: Hi, everyone, and welcome. Today we are talking with Dr Jonathan Bortz. Jonathan graduated in medicine in South Africa before moving to the Mount Sinai Medical Center in Cleveland, Ohio to specialise in endocrinology and metabolism. He then moved on to Washington University at St. Louis, Missouri, and has since 2006 been working with Albion Laboratories, developing considerable expertise in B12 and folate physiology. He joined Balchem Corporation in 2016, where his focus expanded to choline metabolism and its contribution to the one-carbon metabolic cycle. Dr Jonathan Bortz, welcome.

Jonathan: Thank you very much, Mark.

Mark: Well, today, it's exciting for me because we're finally getting away from this MTHFR gene, and we're getting onto something that I think of as a little more interesting. So we're going to talk about non-alcoholic fatty liver disease, and the part played by choline, particularly obviously, phosphatidylcholine. And a particular interest of yours, which I understand is the PMPT, a catalyst for the conversion of phosphatidylethanolamine to phosphatidylcholine. A big topic to cover, and why livers go bad and why we're facing this catastrophe of liver replacement. But before we do that, just tell me a bit about how you got into this area, what your path was from undergraduate.

Jonathan: Well, I'm not sure how much time you have but I've been in the U.S. for some 35 years, trained in South Africa, as you can tell. Actually came to the U.S. in 1984 and went to Cleveland. I think I was probably the only person who bought a one-way ticket to Cleveland, the travel agent had never heard of such a thing. But was trained at the Mount Sinai Hospital and then moved to St. Louis to do an endocrine fellowship. So, I then, after I did my research here and then went into practice in 1989. Practiced as an endocrinologist for about 15 years, a little under 15 years. Developed a Multidisciplinary Diabetes Center, and that sort of led me into developing a sort of online disease management program that I started a little biotech company, go on to raise some money.

And during the process of raising a second tranche for this company, I went to solicit a successful business person here in St. Louis, who was the CEO of a pharmaceutical company, a publicly-traded company. And while I thought he was interested in my diabetes disease management program, he sort of thought that I might fit into his company, which had, a couple of years earlier, started a branded product franchise in two areas that were completely unknown to me. And one was prenatal vitamins, and the other one was for iron replacement. What was different about this was that this company made generic medications mainly, they had a small branded side.

But they entered into a segment of…that was really exploiting an FDA requirement. That if you formulate a nutritional supplement with 1 milligram of folic acid, that requires a prescription, so it needs to be under the supervision of a physician. 

Mark: Oh.

Jonathan: And that way they developed a prescriptive franchise for prenatal vitamins and haematenic products. And I was asked as a physician to help them develop new product opportunities, identify unmet needs. I was completely unschooled in or had no background in pharmaceuticals or the regulatory environment, or so on and so forth. But what was interesting to me is that, as an endocrinologist, I was supposed to know about nutrition and nutrients, and certainly how it impacted liver metabolism and various other areas of metabolism, etc. But found that I actually knew very, very little about nutrition.

So, I began my sort of pharmaceutical career, if you will, with a huge disadvantage, which I think turned out to be a significant asset in the long run. And the disadvantage was that I knew nothing about the sort of physiology of nutrition and nutrients. The advantage is that I knew nothing about nutrition, and therefore whatever I had to do, I had to sort of build-up from the ground up. And how I did that I had to learn about either the area, the physiology, whether it's of iron metabolism, or folate, or anything, any of the major ingredients that were, you know, currently used to constitute, you know, prenatal vitamins or whatever.

Mark: Right.

Jonathan: And that's sort of how I cut my teeth on learning to understand that nutrient physiology, nutrient pharmacology is extraordinarily complex. And this is sort of how I then began, I suppose, my career in the sort of product development side of things that were considered to be pretty pedestrian by doctors and major pharmaceutical companies.

Mark: It is one of those oddities that we learn biochemistry way too early. Well, certainly in my medical degree, it seemed irrelevant, it was more a hurdle you had to pass through to get to clinical medicine. And then you get to clinical medicine and 30 years down the line, you realise, “wow, I wish I'd paid more attention to those early days.”

Jonathan: That's exactly what happened to me. In other words, physiology, not physiology, biochemistry and chemistry was not just an obstacle, or irrelevant, it was the bane of my life. I hated it, didn't understand it. And to this day, don't really understand all the, sort of, the way in which biochemistry or biochemical reactions are portrayed, they mean nothing to me. 

Mark: Right.

Jonathan: Which is one of the reasons that I had to, you know, had to find other ways for me to understand, you know, what's going on. But that has turned out to be exciting because I've actually really been fortunate enough and been given the opportunity to be able to explore, you know, in-depth, and it's a fascinating area.

