Transcription
Welcome, everybody! Thank you for joining us this morning for the first installment of our two-part methylation series. My name is Anastasia, and I'm a clinician educator at Genova Diagnostics Europe. I will be taking you through today's and next week's webinars with the aim of helping to increase your understanding of how methylation works, what kind of imbalances may manifest into various clinical pictures, and of course, how to interpret Genova's methylation panel, which combines both genomic and functional analytes related to methylation pathways. Lastly, I'll also be covering some case study examples in next week's installment where methylation testing has proven to be valuable.
The aim of today is to cover an introduction to methylation, the basics of what methylation actually is, and then we'll be going systematically through each part of the main processes in methylation, such as the folate cycle, the methionine cycle, and the transulfuration pathway, and how each of these pathways interlinks and the role they play in various key physiological processes. Finally, at the end, I'll also be highlighting the type of patient that would benefit from methylation testing and the symptoms that can point to methylation imbalances so that you, as a clinician, feel more confident in recommending the test.
So, what is methylation? Simply put, it is the transfer of four atoms: one carbon atom and three hydrogen atoms from one substance to another. What you're seeing in the middle is what we call a methyl group. Think of methyl groups as substances that act like billions of switches, which do things like turn genes on or off. They help to regulate mood, detoxify hormones, produce energy, and promote healthy aging. Methylation really does play a ubiquitous role in the body, and it's a biochemical process that happens billions of times per second in every cell of the body, where methyl groups are being transferred and donated between different molecules, which change their structure and function.
We also know that nutrients such as vitamins, minerals, and amino acids from the diet are needed to keep these processes running smoothly. Of course, there are also genetic factors at play, as well as oxidative stresses, which can affect how well these pathways work. These are the factors that I'll be covering extensively throughout today's and next week's webinars.
Aside from a few of the physiological processes that I have just listed, the list really does go on, which highlights how important methylation is in the body. For example, we have creatine production at the top right-hand side here. This is the substance that's found naturally in muscle cells; it helps your muscles and cells produce ATP. There's also DNA synthesis and gene regulation. I've already mentioned hormone regulation and detoxification, so we're also talking about the detox processes related to environmental toxins. Foreign cell membrane repair and myelination are also involved in methylation, which is quite important in relation to cognitive function and the nervous system. Finally, you also have other factors such as fat metabolism, immune function, neurotransmitter production, and metabolism in relation to mood regulation. Lastly, we have nitric oxide production and vascular endothelial function, which is essential in the context of cardiovascular health.
As you can see, this just reiterates how globally important methylation is, and I'm sure some of you are already thinking about what kind of patients would benefit from having methylation testing done. To keep these processes functioning optimally, there is a necessary balance between many different biochemical pathways. What is termed the methylation cycle involves an interplay between folate metabolism, methionine metabolism, and homocysteine transulfuration. The body continuously adapts these interconnected pathways in order to maintain homeostasis. However, key amino acid deficiencies, a lack of vitamin and mineral cofactors, genetic enzymatic predispositions, and a wide array of oxidative stresses can impact multiple enzymes, leading to a disruption in a patient's overall methylation status.
Now that I've given this short introduction into methylation and what it actually is, let's move on to the first part, which is the folate cycle. This here really is the first stage of the folate cycle. What actually happens from the minute we ingest folate is just as essential as looking downstream to the end product of the folate cycle, which is a compound that I'm sure many of you are familiar with: 5-MTHF or 5-methyl tetrahydrofolate. In the diet, folates exist as polyglutamate, and these need to be enzymatically converted into folate monoglutamate in the jejunal mucosa, which is in the small intestine, in order to be absorbed.
It's worth noting at this point that synthetic folic acid, which is used in supplements, is actually two-fold better absorbed than dietary folates in the intestine. However, this doesn't mean that it's better or that we should all be supplementing with folic acid, and I'll be talking a bit more about this in the next few slides. Neither dietary folate nor folic acid are biologically active until they are converted to tetrahydrofolate in the liver by the enzyme dihydrofolate reductase (DHR). As you can see, this is illustrated here. Once folate or folic acid are biologically active, they will then enter into the next stage of the folate cycle.
