Transcription
Welcome, everybody! Thank you so much for joining us this morning for the second installment of our two-part methylation series. If you were able to join me last week, you will already know me. My name is Anastasia, and I am a clinician educator at Genova Diagnostics Europe. I will be taking you through today's webinar.
Like last week, there will be a recording sent to you, as well as the slides. You will also find the slides in PDF format in the handout section on the control panel, along with a support guide, a sample report for the methylation panel, and a fee schedule. All of that information is there for you to download at your leisure.
Following on from our previous installment of the series, today's aim is to go over Genova's methylation panel and its structure, as well as presenting two case studies. This will help you gain a deeper understanding of the value of the markers we test on the panel and, of course, feel more confident in using it with your patients and clients. I recommend that if you didn't get a chance to listen in on the first webinar, you make some time to watch that, as it essentially acts as a primer into the panel we offer at Genova, as well as today's webinar.
Today, I will be going systematically through the various sections of the test, starting off with the summary page at the beginning of the test and then moving through the various sections, such as the methylation balance ratios and what they mean for your patient. I will break down the biomarker pathways chart and finally move into a couple of case studies where the test has been useful in elucidating certain health issues. Of course, there will always be a question time at the end, so I will leave 15 minutes at the end of the webinar or the presentation dedicated to your questions. Please do write your questions as we go along in the questions tab in the control panel.
Before I dive into the various parts of the test, let's start with a short introduction to the test itself and an overview. One of the key distinctions of Genova's methylation panel is that it combines both phenotypic and genotypic information in one profile. What this means is that by measuring the functional analytes involved in the methylation cycle, as well as the genetic predispositions for altered enzymatic activity, this profile can help clinicians design more targeted treatment strategies to optimize patient outcomes. Essentially, clinicians are able to go beyond looking at genetic predispositions or SNPs, such as MTHFR, which I'm sure many of you already know about, or only measuring single biomarkers like homocysteine. It's not to discredit those individual markers, as they can still provide significant information, but there's often a lot more at play that's worth looking into.
Just a bit of practical information: because this test combines both genomic and phenotypic markers, it does require both a buccal swab and a blood sample that needs to be centrifuged. I should also add that the genomic markers aren't automatically included in the test; they are an add-on. So, of course, if your client or patient hasn't already done any genetic testing, I recommend that they add that on at least in the initial test so that you can see how certain genetic variants can be at play among the functional analytes surrounding them.
We know that genotypic SNPs do not always translate to phenotypic expression. Genes can be switched on or off, and SNPs represent genetic predispositions that aren't always clinically manifested. That's why it's so important to test the functional analyte in addition to the SNPs. If you're just testing the genes, you're largely playing a guessing game based on the clinical presentation of the patient or client, whether or not those genes are playing out. Many of you, if not all of you, would have heard the term "the genes load the gun, but the environment pulls the trigger." Ultimately, it's the factors in our environment, such as diet, lifestyle, and the quality of our relationships with our loved ones, like family and partners, that can influence our genetic expression.
On the methylation panel, if an enzymatic SNP is present, abnormal metabolite or functional analyte results near that enzyme may reflect an enzymatic SNP abnormality, giving you insight into whether that SNP is being expressed.
Before I go on, I just wanted to give a brief explanation of SNPs and what they actually are. Apologies if this is repeated information for some of you; I'm sure many of you are already aware of what SNPs mean. A SNP is essentially a single nucleotide polymorphism, and it is the most common type of genetic variation among people. Each SNP represents a difference in a single nucleotide, such as adenine, thymine, cytosine, or guanine in the genome sequence. You inherit two alleles, or versions, of each gene—one comes from your biological mother, and the other comes from your biological father. If the alleles are identical, this means that you're homozygous for that specific gene. For example, you could have two alleles for the gene that causes brown eyes. Being heterozygous, of course, means you have two different alleles, having inherited a different version from each parent.
Here is an example of how we display the genes and the SNPs on the methylation panel. As you can see, each enzyme evaluated will have two results representing each inherited allele—one from each parent. A plus sign indicates that a polymorphism has been detected, and the negative sign indicates that no SNP is present. Two plus signs highlighted in blue mean that the patient has inherited two copies of that gene and is therefore homozygous. One plus sign indicates heterozygous, and no plus sign means that they have the wild type, which means that no polymorphisms have been detected on that gene. You can also see that we display the genes in relation to the down-regulating or up-regulating impact that that particular SNP will have on that gene, and it's listed at the very top.
In laboratory reports, the methylation panel results are displayed on the front page as a convenient visual summary of the test findings. This page is titled the "Interpretation at a Glance" page and provides clinicians with a quick overall methylation status assessment, separating the various aspects of the report into key clinical areas. This is also quite a useful summary page that helps facilitate clinician-to-patient communication, allowing them to know where to draw attention to the main imbalances occurring for that person.