Mark: It is. And I suppose the building blocks were put in place all those years ago so that there was, you know, deeply some kind of knowledge that underpins it. But I do agree, again, my medical career began with not a single hour of nutrition. We had no concept in a medical system when we're dealing with diseases, that nutrition played a part in prevention, or even the management or supplementation could have anything to do with it. It was very much a career of, "Here's the biochemistry, here's the conditions, these are the drugs that you use." And it was a dissatisfying feeling that we were missing something in prevention or less invasive ways of intervening in disease.

Jonathan: Correct. Absolutely correct.

Mark: So, how did you get from there to here? I mean, the interest that I have today is where we're dealing with something that's becoming a bit of a catastrophe within medicine, which is non-alcoholic fatty liver disease and the impact that is going to have on our medical system. An issue that is primarily in the Western world, from my understanding anyway, but there are various cultural groups of different countries with different susceptibilities. How did you move from infant formulas, and infant nutrition, or prenatal supplementation to non-alcoholic fatty liver disease and the whole PMPT scenario?

Jonathan: Yeah, so, I think, and again, this might be a longer answer than you want. But, my introduction...I think that what I would describe is that my area of interest is really more in one-carbon metabolism. 

Mark: Right.

Jonathan: And that sort of began, again, surreptitiously. Why? Because, in the prenatal vitamin world, everything was predicated on folic acid or folate. It was about 20 years ago when the first SNP was described. I, like probably everybody else, didn't have a clue what that was, and felt that this was just a little, sort of, technicality that is being exploited by the, you know, pharmaceutical companies.

And, you know, from there, I had a, you know, one of the things that I had to do in formulating these products was to make sure that every ingredient had earned its place, wasn't just on the label because it was expected to be on the label. What was the amount that was needed? How can we improve the bioavailability or absorption? And one of the areas that I moved to next was after folic acid, which is, you know, very well absorbed as you know, how do we improve on vitamin B12? And what the conclusion that I came to pretty rapidly was that the only way to improve vitamin B12 bioavailability is with intrinsic factor.

Mark: Yes.

Jonathan: And literally about a couple of months later I heard of a little company in Denmark that had been successful in inserting the human gene for intrinsic factor in a plant, arabidopsis, and successfully transform that plant, was able to cultivate it, grow it, and extract intrinsic factor. Now, plants don't use, it's a pristine environment, plants don't use cobalamin, so, whatever was taken out was actually human intrinsic factor. And I landed up with, you know, this company was acquired, I landed up acquiring those assets.

And now went into the business of starting my own little biotech company to commercialise the production of a recombinant human intrinsic factor protein as a way to be able to deliver B12 in a physiologic dose. So I went from folate to B12, and then I was doing some consulting for a company that is the sort of leader in chelated minerals based out of Utah. And that company was being acquired by a corporation, who is the sort of supplier of choline to probably, you know, 80% of the world's choline is actually produced by this company.

Mark: Right.

Jonathan: So, I was asked to, you know, to follow that acquisition, and that is currently my place of employ, if you will. So what's happened is, so there's three, you know, two vitamins and one essential nutrient that all play a critical role in one-carbon metabolism as sort of being, you know, in the wings, if you will, and sort of intersected, which is what has brought me to this. So, when I then started learning about choline, it was, you know, with the same, you know, sense that I described earlier, not knowing anything about it.

And I then began to try and understand what does choline do, what is it? Where is it? Why is it? So, that's been a journey that sort of led me into this, the sort of deeper appreciation of the sort of single nucleotide polymorphisms into conditions like non-alcoholic liver disease, into brain development for both child and some prenatal stuff. So, it's been a treat, it almost seems like it's been, you know, relatively well-choreographed if you will.

Mark: It has and it dances around this one-carbon, carbon with three hydrogens and it has seemed to have three different products in three different areas of interest but with one common factor, the methyltransferase. The choline story is not as visible as the folate and the cobalamin story. So, can you fill me in a bit, what does choline do, what's its fate in the body? And is it an essential? So my understanding is, with PMPT, we do manufacture, at least phosphatidylcholine from phosphatidylethanolamine. So it's not an essential nutrient or is it an essential nutrient?

Jonathan: Well, the fact that our bodies make it has taken it out of the realm of a vitamin. It used to be called vitamin B4, that's sort of like Pluto's loss of sort of planet status. And was then designated as an essential nutrient because the body does make but not enough. 

Mark: Right.

Jonathan: But I think that maybe it would be useful to...Let's talk a little bit about what choline is, and then we can describe, to some extent, what impact it has, and why it has such a broad impact across various physiologic, you know, and now even disease states.

Mark: That'd be great.

Jonathan: So, choline is really derived from the simplest amino acid, glycine. And what happens is, instead of the carboxyl group, you know, three methyl groups end up, you know attaching to glycine and then gets carboxylated to betaine. So, betaine is, in other words...another word for betaine is trimethylglycine, so it's basically glycine that's methylated. So, choline really is principally metabolised by phosphorylation in the cytoplasm, crosses the cell membrane as one of the transporters in which there's a SNP a well-known SNP. But about 70% of this gets pushed down, this one is called the Kennedy pathway, to CDP, cytidine diphosphate-choline.