As you can see, there are various steps that determine the conversion of dietary or supplemental folic acid into the end product, which is 5-MTHF. This one here provides a methyl group to the methionine cycle, which converts homocysteine into methionine. The light blue squares here represent specific enzymes that are responsible for propelling the conversion of folate eventually down into methylfolate. You can also see various nutrients and amino acids that serve as cofactors for various enzymes, such as B6, iron, B2, and B3.
It's also worth highlighting that 5-MTHF is the predominant circulating folate in serum, and there are other non-circulating forms of folate precursors, such as 5,10-methylene tetrahydrofolate, which is mainly found intracellularly rather than in serum, and it's also just as important. Before I go further down into the folate cycle and the various enzymes that are responsible for the conversion of folate or folic acid into 5-MTHF, I wanted to do a brief slide on supplemental folic acid.
I mentioned that while folic acid is better absorbed two-fold than dietary folate, it doesn't mean it's better, and this is why. The DHR enzyme has relatively low and variable activity in the liver, and when it comes to folic acid, the rate of that activity can be significantly lower. Not only that, but taking folic acid can increase the amount competing for the DHR binding sites, which can actually crowd out dietary folate. This sets up the possibility that a high intake of folic acid may result in elevated levels of unmetabolized folic acid in the bloodstream. The debate as to whether unmetabolized folic acid levels contribute to disease is ongoing, and the literature is varied. However, these are just some of the potential negative health consequences that may be associated with unmetabolized folic acid: reduced numbers and activity of natural killer cells, which impacts the immune system; a possible increased risk of cancers, specifically the acceleration of cranioplastic lesions; some scientists have hypothesized that unmetabolized folic acid might be related to cognitive impairment among older adults; and lastly, high levels of supplemental folic acid in preconception were associated with lower scores on several tests of cognitive development in children aged 4-5 than in children of mothers that took lower levels, such as between 400 mcg and 999 mcg.
What about phelanic acid? Some of you may have also heard about this type of folate or supplemental folate. The great thing about phelanic acid is that it can readily enter the folate cycle without being reduced by DHR. For those with polymorphisms on the DHR gene, this may be a more effective way of supplementing. It also seems to be anecdotally more gentle than supplementing with methylfolate for those that are sensitive to methylfolate, which is actually quite common, especially in psychiatric conditions where neurotransmitter imbalances are concerned.
Let's go back to the folate cycle to pick up from where we left off. Dihydrofolate reductase converts folate into tetrahydrofolate. Tetrahydrofolate then gets metabolized by SHMT, this enzyme here, using serine as a methyl donor. After donating its methyl group, as you can see, it becomes glycine. However, the arrows are pointing upwards and downwards for this particular enzyme to illustrate the reversible nature of this reaction. Think of the 5,10-MTHF as a branch point in this cycle, and the direction it takes depends on methylation demand.
The first direction is down into converting 5-MTHF for methylation, and the other direction is pure nucleotide synthesis, which plays a role in DNA and RNA formation, as well as ATP and NADH formation, among other important compounds. When methylation demand is normal or low, this acts as a negative feedback loop to NTHFR by slowing down NTHFR creation and allowing folate to be used for purine nucleotide synthesis and therefore DNA replication and repair.
As you can see, this enzyme here, SHMT, is dependent on B6, SAM, and iron. The enzyme gives metabolic priority to nucleotide synthesis over SAM synthesis, which is why it's implicated not only in disturbances in the methylation cycle but also in conditions such as cancer, where polymorphisms on the SHMT enzyme can lead to genome instability and an overexpression of oncogenes and the inactivation of tumor suppressor genes.
Finally, the end product of the folate cycle, as we all know, is methylfolate or 5-MTHF, which provides a methyl group to the methionine cycle for the conversion of homocysteine to methionine. Methionine will eventually be converted into SAM. SAM is the sole methyl donor in more than 100 different reactions catalyzed by methyl transferase enzymes. SAM is found in every cell of the human body and serves an important biological function in activating the transulfuration pathway, resulting in glutathione synthesis and energy production.