The first half of the page is displayed with the three functional pillars to help separate the biomarkers into distinct categories. The methylation and transulfuration pillars on either side of the genetic polymorphism pillar capture the abnormal biomarkers, which are listed again on page two within their reference ranges. The reason we specifically highlight the abnormal biomarkers here is related to an algorithm that provides a suspected degree of clinical impact, referenced by a color-coding system to indicate the severity of abnormality of the biomarkers. This algorithm takes into consideration two factors: one is how far out of the reference range the biomarker is, and the other factor is the clinical importance of that biomarker.
In the middle, we have a genomic polymorphism pillar that provides the results of the genetic SNPs, which are an add-on to the methylation panel. As you can see, the SNPs are categorized based on whether the polymorphism has a down-regulating impact on the activity of that enzyme or an up-regulating activity.
Underneath the functional pillars on that same page is an overview assessment of the methylation status. The three indicators in this section include the SAM to SAH ratio at the very top, the methylation balance ratio in the middle, and the Met to SAM balance ratio at the bottom, which measures the ratio between the methylation, methionine markers, and the transulfuration markers. Unlike the SAM to SAH ratio, which is commonly referred to in literature as the methylation index, the methylation balance ratio in the middle and the Met to SAM balance ratio at the bottom are completely unique to Genova's methylation panel. They are novel algorithms created by Genova to represent a broader attempt to capture functional disturbances within the methylation and transulfuration pathways by comparing multiple analytes related to those pathways.
I'll go into detail about how each of these ratios works and what they can tell you about your client or patient. As you may recall from the first webinar, global methylation is dependent on two key factors: adequate SAM supply and adequate SAH removal. The SAM to SAH ratio has therefore been proposed to indicate the likelihood of hyper- or hypomethylation. As we discussed in the last webinar, S-adenosylhomocysteine is a potent feedback inhibitor of methyltransferase reactions, which is why when SAH is elevated, it often leads to hypomethylation. We also know that poor homocysteine clearance contributes significantly to SAH accumulation, which is, of course, another sign of hypomethylation.
The methylation balance ratio compares four methylated metabolites, including SAM, as you can see listed here, and also compares them to four unmethylated metabolites listed on the right-hand side. The methylation balance ratio can be thought of as an expanded SAM to SAH ratio. The clinical utility of the methylation balance ratio is that it represents a potential way to detect subtle methylation imbalance prior to alterations in the SAM to SAH ratio. It's worth mentioning that this approach is still novel and based on biochemical pathway analysis. However, early Genova data analysis of this biomarker has demonstrated its ability to distinguish a healthy cohort from an unqualified cohort, and Genova will continue to conduct ongoing research on this novel biomarker's clinical application.
The second calculated ratio is called the Met to methylation to transulfuration balance ratio, and it compares analytes between the methylation pathway and the transulfuration pathway. There is a relative balance that exists between the methylation and transulfuration pathways, ensuring that adequate levels of glutathione are produced to counteract oxidative stress and that an adequate level of SAM is made for methylation reactions. You can see the ratios here in the middle that are used to compare the two analytes in the two sections: methylated metabolites and unmethylated metabolites.
The second page of the report consists of the biomarker results, and as you can see, the biomarkers are grouped based on their significance to the methylation panel. You have the ratio section at the very top, the methyl group donors second, then methyl group metabolites, and finally the transulfuration metabolites at the bottom. The patient's results are listed in the first column and are either accompanied by an "L" or an "H" flag if the results fall outside of the reference range.
Just a very obvious piece of information here: biomarkers that are outside the reference range will appear under their corresponding pillar on the first page. The biomarkers that are listed as either too high or too low are the ones on the first page in the pillars on the first half of that first page, and you will see them with a corresponding triangle pointing either upwards or downwards in yellow or red, depending on how far outside of the reference range they are.
Finally, on page three, we have the patient's biomarker results and their genomic results all displayed together in the pathway format. It's quite helpful when communicating to your client or patient the various imbalances that could be occurring to use this page because you get to look at it from a bird's eye point of view. This really allows clinicians to visualize a patient's unique biochemistry based on their individual genomic and phenotypic results all in one place. As you can see, the genomic results appear next to the enzymes that are evaluated and utilize the same sort of positive-negative abbreviations as on the first page. Any abnormal biomarkers appear with either a yellow or a red border, indicating either a borderline or abnormal finding, respectively.
This is really the overview of the panel and the structure of how the panel works. As you can see, you've also got a key on the top right-hand side, which shows how the enzyme is displayed, the cofactors, which are normally listed next to the arrows, and you've also got the genomic results, of course, in another plus or negative sign depending on the SNP that they've inherited.
After that brief overview of the test and its structure, I'm going to move on to the case studies. My aim with these is to demonstrate varying clinical presentations on the test and the various interventions that can be made to support imbalances. I won't go into too much detail on the therapeutics, as there isn't a huge amount of time, but I will go into detail on the biomarkers and what they could be telling us about the individual.
