0:04 Slide #1 Good afternoon. Thank you for inviting me to join you in MEBO Miami Meetup. 0:12 I'm Elizabeth Shepard, and today I will introduce how the gut microbiome and diet are relevant to those who have a condition known as Trimethylaminuria, or Primary Trimethylaminuria, a disorder which is caused by mutations in the FMO3 gene. 0:36 Slide #2 The term microbiome refers to a collection of bacteria that inhabit a particular niche in our body, and we have a number of these microbiomes. 0:50 For example, we will have a skin microbiome, the bacteria that are found in our skins, our nose, in our eyes, and in many different niches, including the gastrointestinal tract. 1:03 If we take all these bacterial cells together, we actually have ten times more bacterial cells in and on our body then the number of human cells that make up our body. 1:14 So, there are 10 to the 13th cells in a human, so we have 10 to the 14 bacterial cells or a hundred trillion cells compared to 10 trillion cells of the human body. 1:26 Now ninety percent of an adult human, then, you could consider is bacterial cells. 1:33 How do we acquire a microbiome? Well, a baby is born without microbiomes, but it will inherit its mother's microbiome, particular if it's born from the vaginal delivery; and baby born by caesarean section will develop its microbiome slightly delayed from that of a vaginal birth. 2:03 Now the microbiome is absolutely essential. It provides functions that people say, feed us, form us, and protect us. 2:16 And what is meant by that is the bacteria typically, for example, in our gut, will digest complex molecules providing us with nutrients. 2:25 They form us; the bacteria helping us to develop an immune system, and they protect us because if we are inhabited by non-pathogenic bacteria, they will protect us from being inhabited by pathogenic bacteria that can cause disease. 2:48 Now the microbiome that I want to discuss very briefly today is the gut microbiome, the bacteria that inhabit all of the regions of the gastrointestinal tract, and this is a huge area inside our bodies. 3:04 We have bacteria that inhabit the esophagus, the stomach, the small intestine, the cecum, and the colon. 1
3:15 Slide #3 So, why is the gut microbiome relevant to Primary Trimethylaminuria? 3:22 When we eat food, there are a number of dietary components that have the general structure that is shown on this slide, where we have N, which is nitrogen, and the group CH3, which we call a methyl group, and this little group is a Trimethylamine group. The R stands for a number of other atoms that might be linked to this molecule. 3:52 Microbial action in the gut will break the bond between what I've shown as R and the nitrogen (N) and will liberate this molecule, trimethylamine (TMA). So this reaction is actually carried out in the gut by bacteria that reside in our gut. 4:05 This [odorous] trimethylamine (TMA) molecule is very quickly absorbed [into the bloodstream] and is taken to the liver. In the liver we have this enzyme called FMO3, which is able to catalyze a reaction, which adds an oxygen (the O here in the diagram) onto the nitrogen (N) to form the molecule called trimethylaminuria-n-oxide (TMAO)[non-odorous], which is then were very rapidly excreted through the kidneys. 4:39 Now the difference between these two molecules is that trimethylamine (TMA) has a very pungent smell and trimethylamine-n-oxide (TMAO) does not. 4:49 And so, the cause of trimethylaminuria is when the FMO3 gene carries a mutation, such that the enzyme that is encoded by this gene, cannot carry out this reaction very efficiently. 5:05 Now the name FMO3, or Flavin containing Monooxygenase 3 [enzyme], it s quite a long name, but it just simply means that this enzyme has to have the help of a small little chemical group called Flavin, and it s a monooxygenase. 5:26 This means that one atom of oxygen is added to the substrate. The substrate for the enzyme in this case is trimethylamine (TMA)[odorous], and the oxygen is added to it to produce trimethylamine-n-oxide [non odorous]. 5:45 Now there are multiple microbiome mediated pathways that can lead to Trimethylamine (TMA) production. 5:56 We know that this trimethylamine (TMA) is produced only by the action of bacteria because if we look at animals that have never been exposed to microbiomem and you can do this by rearing the animals in a sterile environment never touched by human hands, then these animals do not produce trimethylamine. 6:22 2