Some of the methyl transferase enzymes include the following, but aren't limited to: DNMT, involved in DNA methylation and the switching on or off of genes; HNMT, involved in the breakdown of intracellular histamine and therefore plays a significant role in someone's susceptibility to environmental sensitivities, seasonal allergies, etc.; COMT, which I'm sure many of you are familiar with, involves the detoxification of catechol compounds, and therefore compounds such as dopamine, noradrenaline, and adrenaline, as well as estrogen. We know that a deficiency in 5-MTHF and a slow COMT enzyme can result in symptoms of excess estrogen and an amplified stress response; PNMT, involved in the conversion of noradrenaline to adrenaline and therefore plays a role in healthy adrenal function; ASMT, involved in the production of melatonin and therefore involved with healthy sleep cycle function; TEMT, involved in the production of phospholipids, healthy bile composition, gut health, and cell membrane structure, which is important in the context of cognitive health as well as other issues; and finally, GAMT, involved in the production of creatine, which we've already mentioned plays a role in muscle recovery from exercise as well as energy production.
These are just some of the methyl transferase enzymes that transfer a methyl group from SAM to their substrates in order to catalyze a specific reaction. Based on this and the various functions of these enzymes, you can only imagine what the impact would be if there was a lack of SAM production due to issues, perhaps upstream in the methionine cycle or the folate cycle, and how this could manifest clinically from person to person.
Now that I've covered the folate cycle, I want to move on to the next part, which is looking at the methionine cycle and how these cycles interconnect to eventually help produce SAM through the recycling of homocysteine. Here we've zoomed out, and we can see where the folate cycle connects with the methionine cycle. The purpose of the methionine cycle is to convert homocysteine into methionine, which then becomes SAM, the end product of methylation.
As you can see, the first enzymatic junction between the folate cycle and the methionine cycle is MTR, this enzyme here. This enzyme requires methylfolate as a methyl donor and methyl B12 as a catalyst to convert homocysteine to methionine. However, you'll also note that there's an additional enzyme, MTRR, on the right-hand side of this cycle. This enzyme is responsible for maintaining adequate levels of activated or methylated B12. Generally speaking, any SNPs or variations on this enzyme, the MTR enzyme, have an upregulating impact. This means that if this was playing out in real time, we would see an upregulation of homocysteine conversion to methionine.
However, if there is a lack of activated B12 or methylated B12 going into this enzyme, it won't be able to effectively do its job in homocysteine recycling, which could effectively lead to a backup in homocysteine. We also have the middle of the methionine cycle, which is often thought of as the salvage pathway for homocysteine conversion. This is sort of the backup or the plan B pathway because when methylfolate levels are low, whether it's due to a dietary deficiency or whether it's due to SNPs downregulating methylfolate availability, the salvage pathway or the BHMT pathway is there almost as a backup for providing methyl donors for homocysteine to methionine conversion.
As you can see, this pathway is dependent on choline oxidation to betaine, which is just here. Betaine is used as a methyl donor to convert homocysteine to methionine via the BHMT pathway. Zinc is also a cofactor for this enzyme to function optimally. The product of the BHMT pathway is DMG. Once betaine has donated this methyl group to the BHMT enzyme, it becomes DMG. When DMG is elevated, this can act as a negative feedback loop by inhibiting BHMT.
As you can see, there's always this fine balance between the activity of the enzymes and the availability of cofactors going on in order to continuously reach homeostasis. The main point of the methionine cycle is to convert homocysteine to methionine to then eventually produce SAM. As I'm sure many of you know, controlling homocysteine levels is one of the main ways that we gauge someone's methylation capacity. It plays a fundamental role in methylation; it's a major branch point in the methylation pathway.
As we've already gone through, it's either converted to methionine through the methylfolate and methyl B12 dependent pathway or the BHMT or salvage pathway, which is illustrated here in this diagram. There is just one more route that homocysteine removal can go down, and that's the transulfuration pathway, which I'll be covering in the next few slides.
Homocysteine is not a classic amino acid found in dietary protein; it comes from the demethylation of SAM. Under normal conditions, the body will remethylate homocysteine several times before allowing irreversible transulfuration. This means once it's been removed down the transulfuration pathway, there's no going back; it's removed permanently. Being the important biomarker that it is in relation to methylation, homocysteine is often tested to gauge someone's methylation capacity.