This is case study one: a 56-year-old female patient. As you can see, her main health concerns were anxiety/panic disorder. She also had a significant family history of cancer on both her maternal and paternal sides, which was something that was on her mind as well. She really wanted to look into prevention. She was also going through menopause. Her medications and therapies included HRT as well as hypnotherapy, and her most concerning symptoms were anxiety, coupled with low mood, brain fog, and panic attacks. She also suffered from cold extremities, intolerance to the cold, low blood pressure, dizziness, insomnia, and suspected sugar addiction cravings.
I already mentioned she had a family history of cancer, and a particular concern to her was breast cancer on her maternal side. She explained that she had anxiety since a young age, but it got significantly worse during her father's illness and subsequent passing later on in life.
Moving on to her diet, this patient had a diet high in refined sugars, featuring things like chocolate bars, ice cream, cakes, and sweets on a daily basis. She had a low to moderate protein intake, and many of her meals were very snacky, lacking balance in terms of their ratios between the various macronutrients. They often contained things like crackers, hummus, and pancakes. She didn't often cook herself a meal at lunchtime or breakfast; the only real balanced meal in the day tended to be in the evening, which often included some sort of animal protein, as well as some vegetables and complex carbohydrates.
I had her do some initial labs and wanted to look at homocysteine, as well as doing a comprehensive thyroid panel in relation to the symptoms around her thyroid. As you can see, homocysteine was quite high at 15.57 µmol per liter, and her comprehensive thyroid panel came back healthy. She had already had some hormone testing done through her GP, but it wasn't very comprehensive, so I kept that in mind as something to also look into. However, methylation was one of the things I wanted to look into first in relation to anxiety and the cancer in her family.
We did the methylation panel, and this is the screenshot of the first section on the "Interpretation at a Glance" page. As you can see, she is trending towards hypomethylation, judging by the level of SAM, which is moderately low, as indicated by the yellow triangle pointing down, and the level of SAH, which is moderately high. You will recall that SAH inhibits methyltransferase enzymes, indicating hypomethylation along with low levels of SAM. We can also see that many of her proteins are also quite low, which may contribute to global undermethylation. For example, methionine, which you can see on the left pillar under methylation, is moderately low. Methionine is a precursor to SAM, so this may lead to lower levels of SAM if methionine is low, which was unsurprising considering her diet was very low in protein.
I'm going to go into her SNPs in a second, but I just wanted to show you the initial findings here. I've inserted the SAM to SAH ratio screenshot from her test so that you can see what a typical picture of hypomethylation looks like. Below is a screenshot of how the SAH portion of the pathway layout page was shown. I wanted to highlight this, as we know that what normally triggers a high SAH is an accumulation of homocysteine. You can see that her homocysteine in this particular test was healthy. I've also inserted a screenshot of where her homocysteine lay within the reference range on this test. However, because we had done that initial homocysteine test a month prior, we knew that her homocysteine was high, which could have been contributing to her high SAH levels.
In a methylation panel, you may see normal levels of homocysteine and elevated levels of SAH. What this could indicate is that perhaps the homocysteine levels had previously been high, leading to this backup into SAH. That's what I wanted to highlight here, because of that initial high homocysteine binding a month prior to doing a methylation panel.
Now, let's look at some of the genetics upstream in the folate cycle that may also be triggering hypomethylation. As you can see at the top of the folate cycle, she has a heterozygous SNP on the SHMT gene, which regulates the availability of 5,10-methylene tetrahydrofolate to act as a substrate for MTHFR. The impact of a SNP on this gene is downregulation, and one of the results of downregulation is, of course, a reduction in circulating levels of 5,10-methylene tetrahydrofolate and increased homocysteine as a result of that, because there may be less 5,10-methylene THF available.
In addition to the SHMT enzyme, as you can see, she also has two heterozygous SNPs on the MTHFR gene. Genova's panel detects the presence of both the C677T and the A1299C polymorphisms on MTHFR, and in this patient, she has heterozygosity on each one, both triggering a downregulating impact on the activity of the enzyme. You will recall that the MTHFR enzyme converts 5,10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate. This step activates folate to be used for homocysteine conversion to methionine, meaning that if these SNPs are being expressed, this leaves less methylfolate available to convert homocysteine to methionine.
As you can see, there is some explanation on the impact that either one of the SNPs can have on the left-hand side. Research shows that a heterozygous SNP on the C677T polymorphism can result in downregulation; however, a heterozygous SNP on the A1298C polymorphism is unlikely to. But if someone has combined heterozygosity for both those two polymorphisms, this can result in significant plasma homocysteine elevation, and this is exactly what this patient has. Despite the fact that her homocysteine levels are normal in this particular test, we can assume from the elevated SAH that the previously high homocysteine may be stimulating the SAH accumulation.