And that is the major substrate for producing phosphatidylcholine, that becomes the substance of most membranes, and VLDL, for example. Well, there's an alternative pathway, and for this, choline has to get shuttled into the mitochondria, and…where it is then converted to betaine like I described earlier. And it's an oxidation process. So, if choline is oxidised to betaine, and now betaine becomes the way in which one or more of these methyl groups can get separated and therefore donated to a worthy recipient. 

Mark: Right.

Jonathan: And it gets transported back into the cytoplasm, and now is the way in which it will donate that methyl group to homocysteine and thereby making methionine. In fact, homocysteine has got that thiol group that self hydrol group. And the methyl just basically attaches to that, which is I think one of the reasons why homocysteine in larger amounts is relatively toxic to tissues…

Mark: Yes.

Jonathan: …neuro tissues, and others. And the way in which that toxicity is reduced when it gets converted methionine is that self hydro group is just kept by this, you know, methyl group.

Mark: Methyl.

Jonathan: So now you've got methionine, and methionine is a very important amino acid for the start codon and, you know, DNA synthesis. But this now gets channeled with the presence of ATP, gets made into S-adenosyl-L-methionine or SAM, or SAMe. And SAM is an important transporter of these methyl groups, you know, to a variety of functions. Well, it's under the influence of oestrogen in women in which SAM can get trimethylated by the PEMT enzyme. This is where PMPT plays its role, and it sort of goes through three cycles of methylating SAM, and, you know, methylating phosphoethanolamine okay, forgive me, from SAM. And this is an alternative way of producing phosphatidylcholine.

So you'll see that, just in this oxidative pathway, there is some, there is redundancy. In other words, there are two ways to make phosphatidylcholine. Well, it turns out that there's a very good reason for that, Mark, and that is that phosphatidylcholine made through the PMPT pathway or this betaine methionine SAMe oxidative pathway preferentially binds to longer chain fatty acids like DHA and arachidonic acid. 

Mark: Oh.

Jonathan: And it's the phosphatidylcholine, the phospholipids through the PEMT pathway that binds DHA and takes it to the liver, and then let's say it's in a woman and she's pregnant across the placenta to the foetus.

DHA does not bind to phosphatidylcholine made through the phosphorylation or Kennedy pathway. So, you know, there's a T junction right early on with choline, is either you're going be pushed into the majority of your work is making phospholipids that will constitute parts of membranes and myelin and all that sort of stuff. Or you'll get oxidised and now become a methylator. 

So, I think the way I look at the role of choline is it's critical for the integrity of membranes, okay? Through mainly the Kennedy pathway, or it is used for methylating a variety of compounds as well as genes. So those are the two main sort of, say, characteristics, if you will, of choline.

Mark: Can I just interrupt? The role of oestrogen there…I mean, I can see, from the clinical perspective, the upside of oestrogen, escalating the metabolism of phosphatidylethanolamine to phosphatidylcholine, if that carries the DHA to the brain of the developing baby, there's got to be an upside in evolutionary terms for that?

Jonathan: Absolutely. So, just think about this, the development of the foetal brain absolutely takes off in the third trimester. So, I was at a conference...actually I was at a conference in Melbourne a couple of weeks ago, in which one of the speakers described that between 35 weeks and 40 weeks, or maybe just 32 weeks, I forget, but it was at the last months, you know, of gestation. The foetal brain doubles in size and there is a dramatic rise in choline requirements in that third trimester, and there's a dramatic rise in DHA requirements by the foetus in that third trimester. And the PEMT gene is responsive to oestrogen, so it's an oestrogen-responsive gene. 

Mark: Right.

Jonathan: So as oestrogen ramps up in that third trimester, so too the machinery…

Mark: Right.

Jonathan: …ramps up concomitantly. So that, by the way, I don't know when the paper was published, but, you know, a few decades ago, Steve Zeisel from University of North Carolina, who really is the sort of modern forefather of choline. He's sort of done so much of the work that's put choline, you know, on the map. He actually did a study in which he took 10 healthy men, put them on a 10% choline diet, and it was a very choline-restricted diet. And then monitored not just choline levels, but their liver function tests. And I was actually astonished to see the data. And that is that within two weeks, liver function tests are elevated…

Mark: Right.

Jonathan: …significantly increased at three-weeks. And when he gave these healthy folks 550 milligrams of choline, within one-week, LFTs had returned to normal. So, it was that 550-milligram dose that the FDA in May of 2017, I think, said, "This is the recommended daily intake, 550 milligrams for men." And what was extrapolated from there was 425 for women, 450 milligrams for pregnant women. 

Why was it a lower dose? Not because it's on a per-kilogram basis, but because women have got an oestrogen mechanism by which they can produce more choline than men. So, I'm speaking that out just to demonstrate the impact of oestrogen sensitivity on the PEMT gene.