We also know that high homocysteine carries its risks. I mentioned there are some significant risk factors, and here are some of the important ones. Elevated homocysteine can enhance vascular smooth muscle cell proliferation, increase platelet aggregation, and act on the coagulation cascade and fibrinolysis, causing normal endothelium to become more thrombotic. In a nutshell, it can contribute to the development of atherosclerosis as well as other cardiovascular diseases. Elevated homocysteine has been shown to induce inflammatory cytokines and contribute to disease progression. Homocysteine can also impair bone health by interfering with osteoclast activity, and it does that by impairing the cellular and molecular mechanism of bone marrow-derived osteoclasts. Elevated homocysteine increases the central nervous system phosphorylated tau, leading to increased neurofibrillary tangle formation seen in Alzheimer's dementia, which is, of course, the hallmark of dementia. Finally, we also know that elevated homocysteine can interfere with methyl transferase enzymes, so it can actually have an inhibitory impact on methyl transferase enzymes related to neurotransmitter synthesis and/or the breakdown of neurotransmitters. We also know that homocysteine does have some neurotoxic properties, which is important in relation to brain health.
So, what actually causes high homocysteine levels? We already know that a lack of those cofactor nutrients for both pathways in the methionine cycle can lead to a backup. These nutrients include methylfolate, methylcobalamin, some of the B vitamins, zinc, choline, and iron. Then we have the SNPs, or single nucleotide polymorphisms, which may lead to a downregulation in the activity of certain enzymes. For example, the SHMT enzyme, which we've already spoken about, when downregulated can limit the availability of folate to act as a substrate for MTHF. Then we have the MTHFR enzyme itself, which when downregulated will limit the availability of 5-methyl tetrahydrofolate for homocysteine conversion. We have the MTRR enzyme, which when downregulated can limit the availability of methylated B12 for homocysteine conversion. Finally, we have the BHMT pathway, which will prevent the functioning of this backup pathway for homocysteine conversion.
There are also other factors that can lead to an accumulation of homocysteine, and that's related to lifestyle factors such as high alcohol intake, tobacco use, and a high intake of caffeine. I've gone over why elevated homocysteine can be a problem, but what about low homocysteine? It's worth noting that low homocysteine levels and the clinical associations with this aren't well represented in literature. However, because homocysteine is actually used to make glutathione, this could effectively lead to issues if there were low levels of homocysteine, such as a low level of glutathione production.
We also know that there are a couple of factors that can contribute to low homocysteine. One of them is a low dietary intake of protein, and of course, poor absorption of protein. There may also be an upregulation in the transulfuration pathway, which may deplete homocysteine levels, but we'll get on to this in the next few slides. I should also add that if homocysteine is low, then of course the main issue for that is that there will be lower levels of methionine and, consequently, lower levels of SAM. That's probably the most important factor there to highlight.
Once homocysteine is converted to methionine, methionine is then converted into SAM, and this is done via the MAT enzyme or the methionine adenosyl transferase enzyme. As you can see, this enzyme depends on ATP, magnesium, and potassium. A depletion in any of these compounds or nutrients can downregulate SAM production. There's also the possibility that someone can have a SNP in the MAT enzyme, which has a downregulating impact on the activity of that enzyme, and that, of course, can lead to a depletion as well.
We finally get to SAM in the methionine cycle. SAM is tightly regulated in the liver, and when SAM levels are elevated, this acts as a negative feedback to MTHFR, inhibiting the activity of MTHFR as well as BHMT. This is, of course, to maintain homeostasis and prevent a hypermethylation picture. High levels of SAM have also been associated with adiposity and obesity, although the mechanism of this association is still being studied.
The final part of the methionine cycle is SAR, which is the end product of methylation reactions and a metabolic precursor to homocysteine. Once SAM has donated its methyl group to methyl transferase enzymes, it eventually becomes SAR. The enzyme responsible for this conversion is GNMT, which plays an important role in removing excess SAM by catalyzing the methyl group transfer of SAM to glycine, ultimately forming sarcosine. This removal process is downregulated when there are low levels of methyl folate and SAM levels, again trying to maintain homeostasis constantly.