But we are inhabited by a microbiome that has the capacity to produce trimethylamine. 6:25 In the gut we have many different pathways that can be carried out by different bacteria to produce trimethylamine (TMA). So the trimethylamine here is shown as TMA in red. 6:40 Bacteria can actually convert trimethylamine (TMA)[odorous] into trimethylamine-n-oxide [non odorous], and use it themselves. So this would be by bacterial enzyme. 6:51 Actually trimethylamine-n-oxide is a very important molecule for some bacteria, and helps them to survive in our gut. 7:01 But the trimethylamine (TMA) is also produced, and not all of it is converted for the use of the bacteria, and the rest, of course, is absorbed and then move to our liver. 7:15 Now, I ve talked about the diet, and there are a number of different constituents of the diet that have been shown when you culture bacteria to be able to give rise to trimethylamine. These include choline, betaine, carnitine, and also trimethylamine n-oxide (TMAO)[from fish and seafood] itself, and I'll come back to that in a minute. 7:42 The problem that we have is first of all, we have a lot of bacteria, we have multiple pathways that can produce trimethylamine (TMA), and many different bacterial phyla (that is collections of related bacteria), that can produce trimethylamine (TMA). 8:00 So, there isn't one particular bacterial species that we can pinpoint to say this is the one that produces trimethylamine. A lot of bacteria can carry out this reaction. 8:13 Slide #4 I said I would come back to trimethylamine-n-oxide (TMAO) as a dietary source for the production of trimethylamine (TMA). 8:19 Now marine fish are the richest dietary source of trimethylamine (TMA), and the reason for this is that fish that live in the sea, live in an environment that has a very high salt concentration; and as fish move from different concentrations of salt, they need to protect their protein. This is a survival mechanism for the fish. 8:50 So what they do is they actually increase the amount of a fish Flavin containing monooxygenase protein, and they produce a lot of trimethylamine-n-oxide (TMAO). 9:01 This molecule is able to protect their proteins from basically breaking down; it protects the muscles of the fish. 9:18 3
9:10 So certain marine fish produce about three milligrams of trimethylamine-n-oxide (TMAO) per gram. 9:24 Now it's not just the fish that live in the sea that use this mechanism, fish that live, for example, in deep lakes or deep in the sea as well, they are subject to quite strong pressure changes in water pressure changes, and they also use this mechanism, the production of trimethylamine-n-oxide (TMAO) to protect their protein when they are subjected to these pressure changes. 9:54 Now in the human gut, when we eat marine fish or fish from deep lake, the trimethylamine-n-oxide (TMAO) is converted to trimethylamine (TMA) by a bacterial enzyme called trimethylamine-n-oxide reductase [enzyme]. That simply means of enzyme removes the oxygen [to make odorous TMA]. 10:11 And then, I mentioned on the previous slide that bacteria also have this enzyme that can make trimethylamine-n-oxide (TMAO), and in this case, they call the enzyme trimethylamine monooxygenase [enzyme]. 10:29 I'm showing the two reactions, but the reaction that's happening in our gut is very much in favor of the trimethylamine-n-oxide (TMAO) being converted to trimethylamine (TMA), and because there's so much of it, we will then have a lot of trimethylamine absorbed [into the bloodstream to the liver] because we've eaten a diet that would be rich in marine fish. 10:55 Slide #5 Dietary management is recommended for trying to control the symptoms of Primary Trimethylaminuria. 11:07 How do we actually measure the content of a particular foodstuff? 11:19 Well, when you see these tables that tell you what trimethylamine content is in a food, this will generally have been done by a scientist taking the foodstuff and chemically digesting it under quite harsh conditions to liberate as much of the trimethylamine (TMA) as is possible by those conditions. So this will give you one particular value. 11:34 Then other studies have done what we call biological digestion studies on particular food, that is monitoring as a specific food taken in by an individual and then monitoring the excretion of trimethylamine (TMA) and trimethylamine-n-oxide (TMAO) in the urine [as currently used in the TMAU urine test.] 12:03 Now the two methods don't always give the same answer. One is a very harsh chemical condition, and the other one is reliant on the biological digestion of the foodstuffs in the gut by the bacteria that reside in the gut of that Individual. 4