Mark: Does the FDA also acknowledge a higher requirement post-menopause, or, I mean are they aware of the mechanism that they're regulating there?

Jonathan: So, I can't answer for what they are or aren't aware of. Probably they are? I haven't thought about it that way. But, that I think that, yeah, requirements should go up post-menopause, correct.

Mark: They should, shouldn’t they?

But it is the oestrogen that increases the rate of the metabolism of the PE to the PC.

Jonathan: That's right. 

Mark: And the PC then helps the transport of the DHA and other nutrients in the pregnant woman when the oestrogen is at its highest for a prolonged period to the brain of the developing foetus, is that the kind of step-wise mechanism?

Jonathan: So, that is correct. In other words... the same as arachidonic acid…

Mark: Yes.

Jonathan: …which is also required by the foetal brain.

So, there is this sort of selection of these two long-chain fatty acids, which is why...And by the time, you know, the third-trimester sort of is winding down, something like 7 grams of fatty acids are transported across the placenta every day, so it's like substantial. And by the way, this is an obligatory situation. So, when we give pregnant women DHA, we're actually replacing what is sort of being sequestered from their own pools, if you will.

Mark: Right.

Jonathan: That's how important DHA is, you know, for foetal brain development.

But, Mark, I think that what sort of first drew my attention to the sort of impact of these SNPs was the work that was done by Steve Zeisel to look at the SNP associated with PEMT. And I, again, was pretty astonished to see that it had very similar prevalence in the Caucasian population, that the methyltetrahydrofolate SNP has. 

Mark: Right.

Jonathan: And there are many different studies, but it's at least 50% of Caucasian women and as high as 70% who've got, you know, one of the alleles for a oestrogen insensitivity.

So, what that means is that, women who've got this particular SNP don't enjoy the advantage that they should of the sort of high oestrogen environment in the last trimester, which is why they would require more choline for example, in order to encourage that pathway, the PEMT pathway, to drive the phosphoethanolamine sort of methylation process.

So..and in fact, this has been shown. We've seen some beautiful work done by Marie Caudill at University of Cornell. In which she took a pregnant woman in their third trimester...This is an important thing to describe as well. The average intake of choline in women is roughly in the 330 to 350 milligram-per-day range. In fact, when you look at NHANES data and other meta-analyses and other papers from, you know, both developing and emerging, you know, economies, approximately 90% of women do not take the recommended daily intake of choline.

Mark: Right.

Jonathan: So, what she did was, in her cohort, where the average choline intake was about 380, she supplemented a randomised, you know, trial. She randomised half of them to getting 100 milligrams of choline supplemented and 550 milligrams supplemented. So, these two cohorts had either 480 or 930 milligrams of choline. And what she was able to do is...And they took this for the third trimester, and they also took it for...another cohort took it for the first three months of nursing their infants. And she's done a lot of different stuff, you know, on those subjects, one of which was with stable isotopes able to label what the metabolic safety is of this choline. And she's pretty much confirmed a few things.

One is, is that, there is approximately 70% of the choline is metabolised down the Kennedy pathway and only about 30% goes through this betaine methionine pathway.

Secondly, what she showed is that there was no wastage of choline, no excretion in the urine. In other words, even in the folks who were given, you know, nearly twice the recommended daily intake, it was all utilised. 

Mark: Right.

Jonathan: So, therefore, even as we think of 550 for men and 450 for women, that…probably a more accurate description is that that's the amount that is required to keep the liver healthy, not necessarily the optimal dose.

But, with that, along with these clinical theories that she looked at, she was also able to determine whether the higher amount of choline was capable of methylating certain genes. So, for example, she looked at the corticotropin-releasing hormone gene in the placenta and found that the higher supplemented group had a greater degree of gene methylation. And with the actual expression of CRH was significantly decreased. So, methylation as you know, its main epigenetic effect, or one of its main epigenetic effects, is that it stops transcription, it down-regulates the production of the product.

So, she was able to demonstrate that higher choline reduces corticotropin-releasing hormones in the placenta. And when she measured the cortisol levels in the infants, they were diminished. So, once she was able to demonstrate that a higher prenatal choline actually reduced a less metabolic distressed infant…

Mark: Right.

Jonathan: …at the time of birth. But it is an interesting thing about the direct impact of methylation over and above, you know, other effects as well.

Mark: It is arguably the most popular concept in genetics at the clinical level is to methylate or not methylate? To check the MTHFR. I think now, after today's talk probably more of us will be looking at PEMT as another way of trying to influence methylation pathways.

I do have a concern sometimes that it's a little bit like giving kids the controls of a 747 jet that methylation is a very powerful process. We understand little about it, and there is this kind of concept of more methylation equals better. And as you said, you know, there is an influence that is, you know, transcription across the genome, where methylation plays a part with genetic expression. Where we…I don't think yet we know what we're doing as clinicians I think is...would you say that's fair too?