Essentially, homocysteine is then hydrolyzed to homocysteine, which completes the methionine cycle. This reaction is readily reversible, but the dynamics strongly favor the production of SAR. Any elevation in homocysteine will consequently lead to an increase in SAR. SAR is a potent feedback inhibitor in methyl transferase enzymes or reactions. Its pathogenicity lies in its binding affinity for and inhibition of methyl transferase enzymes within many tissue components, including DNA, RNA, phospholipids, and many others. For this reason, plasma levels of SAR have been shown to be a more sensitive marker of clinical cardiovascular disease, renal disease, and Alzheimer's disease than plasma homocysteine. Methylation reactions ultimately depend on SAR removal, and as you can see, it also relies on the functioning of the AHCY enzyme, which is dependent on the availability of B3 for its functioning.
Finally, we come to the last part of this webinar, where I will cover the transulfuration pathway. As you already know, transulfuration is the main route for irreversible homocysteine transulfuration or homocysteine disposal. It connects methylation, mitochondrial energy production, and glutathione biosynthesis. Transulfuration occurs when a sulfur group is transferred to various molecules, such as to cystathionine, as you can see in the first step here in the transulfuration pathway, and then to cysteine on either side here. This ultimately either ends up in the biosynthesis of glutathione as well as pyruvate and taurine. This process is upregulated when needed, such as when the body is under oxidative stress and therefore glutathione on demand is higher. Alternatively, transulfuration will also contribute to energy production when glutathione is not in high demand. One of the molecules created in this pathway is pyruvate, which plays a major role in cell metabolism and fuels the citric acid cycle and therefore ATP production.
As you can see, there is an enzyme that interconnects homocysteine to cystathionine, and that's the CBS enzyme, which stands for cystathionine beta-synthase. This enzyme has three cofactor nutrients: B6, iron, and serine. CBS is upregulated when excess methionine and SAM are present, which causes an increase in homocysteine conversion to cystathionine. The opposite will also happen when SAM and methionine are low.
In general literature, a SNP in this enzyme indicates that it has an upregulating impact, meaning that the removal of homocysteine via transulfuration is sped up. However, population studies show that a SNP can also lead to a downregulation in the enzyme, effectively leading to a backup in homocysteine and a potential lack of glutathione production. There are also a couple of other enzymes here that are important in this pathway, such as CTH and GSS, that also have an impact on glutathione synthesis. You'll note that B6 is a cofactor nutrient here as well for these two enzymes, and any insufficiencies in B6 will, of course, lead to a backup in this pathway and therefore potentially an insufficiency in glutathione, as well as you could also say pyruvate.
In my opinion, this is something that comes up very often, particularly after seeing many reports on the metabolomics and neutral valve panels, in which many of you may be familiar with, and B6 often is highlighted as insufficient for many people. So, I think it's a nutrient that's often overlooked.
I'm sure many of you already know about the importance of glutathione, but I needed to make it part of this presentation because it's such an important outcome of the transulfuration pathway. It is essential for neutralizing hydrogen peroxide and lipid peroxides through GSH peroxidase reactions. Hydrogen peroxide and two markers of oxidative damage and oxidative stress are directly related to the level of glutathione because it's so important in reducing those products. It's also involved in phase two detoxification by conjugating hormones, toxins, and xenobiotics to make them water-soluble for excretion.
With methylation underpinning so many processes, the big question in practice is when to test and what kind of clients or patients would we choose to use this test on. Going back to one of the slides at the very beginning of this presentation, let's look again at some of the processes that methylation plays an important role in. We have creatine production at the top, which is a substance found naturally in muscle cells. As we've already mentioned, it helps your muscles and cells produce ATP. In the case of chronic fatigue or poor exercise tolerance, looking into methylation may be a good idea.
We also have problems with DNA and RNA synthesis, as well as gene regulation. Conditions such as cancer are highly implicated here. Then we have the role that methylation can play in hormone regulation and detoxification of environmental toxins. We have key issues such as hormone imbalances, symptoms of estrogen dominance, for example, PCOS, PMS, and so forth. We then also have the importance of cell membrane repair and myelination when it comes to methylation. In this section, we're looking at neurological conditions, such as Parkinson's disease, as well as other neurological diseases that can be highly related to poor methylation. Those with mental health conditions such as depression, anxiety, bipolar disorder, schizophrenia, and other mood disorders may also have methylation defects due to the role that methylation plays in neurotransmitter production and catabolism. Finally, we have cardiovascular disease because of the fact that methylation plays an important role in endothelial function and nitric oxide production as well.