Now, let's look at the SNPs related to B12 metabolism and the role that they could further play in limiting homocysteine to methionine conversion. As you can see, she has a heterozygous SNP on the MTR enzyme, which has an upregulating effect on the activity of the enzyme. This enzyme is responsible for the conversion of homocysteine to methionine, so we may assume healthier methionine levels if that SNP is being expressed. The MTRR enzyme also has a heterozygous polymorphism on it, and that has a downregulating impact on the activity of the enzyme. MTRR is responsible for reducing oxidized forms of B12 to be reused, which essentially regulates the availability of methylated B12 to convert homocysteine to methionine in addition to methylfolate.
Again, we can assume that this would lead to an elevation of homocysteine, which isn't the case in this particular test. But again, because of the high SAH, it may be that the previously higher homocysteine was contributing to the high SAH levels and hypomethylation.
Finally, we get to the methionine adenosyltransferase enzyme (MAT), which is shown there with the arrow. The MAT enzyme is highly conserved and regulated; it's responsible for converting methionine to SAM. The SNP downregulates the enzyme's activity, which decreases SAM production. As you can see, the availability of cofactor nutrients like magnesium and potassium, as well as the compound ATP, are necessary for the MAT enzyme to function properly. Regulating the availability of these compounds and nutrients is essential. We also know that this enzyme is further downregulated by oxidative stress, and as you can see, peroxidation is low. We know that glutathione is the body's most potent intracellular antioxidant and is able to scavenge free radicals, acting as a detoxifying agent. Her oxidative stress was potentially high because glutathione was low, which could have been impacting the functioning of that enzyme in addition to having a SNP that has a downregulating impact on the enzyme.
You can also see that cysteine was moderately low, and the availability of cysteine is known to be rate-limiting for glutathione synthesis. This could be further contributing to her low glutathione levels.
There are some other factors that could have been impeding her glutathione biosynthesis. As you can see, her cystathionine is elevated here. Cystathionine is a dipeptide that is then converted to cysteine via the enzyme CTH, which you can see in the middle of the arrow. Because cystathionine is an intermediate of the transulfuration pathway, an elevation of this biomarker, in addition to low levels of cysteine, may indicate a backup in the transulfuration pathway. Conversion of cystathionine to glutathione requires necessary cofactors such as B6, as you can see illustrated next to the arrow. There are also other cofactors like zinc and glycine, as well as magnesium. Therefore, transient elevations of this metabolite, cystathionine, may indicate an increased need for these cofactors. As you can see, there is quite clearly a backup going on here.
Cysteine is a non-essential sulfur-containing amino acid that can be obtained either from the diet or endogenously made from cystathionine. As mentioned in the previous slide, it is a very important component of glutathione and has been shown to be the limiting amino acid for glutathione synthesis. The conversion from cysteine to glutathione requires the enzyme glutathione synthetase (GSS), which is the same enzyme that also catalyzes the conversion of glycine to glutathione. As you can see, glycine is quite significantly elevated, which suggests to me that potentially there is also a backup going on here within this enzyme. It's interesting because this is the same enzyme that requires B6, so there could be an elevated need for B6 as a result of this.
Now, as you will recall, this client's chief health concern was her anxiety and panic disorder, which is why having the COMT gene included in the methylation panel is so incredibly useful. The COMT gene is not displayed in the biomarker layout page; however, it is tested because it plays a key role in phase two detoxification of estrogens, as well as the deactivation of catecholamines such as dopamine, noradrenaline, and adrenaline. Of course, you can see that one of the cofactors of the COMT enzyme is SAM. So having adequate levels of SAM produced through the methionine cycle is essential for the activity of COMT. This patient had low levels of SAM, and as you can also see, she has a homozygous SNP on the COMT gene, which means that there is most likely a downregulation in the activity of this enzyme, leading to a backup in catecholamines, as well as estrogens. This could be why she suffered from such a terrible anxiety disorder.
Because her other chief health concern was preventing breast cancer, this was also useful, as we know that having prolonged estrogen exposure can increase the risk of breast cancer. This client was also on HRT, so it made it even more important to know her risk and potentially go on to do some hormone testing to see how well she is metabolizing the HRT.
With that in mind, I know this is going outside the methylation panel, but because of her family history, I wanted to check her hormone metabolism. As you can see, her parent estrogens, estrone and estradiol, are very high, which is unsurprising as she is on HRT. However, what I'm concerned with is which pathways she is methylating or metabolizing her estrogens in phase one, and how well she is methylating her estrogens. As you can see, she's quite elevated on all three phase one pathways. The two-hydroxy, the 16-alpha-hydroxy, and the four-hydroxy are all elevated. What we never want to see is higher than normal levels of the four-hydroxy pathway or metabolite, as this metabolite has the greatest estrogenic and genotoxic potential of all three. We know that four-hydroxy metabolites may induce DNA damage, generating reactive oxygen species, especially if they don't go through phase two detox methylation as well as they could.