Jonathan: We absolutely do not know what we're doing, okay? However, however…so, in fact, as I've read on this, first of all, it's always illuminating to read a paper by a researcher who has a certain view and as you know, people develop a vested interest in not just their research topic, but NIH grants and all that sort of stuff. But it's sometimes easy to lose one's objectivity.

But it's always refreshing to come across a paper that says, "You know what? We expected this and we found the exact opposite. And we don't quite know why.” So, we all have a natural tendency to want to sort of tie things up into nice little neat packages…

Mark: Yes.

Jonathan: …and this area is anything but neat. And I think what it says to me is...I don't see the danger signals, okay? To say that we are sort of now zapping everything with a laser, for example, how do we know that we're not breaking things, you know, etc, etc. I think that I have an inherent sort of trust in the fact that it's important for us to have methyl groups…

Mark: Yes.

Jonathan: …that we ingest. Choline is the methyl donor to the body from a nutrient perspective essentially. Folate and B12 are methyl carriers. 

Mark: Yes.

Jonathan:  So, how do you expect either folic acid or folate to work well or cobalamin to work well if we don't provide enough of the methyl batons, you know, in the relay race, if you will?

So I think that that's one...And there are controls that are so exquisite and so beyond our ken at this point that I think that the best we could do, like anything, is to approach things with some degree of moderation. But one of the advantages of, or one of the safety valves, if you will, is that, even to meet current dietary recommendations, let's say in a supplement, you need a reasonable amount. 

Mark: Yep.

Jonathan: So it's not as if people are going to be massively overdosing on choline for example. There's a much higher chance of overdosing on B12 because it's measured in micrograms, or overdosing in folate. Marie Caudill who’s previous…who’s interest is also folate, you know, maintains that we give supra-pharmacologic doses of folate. 

Mark: Yeah.

Jonathan: Choline is in milligrams, so, when we talk formulators and they say "How are we going to get 500 milligrams into a tablet?" Because the cation, you know, is anywhere between 40 to 70%. So, you know, we have to give huge or several, you know, tablets or capsules. And what I advise is to say, "Look, why don't you just get to the recommended daily intake?" 

Mark: Yes.

Jonathan: So a dose of 100 or 200 milligrams will actually get you into that zone along with your normal diet.

I think a bigger challenge is to persuade people who've been brainwashed over the last 60 years that eating eggs and red meat and liver is the equivalent of, you know, consuming daily doses of cyanide. I think there is a challenge for us to say, "You know what? Eating eggs is actually healthful and doesn't necessarily, you know, have the negative, you know, impact that we all were raised to believe that it does."

Mark: So can vegans and vegetarians...So the choline is available and is bioavailable from a vegetarian source as well isn't it? So it's not purely meat and animal product eaters?

Jonathan: Right, so the choline that is available now is actually synthesised, and so it's a chemical reaction, number one. 

Mark: Yep.

Jonathan: Number two is that the salt, you can either get choline bitartrate and the salt is obviously from tartaric acid, it's a derivative of grapes. And the other one is, what you find in infant formulas mainly, is a choline chloride. So, yeah, for vegans or people who do not want to have animal sourced, you know, nutrients, certainly supplementation should meet their needs.

Mark: I would like to just address this issue of the prevalence of the A allele, I think this is the G523A of the PEMT, the A allele is effectively a low function allele, it's highly prevalent. From what you were saying, I think it's almost the majority of the population carries one or both of the A alleles? That means, does it not, that there is a population at risk from either low choline in the diet, or there is a population at risk for not producing the betaine or not producing the quality of the cell membranes? That's a huge proportion of the population that we have to look at.

And the second issue I want to address is, we're seeing fatty liver disease, non-alcoholic fatty liver disease increasingly happening. Are these two things linked at all? Is non-alcoholic fatty liver disease and the choline question, this kind of genetic variant, are they linked together in populations like the white American and Australian populations who get overweight, or are they distinct issues entirely?

Jonathan: Okay. So, maybe if it's okay, I'm going to start with the last part of your question first. And this is sort of what I was referring to earlier when I was saying that it's actually refreshing to see work that doesn't necessarily come up with nice neat answers.

What has clearly been demonstrated is that, if we look at the various SNPs that would impact both the choline sort of transport across either cell or mitochondrial membranes, or…so the solute carrier family for example. Or in the folate metabolism, or the folate-B12, you know, cycle. What you find is that there is obviously tremendous population-to-population variability, it's very hard to pick on things. Clearly I think that the Caucasian group from my reading has sort of, across the board, got the highest prevalence of most of the sort of important SNPs, you know, associated in this pathway.

By the way, they are probably, you know, the last I saw was 300 and counting, you know, SNPs that'll end up having an effect sometimes mild, sometimes significant, you know, in the PEMT pathway…

Mark: Right.

Jonathan: …and many many more in the folate and B12 side as well, many, many more.

