**3. Synthesis and metabolism of gut microbial metabolite TMA and TMAO**

## **3.1 Production of trimethylamine by gut bacteria**

Trimethylamine (TMA) is the source of TMAO in humans. TMA is derived either directly from meals high in TMA, such seafood, [68, 69] or indirectly from the bacterial metabolism of dietary choline and choline-containing substances in the colon, such as phosphatidylcholine [16], betaine [70], and dietary L-carnitine [33, 71]. The ability of different gut microbes to produce TMA from food precursors varies. This is because it is produced in the gut via a variety of microbial mechanisms (**Figure 1**). As a result, the composition of an individual's microbiota influences the magnitude of TMA production. It's worth noting that the genes essential for TMA formation are found in just a small percentage of the microorganisms in the intestine (less than 1%) [72]. TMA formation appears to be possible even at extremely low concentrations of these microorganisms, highlighting the importance of the gut microbiota in this context [73]. TMA and TMAO levels have been linked to increased activity in bacteria belonging to the phylum Firmicutes and Proteobacteria, which are known producers of this metabolite. Furthermore, because Bacteroidetes are unable to make TMA [74], TMA and TMAO levels have been connected to an enhanced Firmicutes/Bacteroidetes ratio, with higher levels of Firmicutes and lower levels of Bacteroidetes [57, 58]. The genes coding for the glycyl radical enzyme choline TMA-lyase (CutC) and its related radical S-adenosyl-L-methionine (SAM)(CutD) activating protein were discovered in the Choline Utilization (cut) gene cluster in gut bacteria, which is responsible for the anaerobic breakdown of choline into TMA [75]. A two-component CntA/CntB oxygenase/ reductase system capable of cleaving L-carnitine into TMA and malic semialdehyde is another microbial metabolic route that generates TMA from L-carnitine [76]. The yeaW/X gene products (YeaW/X TMA lyase) are a closely similar bacterial lyase. Choline, betaine, L-carnitine, and -butyrobetaine can all be converted to TMA by this promiscuous lyase [74, 77]. Aside from these TMA-generating processes from dietary trimethylamines, some gut microbes like *E.coli* have also been found to have another pathway that converts TMAO to TMA via the activity of a torA-like gene product which acts as a reductase [78, 79].

*Gut Microbial Metabolite Trimethylamine-N-Oxide and Its Role in Cardiovascular Diseases DOI: http://dx.doi.org/10.5772/intechopen.107976*

#### **Figure 1.**

*Chemical formulae of TMA and TMAO's principal dietary precursors. The key metabolic pathways for the synthesis of TMA by the gut microbiota and endogenous enzymes, as well as the conversion of TMA to TMAO by hepatic FMOs, are depicted in this diagram.*

#### **3.2 Conversion of TMA into TMAO and its regulation**

TMA generated from a choline -rich diet through various metabolic pathways is absorbed from the gut into the hepatic portal circulation and oxidized by the enzymes flavin-dependent monooxygenase isoforms 1 and 3 (FMO1 and FMO3) in the liver to create Trimethylamine-N-oxide(TMAO) (**Figure 2**) [80]. TMAO is excreted out of the body, usually through urine [81]. Sweat, feces (4%), exhaled air (less than 1%), and other body secretions are some of the other ways TMAO is excreted [82]. TMAO can be metabolized to DMA(Dimehtylamine), formaldehyde, ammonia, and methane by methanogenic bacteria that carry the TMAO demethylase enzyme [83]. Furthermore, it has been demonstrated that TMAO derived from food can be absorbed directly in the gut [84]. As a result, plasma TMAO levels are regulated by TMA synthesis and degradation, as well as the rate at which TMA, and TMAO are secreted [85].

## **3.3 Dietary precursors of TMAO and the relationship between TMAO levels and dietary habits**

As discussed in Section 3.1, seafood is a rich source of dietary TMA/TMAO and various dietary precursors like L-carnitine, choline, ergothioneine and betaine (**Figure 1**) equally contribute to the generation of TMAO in the body. Free TMAO present in seafood is not metabolized by gut microbiota and is directly absorbed into the systemic circulation [86]. L-carnitine is present in high concentrations in meals derived from animals (meat and dairy products), and in smaller amounts in grains