As you can see, if you look at the arrow from the four-hydroxy to the four-methoxy metabolite, which is metabolized through the COMT enzyme, this is exactly what is occurring for her. Her four-hydroxy is at 6.1, and her four-methoxy is below detectable levels, which suggests to me that this homozygous SNP on her COMT gene was playing out in real time. So she's not methylating her estrogen very well.
So why is this so important in the context of the methylation panel? Aside from affirming that her COMT SNP is indeed most likely being expressed, when looking at her poor estrogen methylation and anxiety disorder, we also know that her glutathione is low. Glutathione is a key antioxidant for reducing the toxicity of the quinones and the reactive oxygen species generated by poorly methylated four-hydroxy metabolites. So having low glutathione means that she's at a higher risk of DNA damage and therefore breast cancer.
Here were my interventions. One of the tricky aspects with this particular client is that she was incredibly sensitive to B vitamins. She had already been on a journey of trying to find solutions for her anxiety. She was very well-versed in supplements and had already tried B vitamins in various different forms, both liquid and capsules, and she always felt incredibly anxious after taking them. This is quite a common issue for people with anxiety disorders.
What I have found is that because methylation plays such an important role in detoxification processes, if there are high levels of toxicity, such as an accumulation of steroid hormones or potentially environmental toxins, and then we start supplying the person with high levels of methylfolate and methyl B12 without addressing other aspects of detox pathways, this can lead to an increase in symptoms, including anxiety and other issues. So I didn't recommend methylated B vitamins; I didn't think it was worth trying that. My aim was really to focus on dietary approaches.
Here are some of the dietary changes: increasing her green leafy vegetables for folate, increasing fiber, especially from the cruciferous family of vegetables for obvious reasons, supporting the estrogen detox pathways, improving her protein intake because it was so low, and reducing the high glycemic load intake of foods that were high in sugar. She was also happy to have liver on a weekly basis, which is a really important nutrient for choline as well as vitamins. These are unlikely to have such an impact on her anxiety and will help to provide the raw materials without going in with concentrated doses.
I also worked on other aspects of methylation. I provided her with a complex that contained choline and betaine, which helps with the salvage pathway of homocysteine to methionine conversion. Obviously, this is gentler. B6 is a very important nutrient for her; I suspected that there was some depletion going on there because of the backups in the transulfuration pathway. Then, of course, liposomal glutathione, as well as NAC. I also gave her some botanicals in relation to her acid and detox pathways, and magnesium to support the COMT enzyme, as well as helping with the anxiety.
This client is ongoing, as is the second case study. She has seen quite significant improvement; however, it's still very much ongoing. As I'm sure many of you can appreciate, these things do take time. They are not overnight changes, but we are in that process, and I do believe that this approach is far more gentle than introducing methylated B vitamins from the get-go.
Let's move on to the second case study. I'm just aware of time; I don't want to run out of time here. This was a male client, aged 44. Again, his main concerns were anxiety and anxiety disorder, as well as OCD, depression, and obesity. He was on amitriptyline, which is a tricyclic antidepressant, and had been on that for many years—at least 20 years. He had been seeing a somatic experiencing therapist to help support him with some trauma he had experienced in his life. His most concerning symptoms were social anxiety, brain fog, low mood, hyperactivity, and an interesting thing, which I've only come across a couple of times in my time as a clinician, which was post-orgasm illness syndrome. He was very sensitive in the days following intimacy with his partner; he said he felt very unwell and down, which was quite an interesting part of his clinical presentation.
He did have a high intake of alcohol, which really helped him to relax, along with sugar cravings, insomnia, and obesity. In terms of his family history, there was significant history on his maternal side regarding mental health. He explained that when his mother was pregnant with him, she suffered from severe depression and had some quite invasive therapy at the time. The most pivotal life stage for him, when he remembers his mental health really taking a turn for the worse, was during university, where he began binge drinking and was in a very toxic relationship. It was at this moment that he started taking antidepressants, which he said were a lifesaver at the time. He also had a lot of stress in his environment, which I haven't mentioned here. He has two sons with autism, one of whom is quite severe and non-verbal, so those were very significant factors in his stress.
In terms of his diet, he described himself as an emotional eater. He goes through cycles of restriction and binging very frequently. He is a massive snacker, tending to restrict during main meals and then snack around meals on things like crisps, fruits, nuts, and biscuits. In his restriction phase, he would stick to things like chicken salads and salmon salads, which he would buy ready-made from places like Marks and Spencer. He would buy a packet of chicken fillets or cooked salmon and a salad separately, which wouldn't contain much—just salad leaves and tomatoes, with no fats or complex carbohydrates. For breakfast, he would often have fruit and walnuts, and that was it. In his binge phase, he would go through a phase of very high intake of alcohol, cakes, pizza, crisps, and high coffee intake on a general basis. So there were quite a lot of things to change there.