So, I think getting back to your earlier statement about, you know, this is the Wild West, it's often very difficult for us to wrap our head around the fact that some SNPs are more important in a certain population group, but even in a population group, it lands up becoming sort of any disease state, fatty liver is a great example, is multifactorial. 

Mark: Yeah.

Jonathan: So, we always have a predilection for wanting to blame one thing, right? That's what the whole cholesterol story, right? Want to blame one thing and then we'll blame something else, then we'll blame something else.

So, one of the publications that came out of the University of North Carolina, Steve Zeisel's group, was trying to identify what are the SNPs that really, you know, have an impact on, let's say fatty liver? And what they found is that there are a whole bunch of them. And they looked at, for example,…And, again, you're limited by how many SNPs you can look at in a reasonably funded clinical study that isn't taken on by a sort of like a genomic, you know, project if you will. 

Mark: Right.

Jonathan: And what they sort of concluded was, yeah, they are... all of them are associated, some more than others. What Dr. Zeisel ended up doing was saying, "You know what? We shouldn't even blame one SNP." Because I think he might have found one SNP that had a consistent, sort of negative effect, okay, on fatty liver. 

Mark: Right.

Jonathan: And that is a SNP that I've never heard of until I'd read the paper, and that's called the PNPLA3, okay.

Mark: Right.

Jonathan: Which is responsible for, I think that was it, which is responsible for making VLDL. But the others become associative. So what he started doing is looking at panels, so looking at associations. Let's look at 5 or 10 SNPs and put these into two or three different or four different categories. And see can we figure out a panel that will end up being predictive of?

So I think that he came to the conclusion was that that is probably a better way to go. But it also recognises the fact that there are certain...African Americans for example, US African Americans have a much lower incidence of fatty liver. Some of them have, you know, some of the common SNPs associated, but they don't seem to be playing a role. Caucasians have those SNPs, and it seems to play a role.

So, what he was coming out to say was that "You know what? We might be able to use these sort of groupings of SNPs to identify vulnerable individuals where lifestyle... if I remember correctly, he looked at a morbidly obese group who were undergoing bariatric surgery. Then he looked at a group from a liver clinic. So, these were liver people and then morbidly obese people, okay, who, yes, they had a high incidence of fatty liver, but it wasn't the same as the liver people cohort. 

Mark: Right.

Jonathan: So, I guess the message is, is that again, it gets back to your earlier comment, this is such early days.

Mark: Right.

Jonathan: So I think…

Mark: I was hoping you were going to give me this solution and say "No, it's just one SNPs and if we cover this with trimethylglycine or..." I thought there would be an easy answer. And I know there never is, but every so often, I just think, "Yeah, that SNP looks..."

Jonathan: No. But I do think that, in a sense, it does provide us with a concept to say, if we increase the availability of choline, we know that there are key functional SNPs. He calls them, you know, effect alleles that really do have an impact. As we know, most SNPs, you know, are silent, we don't know what they do or whether they do anything or whatever it is.

But we do know that there are several important SNPs that do land at decreasing the efficacy of the enzyme that they are, you know, designed to produce. And therefore that reduction in enzyme capability is telling us that we should be increasing substrate in order to offset that. That is part of the story of the methylenetetrahydrofolate reductase, you know, SNP, right?

Mark: Yes. Right.

Jonathan: You know, we have a challenge. So there is a way to compensate. And therefore, if we do so on a more blanket basis, and I go back to, you know, Dr. Caudill's study where she says that, "Hey, 930 milligrams in a pregnant woman doesn't get lost in the urine," tells me we've got some room.

Mark: Okay, some wiggle room. So, is overdosage even known? Is there such a thing as overdosage with choline?

Jonathan: The answer is no. The upper limit that is recommended by the FDA is somewhere in the range of I think 3,000 or 3,500, okay.

Mark: Right.

Jonathan:  So it's substantially above what our current dosing capabilities are. The toxic level is described as 10,000 milligrams, and that degree of toxicity is a fishy odour. But the answer is no.

There was some concern years ago, there were some papers published about an impact on prostate but that has been disproven. But the answer is, we've got quite a length of rope to deal with here.

Mark: And so that means a bit of exploring to be done about the potential impact of intervention with choline. Just…you know, we've dealt with the other cofactors, the trimethylglycine, S-adenosyl methionine, they're all parts of this story, aren't they? And so are we kind of looking at the manipulation of the various cofactors of the S-adenosyl methionine itself, which seems to be a critical factor in the phosphatidylcholine story, or is it primarily a choline issue? That if you deliver enough substrate, the body will sort it out itself no matter what the SNPs?

Jonathan: No. I think it's a great question and I think that...This is why I like to think of this as a one-carbon story. 

Mark: Right.

Jonathan: Because there's been ample evidence to suggest that you really need adequate amounts of the appropriate, you know, three players here. The choline, the B12, and the folate. And, you know, I mentioned some of the redundancy associated with making phosphatidylcholine two ways. There are also two ways to methylate homocysteine and one is through the choline pathway, and the other one is through folate. And folate can't methylate directly, folate has to pass its methyl group…

Mark: Right.