12:26 Slide #6 So on this slide, I've included the table of different foodstuffs, indicating the amount of time of trimethylamine (TMA) generated from a certain amount of the foodstuff by chemical digestion, and the amount of trimethylamine released by biological actions by feeding to a volunteer the same amount of that food. 12:40 This was a study that was carried out by Mitchell, Zhang, and Smith, and if anyone would like to consider this publication in more detail, I've included the digital object identifier (doi) at the bottom of the slide. 13:05 So on this slide, we're looking primarily at fruits and vegetables, and you can see, as I said, the answers are not always the same from the chemical and the biological digestion. So just looking at a table doesn't necessarily give you the amount of trimethylamine (TMA) that's might be released when a human eats this particular foodstuff. 13:34 All of these numbers are quite low for these particular foodstuffs. 13:37 Slide #7 Here I've included some of the values from the same study by Mitchell, Zhang, and Smith, where they've now looked at foodstuff, such as chicken, mushrooms, pork, eggs, beef, soya, lamb mackerel, and cod. 13:56 I think what you can see that is very striking is for example, chicken, which chemically would give a certain value for trimethylamine, biologically gives a lower value. 14:07 Mushrooms actually from this study shows a greater value of trimethylamine both chemically and biologically than is found in chicken. As we go down at the table, what is very striking is that when we get to the seas fish, the seafood, mackerel and cod, there s an extraordinary amount of trimethylamine produced by these particular fish. 14:39 This is for the reasons that I explained about the seafood and fish having to produce trimethylamine as a survival mechanism because of the salinity of the sea in which they reside. 14:55 Now I don't have a value here for river fish, but river fish are not subjected to have the high salt concentrations, and therefore, they do not produce a high amount of trimethylamine-n-oxide. 15:14 So definitely, what one can say from looking at this table is that definitely to be avoided is any form of seafood, if one suffers from trimethylaminuria. 15:33 Slide #8 I've included this slide not because it has anything to do with the gut microbiome, but just as a cautionary tale from a study that was done on volunteers that were fed 300 grams of brussel sprouts a day for several weeks. 5
15:55 This study showed a reduced amount of trimethylaminuria-n-oxide [non-odorous TMAO] urinary excretion. In other words, the eating of this large amount of Brussels sprouts increased the amount of trimethylamine excreted in the urine. 16:12 Then the researchers went on to identify by quite a complex procedure, chemicals within the brussel sprouts, and then they used these chemicals to identify the ones, and to see if they actually were directly inhibiting the activity of the enzyme. 16:31 They were able to do this; and we call this chemical an indole. 16:38 And these dietary indoles found in Brussels sprout actually inhibit the enzyme. So this is also something to be avoided in that one would not want to eat a foodstuff that is actually going to inhibit the enzyme activity if a person is already compromised by either the amount of the activity of the enzyme that they possess. 17:06 Slide #9 So for summarize, the diet includes precursors for trimethylamine, which is liberated by gut bacterial action. 17:16 The gut microbiome is person-specific. So the liberation of trimethylamine from a particular food to us may differ from person to person. 17:27 The gut microbiome itself is not static. It will change depending on our diet, and this is because what we eat feed the bacteria that inhabit our gut. 17:39 So a diet that reduces trimethylamine in one person, may not be as reliable in another person because of the interplay between what they have eaten and the bacterial species that reside in their gastrointestinal tract. 18:08 Slide #10 So here I've included a link to the human microbiome project this is a huge project that is revealing extraordinary things that our reliance and how our interactions with the bacteria that inhabit our body. 18:23 The second link is to an exhibition, which is called, Invisible you, and that means the bacteria that inhabit us, but which we can't see. 18:39 This can be accessed at the URL that I ve shown, which is part of the Eden Project in the United Kingdom; but their website has a lot of information and about the microbiome. 18:50 This project is has been sponsored by the Wellcome Trust. 6