Here were his methylation test results. This patient's test results were quite interesting. While his homocysteine was significantly low, his SAH was very high. We would normally expect to see homocysteine elevated in addition to SAH, or at least normal. This was an interesting finding. In any case, we know that high levels of SAH, in and of itself, are very inhibitory to methyltransferase enzymes. If we quickly look at the COMT SNP, he is also homozygous here. So again, we have another case where chronic anxiety is potentially being impacted by having a homozygous SNP on the COMT gene, additionally downregulated by high levels of SAH.
This here is just to highlight this. While his levels were good, as you can see, well within the reference range, I've provided a screenshot there. Because the SAH is so high, the standard SAM to SAH ratio is also low, which indicates hypomethylation. I've added a screenshot at the bottom here on some of the factors that can be employed when the SAM to SAH ratio is low, and you'll find that in the support guide.
You may recall in the previous webinar, I spoke about how the complex interplay between the availability of methyl donors and the activity of the enzymes is constantly trying to find a balancing act to prevent hypo- or hypermethylation. Here is an example of that. While there are low levels of homocysteine, which we've seen on previous slides, and an unsurprisingly low level of methionine, in addition to that, you have a SNP on the MAT enzyme, which would normally downregulate the conversion of methionine to SAM. Despite all of those factors, there are still healthy levels of SAM. This really drives home the point that it is essential to not only test genes but also test the functional analytes to see how those genes are playing out in real time.
Despite all of those factors, there are some compensatory mechanisms that help to keep things in balance, and this is what's playing out for him. His SAM levels are healthy despite the fact that all of those other factors would normally lead to lower SAM levels. There is a relative balance that exists between the methylation and transulfuration pathways. I've mentioned before that this is really to ensure that there are adequate levels of glutathione to counteract oxidative stress, as well as ensure an adequate level of SAM is made for methylation reactions.
In the presence of oxidative stress, more homocysteine is used for glutathione production because oxidative stress induces enzymes in the transulfuration pathway. As you can see, this could be the case for this patient. His glutathione levels are very low, and while his homocysteine is also low, his cystathionine levels are healthy. This is potentially giving me insight into the transulfuration pathway being upregulated and shunting that homocysteine down that pathway in order to address the low levels of glutathione.
Again, with this particular patient, you can see that there is a potential backup going on. His cystathionine, glycine, and cysteine are all healthy, but his glutathione is incredibly low. There could be a lack of availability of those cofactor nutrients like B6, as well as the other factors that I mentioned previously, that could be preventing the conversion of cysteine and glycine to glutathione. So B6 was a key nutrient for him as well.
One of the things that I really wanted to look into was why his SAH levels were so high, particularly because his homocysteine levels are so low. There are a couple of things. We know that his alcohol consumption was very high, and in my research, I found that chronic alcohol consumption in mice, at least, increased SAH levels quite significantly by impairing the binding cycle. We can also see that B3 is a cofactor nutrient for the enzyme responsible for the hydrolysis of S-adenosylhomocysteine. B3, as I'm sure many of you already know, is used to form nicotinamide coenzyme (NAD), which is essential for supporting redox reactions that are important for mitigating oxidative stress.
Interestingly, NAD is an important coenzyme of the glutathione reductase enzyme, which converts oxidized glutathione to its reduced form. Alcohol can deplete B3 and the pool of NAD, so there seems to be this vicious cycle of alcohol increasing SAH and depleting B3, as well as potentially driving low glutathione, which is increasing his oxidative stress levels. There are also other factors going on, such as the stress and trauma he experienced in his life, as well as his diet, which could have also been contributing to the depletion of his glutathione.
Just very quickly, we did do some other labs with this patient. He did some mineral testing. Selenium, as you can see at the very bottom, was very low, which is an essential cofactor in glutathione biosynthesis. Magnesium is a cofactor for COMT, and while he is not deficient, he was on the lower end of that reference range. As you can see, his zinc was high, and he was already supplementing with zinc, so we did lighten the load on that. Lastly, potassium was low, and of course, that is a cofactor for the MAT enzyme that catalyzes methionine to SAM. However, I wasn't overly concerned about that, as there was no sign that it was impacting the activity of that enzyme. I did suspect that his high alcohol and coffee intake may be depleting his potassium levels.
Here are my interventions. Because of his susceptibility to emotional eating, cravings, and binging, my first priority was to balance his blood sugar levels. I suspected that in his restriction phases, the carbohydrate and fat avoidance was fueling adrenaline levels and overstimulation of his limbic brain. It was very important to introduce complex carbohydrates in addition to increasing his protein and healthy fats. My hope was that by doing this, we would be able to send messages of safety to his nervous system.