Jonathan: …to cobalamin, and it's cobalamin that's through methionine sulphate can methylate homocysteine. So, again, there are two ways, so, that again shows you how critical the methionine generation is. 

Mark: Yes.

Jonathan: That if you have a deficiency of the one, you rely more heavily on the other. And I saw some papers a few years ago that actually looked at seasonal differences where people may land up having higher animal food intake for certain maybe hunting season, you know, times, and then more of the non-animal food or vegetable. And it turns out that their pathways land up adapting to this flux.

Mark: Right.

Jonathan: Number two is that, we've also noted that the same happens to the genes. The genes get up-regulated or down-regulated, according to what the adequacy is of these other, you know, methyl carriers are. And that's also work that's come out of Cornell.

So…and then there's the other aspect, and that is, everyone talks about folate or the methyltetrahydrofolate. There's something called the methyltetrahydrofolate trap. And what that means is that, the step between tetrahydrofolate and methyltetrahydrofolate is irreversible. The only way you can get back to tetra and therefore go back into folate and then produce all its other effects in other synthetic pathways, is in the presence of cobalamin. So if you've got a B12 deficiency, which by the way is a lot more common than people think...

Mark: Yes, it is isn't it?

Jonathan: A lot more common. If you've got a B12 deficiency, you can't get rid of that methyl group. And now, you land up having potentially a functional folate deficiency. So, you need cobalamin to make sure that you're metabolising folate properly. You need folate to be able to receive its methyl group, you know, cobalamin. And then if you land up paralysing that system, because you've got a deficiency of one or both, then you lean more heavily on choline.

And choline, its primary sort of there's a pecking order, and methylating the gene, the epigenetic effects are so important that choline, if it's deficient or if it's in inadequate supply, will start pulling choline from the membranes, from the phosphatidylcholine. 

Mark: Ah.

Jonathan: Which is what leads to the leakage of enzymes out of liver, out of muscle, CPK. You start having inadequate choline to produce VLDL, so your phosphatidylcholine is not being incorporated in your triglyceride carrier and VLDL is critical for exporting triglycerides out of the liver. 

Mark: Yeah.

Jonathan: Phosphatidylcholine is about 40% of mitochondrial membrane. So you take inability to export triglycerides from liver, mitochondrial membrane dysfunction, therefore energy, you know, challenges or deficiencies, together with liver cell membrane integrity issues, and now you're set up for fatty liver.

Mark: Right.

Jonathan: So, that is a fairly simplistic view but is not far from what goes on in early stage. A lot of the drug development that's been focused on fatty liver is actually looking at late stage, anti-fibrosis stuff. 

Mark: Right.

Jonathan: Whereas the nutrient basis of actually how to protect and provide enough, both for VLDL membrane mitochondrial, there's substantial literature support for that.

Mark: I mean, I do appreciate that. The fatty liver story also has something to do with inflammatory issues on the gastrointestinal tract and the liver dealing with that, so, probiotics tend to also have an impact on fatty liver disease and progression. It sounds like we should be focusing more on the whole story if we're going to look at the fatty liver depositions, the methylene... the methyl group transfer is a choline donor, adequate B12, adequate folate. And that gives you the ability to donate the methyl groups and transport them and to keep the liver out of our non-alcoholic fatty liver disease. Is that likely to be the story?

Jonathan: Absolutely. No, that is the story.

Mark: Right.

Jonathan: That is the story.

Mark: Okay. So, in practical terms, we have practitioners primarily who listen to this podcast, what do we do practically? I mean, we try to reduce gastrointestinal inflammatory disorders, maybe increase absorption and nutrition. Try to keep the B12 levels high, and the folates from dietary and supplemental sources high. I think a missing piece has possibly been we fail to look at the choline, partly because we're so fascinated with MTHFR as the early SNP that everyone knew the name of. 

Jonathan: Right.

Mark: And now there's another player in the field. So, practically a doctor who's seeing a person with those transaminitis we call it. 

Jonathan: Right.

Mark: You know, the transaminase is just a bit out to the person going the wrong direction. Apart from weight loss, which everyone kind of gets, what can we do in nutrition or in supplementation to sabotage the non-alcoholic fatty liver story, the progression?

Jonathan: So, look, there are, and certainly in the US and I'm not sure what's in the market in Australia, other parts of the world. But, in the US there are several standalone supplements that contain anywhere from 250 to 500 milligrams of choline. 

Mark: Right.

Jonathan: So if you think that the average diet is going to be in the let's say 350 to... let's say 350, okay? I personally take 700 milligrams, I take two 350 milligrams capsules. 

Mark: Right.

Jonathan: So I'm saying to myself, I'm taking about 1,000 milligrams of choline. And by the way, I did have somebody who...And, again, you know, I'm not in practice anymore, but I used to see these elevated, this transaminitis, as you described, and I used to do an ultrasound and see if there were gallstones and this and that, but we didn't do anything about it, right?