Before I open the floor up for questions, I wanted to go over some of the common barriers to optimal methylation to further elucidate what kind of patient you may want to be offering this test to. At the top, we have GI dysbiosis. Studies have shown that those with microbiome imbalances have a consequent impact on DNA methylation, as well as the role that certain organisms, such as Lactobacillus and Bifidobacterium species, play in the role of folate production and therefore the contribution to circulating folate levels in the body, which relates to methylation capacity.
We also then have increased oxidative stress, which we know has been shown to affect the function and activity of enzymes responsible for regulating DNA methylation. Stress is a huge one as well, particularly early life stress, such as childhood abuse and trauma, adverse childhood events, etc., and the lasting effects on methylation that can persist into adulthood. We then also have exposure to environmental toxins, which I wanted to do a separate slide for here to illustrate the impact of environmental chemicals on methylation.
I found this great illustration, and the illustration is referenced here. This highlights the effect of common toxins on our methylation capacity. Environmental chemicals have been linked to aberrant changes in epigenetic pathways, both in experimental and epidemiological studies. This is an overview of possible mechanisms of action for environmental chemicals on DNA methylation based on reviews of these experimental studies.
As you can see, we've got heavy metals, persistent organic pollutants such as pesticides and PCBs, and polycyclic aromatic hydrocarbons, which all increase reactive oxygen species. This leads to an elevated demand for glutathione conjugation. Glutathione will conjugate the reactive oxygen species, chemicals, and their metabolites. Therefore, what happens is homocysteine is shunted towards glutathione production instead of methionine synthesis, effectively leading to a reduced synthesis of SAM. Of course, some depletion thus inhibits or potentially inhibits DNA methylation and therefore leads to a picture of hypomethylation.
Exposures to specific environmental chemicals, like short-term exposure to cadmium, polycyclic aromatic hydrocarbons, lead, and mercury, can actually directly reduce the enzymatic activity and concentrations of enzymes like DNMT, which is the enzyme responsible for DNA methylation. This is quite an important thing to highlight, really, just how impactful our exposure to these common toxins can be on methylation capacity.
Coming back to this slide, we've already spoken about the impact of nutrient deficiencies, especially those that are cofactors for methylation enzymes. What about sleep? There is substantial evidence to suggest that DNA methylation is critically affected by sleep. Individuals subjected to both acute sleep deprivation and chronic sleep deprivation have changes in their epigenetic landscape, and altered DNA methylation patterns have been observed in genes involved in metabolism, as well as circadian genes. Finally, we have medication use. Common medications such as antacids, non-steroidal anti-inflammatory drugs, corticosteroids, and methotrexate, for example, have all been shown to inhibit DNA methylation.
In summary, to condense the information from the last few slides, these are the types of patients that can benefit from the methylation panel. Because of the all-encompassing role that methylation plays in basic human physiology, the range of symptoms associated with methylation defects is very broad. Those with questionable detoxification, those with a risk of cancer, perhaps a family history of cancer, cardiovascular disease, psychiatric and mood disorders, neurological disorders, as well as chronic fatigue, may all be interesting case studies to test methylation on.
We finally come to the end of the presentation. Please write your questions in the question tab on the GoToWebinar control panel, and I'll start to sift through the questions as soon as I see them coming through. If you have any questions that you want to ask the team personally, you can get in touch via the email that's given here: clinicuk@gdx.net. Any feedback would also be very much appreciated.
Let me have a look and see what questions we have so far. Okay, just so you all know, there is a recording for this webinar. For anyone asking that question, you will receive a recording, and the slides will also be sent to you in PDF format. You may also note that the methylation support guide is in the handouts section on the control panel, but you will receive the slides separately in a separate email.
Okay, I have a question here. Someone is saying they have a very high cystathionine with a potential upregulation of CBS. Cysteine levels are just below the reference range, and homocysteine is just in range. Would this indicate an issue with CTH? Any suggestions on how to reduce cystathionine and increase cysteine? The junction between cystathionine and cysteine is the CBS enzyme, and that enzyme, as you would have noted, is dependent on the availability of B6, particularly. Then you've also got iron as well as serine, but B6 is an important nutrient. If you have a very high level of cystathionine but low levels of cysteine, this suggests that there's a backup in that pathway, potentially because of a lower availability of those cofactor nutrients to help the activity of that enzyme function properly and continue the flow downwards eventually into glutathione production.