I also removed alcohol and caffeine as much as possible, given the fact that there is a huge component of his psychology impacting his cycle in all of this. I did offer him some alternatives to help replace those things with healthier options. I had also read into post-orgasm illness syndrome, and one of my takeaways was the possibility of opioid withdrawal having an impact on his brain in the days following intimacy with his partner. A suggestion was to introduce consistent exercise into his routine to support the balance of opioids in his brain. He really enjoyed walking and running, so he started incorporating that on a daily basis.
We worked on a gentle introduction of B vitamins, as he wasn't sensitive to them, which was great. I also introduced additional B3 on its own, as well as glutathione support through NAC, liposomal glutathione, and selenium. I added magnesium to support the MAT enzyme, and his improvement was pretty dramatic. However, what kept stalling his progress was the psychology around food. He had been in this vicious cycle of binging and restriction for such a long time that, at this point, it was important to have him referred to a therapist who could deal with a potential eating disorder that was at play here.
In general, things really did improve, and it is still an ongoing case. We haven't done a follow-up methylation panel, but that is on the cards at some point.
Apologies for going over time; as you can imagine, there was a lot of information to get through. I'm going to start looking at your questions. Please do start writing them if you haven't already in the questions tab in the GoToWebinar control panel. As always, if you have any further questions that won't be answered today or that you want to ask separately, you can get in touch with us through the email provided there: cleaneduk@gdx.net. Any feedback is also really appreciated.
Let's have a look at the questions.
Okay, so Tara is asking if the inability to metabolize caffeine, medications, and sensitivity to alcohol is indicative of a methylation issue. So yes, potentially. Any sort of questionable detox processes or signs of questionable detoxification can be indicative of poor methylation. Many of the methyltransferase enzymes are related to detox pathways, so if there is hypomethylation going on, then there will be less SAM available as a cofactor for those methyltransferase enzymes, and therefore, potentially, that can lead to poor detox pathways, particularly in relation to things like medications, alcohol, and also potentially caffeine. So yes, absolutely.
This is a really great question. Should the client stop taking supplements prior to the test, and for how many days? I guess this depends on what you're wanting to look for. If you want to see whether the supplement program you have provided for your client is supporting them, then you may want to keep them on that supplement program. If you've previously tested the client's homocysteine levels, for example, and know that they were really high, and you want to check again to see whether the B vitamins you're supplying them with are supporting the homocysteine, as well as other pathways like SAM production, then you may want to keep them on that supplementation program.
However, if you're wanting a baseline reading, then of course you do want to stop them taking their supplements. I would recommend stopping them for four to five days, up to a week, prior to taking the blood sample.
Okay, this is a great question by Madeleine. If you had not done the homocysteine test one month prior, would you be mistaken into thinking homocysteine was not elevated? We can assume that if SAH levels are elevated, at some point, homocysteine may have also been elevated because the most significant driver of high SAH is an elevation in homocysteine and the backup in that pathway. So, yeah, I would just say that if you see healthy levels of homocysteine, it doesn't necessarily mean that they've always been healthy.
I guess this drives home the point that it's important not just to test homocysteine. The pathogenicity lies in the inhibition of methyltransferase enzymes, so ultimately we depend on SAH removal to support methylation. This is why it's really important to test SAH. High levels of SAH are normally indicative of previously high levels of homocysteine if they're not currently high in that particular test.
I've got a question here from Paul. Quercetin is rapidly metabolized by COMT and increases SAH. Would you avoid it, as well as luteolin? To be honest, Paul, I haven't come across that. I didn't realize that quercetin can increase SAH, so this is completely new to me. But if that's the research you've done and you've established that, I would say, of course, remove that prior to doing a test because it may lead to discrepancies in the test results.
Maha is asking a question: Why liver versus eggs for choline? Eggs are great as well; I didn't put that in the slide, but she also increased her egg intake. Eggs, of course, are a concentrated source of choline, but the liver, in particular, is important because of the other nutrients in there, like the B vitamins. The bioavailability of those B vitamins in liver is so great that I wanted to add that in as well, but I didn't exclusively just say liver; I also mentioned an elevation in her diet.
Paul is asking again: When someone is sensitive to methyl B, what are your thoughts on using folinic acid? Folinic acid is an excellent alternative. In her case, she was still sensitive to that, so she's a particularly sensitive client. However, it has been recommended as an alternative to methylfolate because it has a gentler impact. What's great about folinic acid is that it bypasses the DHFR enzyme, so it doesn't need to be activated by that enzyme to enter the folate cycle, and it is readily used to create 5-MTHF. I do think that it's a great option for those who are sensitive, but with this particular client, she was so sensitive that even using folinic acid was not good for her.
Paul is asking again: Would you want to look at SNPs on the VDR gene when you have homozygous COMT? Potentially, yes. I believe the VDR gene is important in relation to the activation of certain neurotransmitters, so it could be significant in relation to COMT. However, I'm not sure in what angle you're looking at—whether that would be a negative thing or not to have SNPs in VDR in relation to homozygous COMT.