Mark: Yep.

Jonathan: You know, whatever. So I think the easiest thing to do at this point, literally is to say to your patients, this is a supplement, and I would recommend, you know, probably in the range of 500 plus, I'm not worried about an upper dose here.

Mark: Yeah.

Jonathan: If somebody wanted to take two 500s, you know, I think that's fine and dandy. I think that this should be the sort of the cornerstone of what practitioners should be looking at. We'll see subjects whether they've got elevated, you know, LFTs or not, it's the same applies to elevated CPK, sarcopenia, you know. So those are certainly two areas.

And I think, you know, the other aspect here is, look, it's difficult to, certainly in a country like the US that has got folate fortification, folate deficiency is much less common. Vitamin B12 deficiency though, there's a study published about, I think 18 years ago, that showed that 39% of the population had a subclinical B12. 

Mark: Yeah.

Jonathan: You know, measuring B12 in the lab, the range is anywhere from 300 to 900 picomoles per liter. 

Mark: Yes.

Jonathan: And if we see someone's got a level of 350 we say that's normal…

Mark: Yeah.

Jonathan: …but that's not normal. And B12 levels are not the best way to measure B12. So I would encourage practitioners to measure MMAs, methylmalonic acid which is a very sensitive way of telling whether there is tissue B12 deficiency.

With patients on proton pump inhibitors, other antacids, people over the age of 50 have decreased acid production, so more tending towards not necessarily achlorhydria, but, you know, gastric atrophy. And reduced intrinsic factor, reduced acid production for any reason that ends up decreasing the ability to split B12 from its protein moiety from the diet.

Mark: Yeah.

Jonathan: I would also caution physicians that giving huge doses of B12 is also problematic. You know, people say, "Hey, even if you get 1% absorption by diffusion, that is, you know, that we're okay with that." Well, we should not be okay with that. 

Mark: Right.

Jonathan: B12 is a huge molecule, gets broken up into little bits and pieces called analogs, they can get absorbed, and some of these analogs have been shown to inhibit B12. 

Mark: Right.

Jonathan: It increases gut microbiome growth and a variety of things. It's been shown, with high doses of B12, increases various solid tumours in a 10-year epidemiologic study of 300,000 people who were followed in Europe published about five years ago.

So, big doses of B12 is not benign. Big dose of folic may not be benign, particularly for pregnant women. 

Mark: Yeah.

Jonathan: And not enough choline is not benign. 

Mark: Right. 

Jonathan: So, there's a sort of a recalibration here that I think we should look at.

Mark: Okay, is there any place for betaine supplementation or SAMe supplementation in cases of already existing non-alcoholic fatty liver disease? Do they play a part in the story or is it enough that the folate, B12 and the choline are more than adequate?

Jonathan: So, I think that the choline-B12-folate story is certainly adequate.

Mark: Right.

Jonathan: There is data to suggest that betaine could be helpful. There are some papers that describe that. I guess, as an endocrinologist, I come from the sort of school of thought that was more inclined to give thyroxine for hypothyroidism because there is a natural conversion to T3. 

Mark: Yes.

Jonathan: Even though that's the active thyroid, you know, hormone, there's a sort of a regulatory component that I don't have control over. 

Mark: Yep.

Jonathan: So, I guess I'm saying, let's put in the funnel, that doesn't mean that giving betaine, you know, is problematic. Some papers support it as being more effective and you obviously, you know, need less, I think it's about a 10th of the amount that's required for choline. But, you know, 70% of choline also goes to membrane synthesis…

Mark: Yeah.

Jonathan: …and myelin, you know, synthesis. So there are lots of...Betaine is purely for methylation.

Mark: Yes.

Jonathan: Choline can be used for membrane, myelin, etc, etc as well.

Mark: That has been a fascinating story. I think some real pearls there about maybe the unacknowledged area of the methylation cycle, is the choline component of it. I'm hoping now that we all as practitioners kind of have that sense that you have to have the three parts of it. It's not a two-part story of folate and B12. It's a donor and it's a dietary component that we can actually move in and do something about, significant supplementation to the diet makes a difference.

I don't think we can put everyone on oestrogen to improve it, but, for those lucky enough to have their natural oestrogens, they seem to have a bit of advantage over the rest of us, and they live a little longer than us, say, expendable males.

But I want to thank you for your time, Jonathan, it's been great to get into this area and to add one more building block to the whole methylation story. Thank you very much.

Jonathan: Been my pleasure. Thank you, Mark, thanks for the honour, the privilege of the call.

Mark: This is FX Omics, and I'm Dr. Mark Donohoe.


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FX Omics Podcast

The FX Omics podcast is hosted by renowned Australian integrative medicine expert, Dr Mark Donohoe. Join us as we explore the emerging research and clinical application of genetics, epigenetics and metabolomics and why this is the future of personalised medicine.