**Figure 2.** *Gut flora mediated synthesis of TMA and hepatic conversion to TMAO.*

and vegetables [87]. The most common sources of choline in the diet are eggs and liver, followed by meats and fish, whole grain, cereal, vegetables, fruits, milk, fats, and oils [88]. One of the most important sources of betaine is cereal-based foods [89]. Betaine can also be found in spinach, beets, crabs, and finfish [90]. Dietary sources are the only way to get ergothioneine. Ergothioneine is found in only a few foods, with the largest quantities found in boletus and oyster mushrooms, as well as to a lesser level in chicken and pork liver and kidney, oat bran, and black and red beans [91]. As discussed in Section 2, a westernized lifestyle and diet full of junk fatty foods and refined sugar, devoid of fiber and important nutrients, predisposes one to increased CVD risk and other chronic diseases. Plasma TMAO levels have been observed to rise when people eat Western-style or high-fat diets [92–94]. However, conversely, epidemiological studies have linked the Mediterranean diet to a lower risk of cardiovascular disease (CVD) [95]. A typical Mediterranean diet is defined by plant based foods (vegetables, fruits, nuts,), olive oil based fats and moderate to low amounts of seafood, eggs and meats [96, 97]. This makes this type of diet high in fiber and low in choline -rich food. The importance of fiber- rich foods has already been mentioned in Section 2. High dietary fiber consumption, followed by gut microbiota-mediated fermentation, appears to reduce TMAO levels in experiments on animal models and clinical medicine [98].

## **4. Role of TMAO in increasing cardiovascular disease risk**

A choline-rich diet puts a person at risk of increased TMAO levels [16], which is directly correlated to an increased CVD risk [99]. Angiographic markers of coronary artery atherosclerotic burden and cardiac risks have strong relationships with systemic TMAO levels, and higher levels of TMAO in the blood are linked to an increased risk of incident cardiovascular events such as myocardial infarction, recurrent stroke,

*Gut Microbial Metabolite Trimethylamine-N-Oxide and Its Role in Cardiovascular Diseases DOI: http://dx.doi.org/10.5772/intechopen.107976*

#### **Figure 3.**

*TMAO- mediated platelet hyper-responsiveness and increased thrombosis risk.*

and even cardiovascular death [55, 100]. Gut microbes play a role in modifying platelet reactivity and generating a pro-thrombotic phenotype in vivo by producing TMAO (**Figure 3**) [101]. Zhu *et al.,* has shown that direct exposure of platelets to TMAO, caused activation of the platelets by the release of intracellular calcium. This modulates the platelet hyper-responsiveness and the potential of thrombosis and causes thrombosis and atherosclerosis [101]. Rebecca *et al*., states that knockdown of FMOs can protect mice from obesity, which is a major cause for cardiovascular diseases [102, 103]. Increased amount of TMAO, obtained from the diet, causes monocytes to enter the subendothelial space and differentiate into colony -stimulating factors when they encounter the growth factors. These form large cells known as dendritic cells and macrophages which possess high expression of SR-A1 and CD36 [104]. These cells take up oxidized, low- density lipid particles to create foam cells that are irregular in the uptake of cholesterol with fatty acids and ester bonds, thus stimulating atherosclerosis [105]. It is suggested that CD36/MAPK/JNK pathways play a vital role in the formation of foam cells [106]. Research studies show that apoe−/− mice fed with choline diet for 8 weeks, gradually exhibited an increase in TMAO, which further recruited macrophages and pro – inflammatory cytokines [107]. Another study by Boini et al. indicates the link between TMAO and inflammation, where TMAO induces NLRP3 inflammasome formation and causes other immune responses [108]. An imbalance of cholesterol transport is observed in individuals with high TMAO, and studies show that mice with administered TMAO inhibited the synthesis of hepatic bile acid by downregulating the expression of Cyp7a1, which promoted atherosclerosis [109]. The activation of oxidative stress pathways following exposure to TMAO, which triggers inflammatory cytokines, is the molecular basis for increasing cardiovascular illnesses. It can also activate the p38 MAPK and NF-kappa beta signaling pathways, which enhances NLRP3 production in the inflammasome and promotes vascular calcification and endothelial cell damage [110]. High administration of TMAO causes oxidative stress, inflammation and suppressed cellular functions, while low levels exhibit a contrary response [111]. A recent study proved that patients with aortic stenosis, had their TMAO levels as 5.5 μM, when the control was 3.6 μM. TMA is also associated with cardiovascular diseases as the levels of TMA in these patients were 59.5 μM and the control was 23.2 μM [112]. Thus, TMAO is considered to be an independent risk factor for cardiovascular diseases.