Elena is asking about hypermethylation. That is something that can be established from the test as well, of course, and you would see that on the SAM to SAH ratio. You would see that on the upper end of that scale. I didn't have any case studies that demonstrated hypermethylation, and to be honest, in all the methylation panels that I've done, I've never seen that before. I do believe that it's much more infrequent than looking at hypomethylation pictures, but it can be established by the test, and of course, in that circumstance, you would see an elevation in SAM.
Let's see. Claire Thompson is asking if I can touch on potassium as a cofactor a little bit more. Potassium is a cofactor for the MAT enzyme, which is responsible for converting methionine to SAM. It's listed there in that pathway in the biomarker layout page; you can see the cofactors next to the MAT enzyme, one of which is potassium. That's why we're touching on that, but because his SAM levels were really healthy, despite the fact that he had low methionine, a SNP on the MAT enzyme, and low potassium, it wasn't something that I was overly worried about.
Do I have a preferred type of magnesium to support methylation? This question is from Maha. I think, and I don't know whether other clinicians feel this way, but I believe magnesium glycinate to be the best magnesium to use in this situation for methylation. I tend to use other types of magnesium for other presentations. For example, if someone has a lot of anxiety, I may also give magnesium threonate or magnesium taurate. But in general, with methylation, I'd say magnesium bisglycinate or glycinate should be a good option. Correct me if I'm wrong.
Tanya is asking why there aren't any gut tests in the case studies. This is a methylation webinar, so this is why we're exclusively looking at methylation panels here. I did branch out a little bit with one of the case studies in relation to estrogen detoxification because of the fact that that particular case study had a homozygous COMT SNP and a family history of breast cancer. I wanted to check the estrogen detox pathways, but we don't have much time, and going completely out there and taking the focus away from methylation was not a priority.
Rebecca Hughes is asking if she was so sensitive to the B vitamins because she had high heavy metals or toxins. I didn't test for heavy metals or other toxins. It could be that she did have high levels of environmental toxins. We know that oxidative stress can have a negative impact on the COMT gene, and she had very low levels of glutathione. She did have an accumulation of steroid hormones, as you will remember, and one of which is the elevation in the four-hydroxy metabolite, which is quite damaging to DNA and increases oxidative stress. So that, for me, was a sign that there was increased toxicity at least from that point of view, but no, I didn't do any testing for heavy metals.
Fakira is asking what percentage of functionality is affected by a downregulated homozygous SNP. That's a very difficult question to answer because it very much depends on the SNP and the gene, as well as the patient and their presentation, and the functional analytes surrounding that SNP. It's a very difficult question to answer with a black-and-white response. I would just drive home the importance of testing the SNPs in relation to the analytes, as well as piecing that up with the clinical presentation, and really just using your skills as a clinician to understand what could be playing out based on the symptoms.
I'll just answer a couple more questions. Kim is asking if he was snacking a lot to be so overweight on what you describe as his diet. I wouldn't say that just the snacking was causing his weight gain. I do believe it was the binge and restrict cycle. When he was binging, he really was going all out—he was going all out on the alcohol as well as the processed foods. I think that was a large part of what was contributing to his weight gain. I should also add that there were genetic factors on his paternal side of the family, where there was quite a lot of obesity, so there was that side of things too.
Deb is asking whether homocysteine could fall through the plug hole into the transulfuration pathway, making it a potential unreliable measure in isolation. Yes, potentially. I guess with the second case study, his was very low, so that could be a sign that it's going overly down the transulfuration pathway to meet his glutathione needs. So, yeah, I do agree with you. I think that just testing homocysteine in isolation can be useful in some circumstances. Of course, that is actually what drove me into doing the methylation panel for the first case study because hers was so high. But I do think it is important to test other factors rather than just doing the homocysteine test.
Yes, that is important.
Okay, just one more question. Does the methylation test include the SNPs? I think I mentioned this already in the beginning of the presentation. The methylation panel includes the SNPs as an add-on, so they're not automatically included. You'll need to add them onto the panel. I explained that if your client or patient hasn't done any genetic testing in methylation, it is really important that they do that in addition to the functional analytes, at least in the first test. Of course, you won't need to test the genes again, but it is important that you do add that on the first test at least. So no, it's not automatically included, but it is an add-on option.
Okay, I think I will leave it there. I'm sorry if I haven't managed to get to your question. There are so many different aspects of methylation that are just so interesting, and it's a very complex topic. I hope that this has helped to some extent in highlighting the main points and helping you understand how it can be valid for your clinical practice. If you have any further questions, again, please do email us with feedback. I will be sending the recording to you later on in case you want to watch it again.
Thank you again for your time; I really appreciate it, and I look forward to seeing some of you soon in future webinars.