Thank you. Just sifting through the questions here.
Okay, someone's asking about preparing a client for the metabolomics plus test. The metabolomics plus is a urine test, and there is a page on our website. The page for the test, the metabolomics plus, will give you some preparatory guidelines, which I would recommend that you look at. Alternatively, please do send us an email.
Someone's asking an interesting question here: Do you commonly see an increase in ammonia when people have CBS SNPs and SNPs to BHMT? Would this also put pressure on the urea cycle, resulting in BH4 deficiency? In our methylation panel, we don't test ammonia. However, theoretically, upregulation of CBS may lead to an increase in ammonia, and of course, yes, this can have an impact on BH4 and the production of BH4, as well as putting pressure on the urea cycle. I often find that when you're getting symptoms of high ammonia, for example, you're suspecting that that's going on for a client or patient, that can be mainly related to GI dysbiosis and the overproduction of ammonia through the presence of dysbiotic bacteria. So that's definitely something to consider in relation to that and not just methylation.
I should also add that with the CBS, there are research studies suggesting that when there is a SNP on that, there is upregulation, but we can also see a downregulation. That's why it's quite important to see what the functional analytes around that enzyme are doing, such as cystathionine and cysteine. I would recommend, if you haven't already, considering doing this test so that you can have a look at whether that SNP is actually playing out in real time.
For clients that present with chronic fatigue syndrome, how can we use the methylation panel to help them? If you recall, there are various methyl transferase enzymes that are important in relation to various aspects of health, energy production, as well as cellular membrane health, nervous system health, and creatine production, which is important for ATP as well as muscle recovery. If you were to do a methylation panel on a patient that has that clinical presentation and they were to show a picture of hypomethylation, this would mean lower levels of SAM. You would be looking at supporting them through making sure that they have the right cofactor nutrients present for both the folate and the methionine cycle to support them in producing optimal levels of SAM, as well as making sure that there aren't any SNPs that are potentially going to slow down or downregulate the production of SAM. That's roughly how you would approach it, and of course, there are other means as well, but specifically related to methylation, that's what we would look at.
How is the test taken? That's a very good question. The test is a combination of a blood draw as well as a buccal swab. It combines both the functional analytes in a blood sample, and the buccal swab is, of course, there to test these SNPs related to various parts of methylation.
I should add that the genomic markers are an add-on to the methylation panel, so they're not automatically included. If your client or patient hasn't done their genes yet, I would highly recommend doing that because they will be able to see which SNPs are having an impact on which parts of the methylation cycle and how they may be impacting certain analytes. The test does require immediate centrifuging, so that's important to consider when organizing a phlebotomy appointment for your client or patient.
Someone's asking if I would consider or recommend testing for Mast Cell Activation Syndrome, and that's absolutely yes. You may recall that one of the methyl transferase enzymes is the HNMT enzyme, which is related to histamine detoxification and metabolism. We also have other things as well, so poor detoxification can be related to methylation, and that is highly related to MCAS. So yes, absolutely, I would recommend testing methylation for that clinical presentation.
Another question is related to the supplementation of folic acid. Someone's asking how much dietary folic acid is too much. There isn't actually an upper limit for dietary folic acid; you can never really get too much in the diet because dietary folic acid or folate, should I say, doesn't have a negative impact on the DHR enzyme. It's supplemental synthetic folic acid that has a downregulatory impact on that enzyme, but dietary intake won't do that, so there is no risk there whatsoever. In terms of the upper limit for supplemental folate or folic acid, it's 1000 mcg. At least, that is the sort of governmental set guidelines. You may recall back at the beginning of the presentation I spoke about the risks of supplementing with folic acid; the risks of unmetabolized folic acid are mainly related to those supplementing with higher than 1000 mcg.
I've got a question here related to supplementation around glutathione or NAC to help improve glutathione. A practitioner here has done a nutrival test for their patient, and her glutathione came back very low, with oxidative stress very high, which is unsurprising. The question is whether to prescribe glutathione directly or NAC. This is just my personal opinion, and it may be different for others, but I would say both. If someone has very low levels of glutathione and high levels of oxidative damage markers, then supplementing both NAC as well as glutathione is probably a good therapeutic focus, at least initially, to try to get levels up to a healthy amount. Then you could switch to one afterward once their levels have improved.
How can you evaluate the SAM to SAR ratio with a client? We'll be going through this in next week's presentation. One of the ratios that we highlight in our methylation panel is the SAM to SAR ratio, which is a universal way of testing the risk of hypo- or hypermethylation according to the levels of both of those compounds. That is measured in our methylation panel, and you will be able to see a little bit more about that in next week's presentation.
I've got a question here to clarify the relationship between cancer and methylation. You will recall that there was a short description or insight into the enzyme SHMT, which is in the folate cycle. This particular enzyme does two things. Depending on the demand for methylation, it will either help to produce 5,10-methylene tetrahydrofolate for the production of 5-THF or it will be shunted down the pure nucleotide synthesis pathway. That pathway has an impact on DNA methylation, DNA regulation, and repair. Essentially, what I was explaining is that any SNPs on that enzyme, which has a downregulating impact on the enzyme, can be related to issues with DNA repair and DNA regulation, which has a pivotal role in the development of cancer or the risk of cancer. I hope that's clarified the question.
The safe upper limit of 5-MTHF is the same; it's 1000 mcg or µg, depending on how you measure it, as the same as folic acid. I've got a question here on my opinion using molybdenum to help the CBS pathway. Molybdenum is an important nutrient for detox pathways and the liver, particularly sulfuration. I think it goes back to the question around whether a CBS SNP will lead to an upregulation and therefore poor ammonia detoxification. Molybdenum will help to improve that, but again, I think it's important to not just go by what the genomic markers are saying but to look at the functional analytes around those enzymes to see whether that marker, or that SNP, should I say, is being expressed and what's actually happening in that dynamic interplay between those pathways.
Another practitioner is asking about a client with high sarcosine and what the potential solutions for GNMT SNPs are. High sarcosine can often be indicative when there is an upregulation in methylation and potentially because of an upregulation in GNMT. What I would say is that this can also be related to pathways a bit further upstream. If you're looking at, for example, the BHMT pathway and how that's functioning, that might help to elicit a little bit more about why there may be an upregulation in the GNMT pathway. The short answer is to check what the SAM levels are like because if they are normal, then this may just be a compensatory mechanism to prevent hypermethylation from occurring. So check the SAM and check the SAR because ultimately it's the outcome of those two compounds that are going to determine whether there is an issue with the outcome of methylation.
I've got another question about whether this test can be performed on children or teenagers. The reference ranges are for an adult population, so that's something to mention—18 and upwards. Depending on the age, of course, it also depends on the child's sensitivity to having a blood sample taken. That's always going to be a bit of a hindrance in testing. I would say that because the reference ranges are for an adult population, you may need to adjust accordingly to what it would be for a child if they were younger than 18, for example.
I've got a question here: Why is the enzyme AHCY not assessed in your DNA add-on? Good question! Thank you for asking. I do not know, as a short answer to that, but I can find out, and I'll see what the outcome of that is. I'm not sure why; I'm sure there is a good reason, but I will find out and come back to that definitely.
Okay, I'm going to just answer one more question here, and then we'll wrap up for today. I've got a question here: Which supplements do you recommend for a patient with both MTHFR and COMT enzyme SNPs? We know that the cofactors for COMT are magnesium and also B2, zinc, and SAM. You can supply them with the nutrients, but you'll need to make sure that they are producing enough SAM. If they're not, it may be related to the MTHFR SNP that's downregulating the availability of 5-MTHF, in which case you could supplement with methylfolate if they do have low levels of SAM. Again, definitely test the functional analytes to see whether those SNPs are being played out with the COMT enzyme. If they have a SNP on the COMT enzyme, then you will know that it's being played out because they will have elevated levels of anxiety and problems with downregulating their stress response, as well as potentially, if they are female, issues with estrogen detoxification.
Thank you so much for your time today! It is much appreciated. I hope that was an informative webinar. Again, you'll receive the recording later on today, as well as the slides. Please do join me again next week, where I'll be talking more specifically about our methylation panel, and then we'll be discussing a couple of really interesting case studies where methylation testing has proven to be very valuable. Thank you once again, have a lovely day, and see you next week!