By Robert Schinetsky

TMAO is a metabolite generated by our gut bacteria from certain nutrients, such as choline, carnitine, or betaine -- all of which are prevalent in animal-based proteins.

Over the past few years, an increasing body of research has found an association between elevated levels of TMAO and an increased risk for clot-related events, including heart attack and stroke and well as higher rates of premature death.[1,2,3,4]

Interestingly, certain varieties of fish and seafood (which are often heralded for their heart-health benefits) can also contain high levels of TMAO.[5] Despite TMAO levels rising after seafood consumption, the effects were short-lived. Within one day, circulating TMAO concentrations returned to baseline levels.

Further muddying the waters is a small body of research in rats, indicating that moderate TMAO levels may not be related to adverse cardiovascular events (or potentially support cardiovascular health[6]) and that other factors, such as impaired renal function, may be impacting poor health outcomes.[7]

How Does TMAO Adversely Impact Cardiometabolic Health?

TMAO is believed to promote atherosclerosis by altering the clearance of liver cholesterol, promoting inflammation and oxidation of LDL (“bad”) cholesterol, and the formation of foam cells.[8]

To better understand the relationship between choline, TMAO and cardiovascular concerns researchers continue to investigate potential links between the intake of certain nutrients (choline, carnitine, GBB, betaine, ergothioneine, etc.) and their respective impacts on TMAO levels and an increased risk for cardiovascular-related events.

Today, we’ll recap the latest research findings specifically as it relates to choline intake, its impact on TMAO, and whether or not it adversely affects the heart and brain.

Choline & TMAO Research Update

Choline Levels & Cardiovascular Disease

First, we’ll look at a meta-analysis published in October 2023 titled Association of Choline and Betaine with the Risk of Cardiovascular Disease and All-Cause Mortality: Meta-Analysis.[9] 17 studies were included in the meta-analysis with a total of 33,009 participants. Higher levels of circulating choline were associated with a higher risk of CVD and all-cause mortality but not betaine.

The second study we’ll look at was recently published in The American Journal of Clinical Nutrition. Researchers analyzed data from nearly 3,500 men and women (aged 33-45) who attended the year 15 follow-up exam and did not have prevalent cardiovascular disease (CVD).[10]

Following their analysis, researchers identified a positive association between plasma choline levels and incident CVD. Among participants with lower kidney function (identified by lower glomerular filtration rate, GFR), TMAO was positively, and betaine negatively, associated with CVD. For individuals with normal kidney function, TMAO was not associated with incident CVD.[10]

Yet another recent cohort study, including 429 newly diagnosed coronary heart disease, found a positive linear correlation between TMAO levels and SYNTAX score.[11] SYNTAX is used by cardiologists to assess the severity of coronary artery disease in complex, high-risk patients.[12]

Finally, a recent study in Bioscience Reports evaluated the relationship between TMAO and cardiovascular risk. Researchers examined 95 patients (avg. age 58.5 years): 40 with clinical atherosclerotic cardiovascular disease (ASCVD), 40 with atherosclerosis risk factors (RF), and 15 controls.[13] The team concluded that TMAO levels are increased with expanded CVD risk and are associated with atherosclerotic changes.

TMAO & Cerebral Stroke

As the research has expanded around TMAO, researchers have learned that TMAO’s potential deleterious effects may not be isolated to the cardiovascular system. It may, in fact, impact multiple systems throughout the body, including the brain.

Animal studies from 2018 noted that exogenous TMAO administration may induce brain aging and age-related cognitive dysfunction.[14] Specifically, TMAO:

• Increased the number of senescent cells, primarily neurons
• Increased superoxide production
• Enhanced mitochondrial dysfunction

Researchers noted that TMAO increased synaptic damage and reduced activity of synaptic plasticity-related proteins (proteins that keep your brain young and “flexible”).[14]

A recently published review shows that TMAO can influence the cognitive impairment caused by Alzheimer’s and Parkinson’s by increasing the accumulation of key proteins that affect inflammation and thrombosis (clotting).[16.,17]

Researchers do mention that while there is a connection between gut-brain axis and vascular cognitive impairment, the exact molecular mechanism by which TMAO exerts its effects needs further examination

Additionally, TMAO has been shown to promote platelet hyperreactivity and increase thrombosis risk, and a growing body of research identifies an association between TMAO and ischemic stroke (IS).[18]

A 2023 meta-analysis of eight studies confirmed an association between ischemic stroke individuals and elevated TMAO levels. However, the pooled data did NOT support a causal role of TMAO in IS occurrence.[19]

More recently published in Frontiers Neurology, a meta-analysis of 15 studies, including 40,061 patients from China or Germany, concluded that TMAO is an independent risk factor affecting the onset, prognosis, and severity of cerebral infarction.[20]

Choline & TMAO: Foe or Friend?

As we mentioned in the introduction, there is some research disputing TMAO’s impact on cardiometabolic health.

For instance, a large-scale July 2017 meta-analysis that included over 184,000 participants, where there were 18,076 incident CVD events and 5,343 CVD deaths. did NOT see evidence to support an association between dietary choline/betaine with incident CVD.[21]

The authors point out that studies showing a positive association between TMAO and CVD risk increase is typically based on samples from populations at high risk for CVD or in participants who have pre-existing conditions, such as chronic kidney disease.

Another previous study indicates that TMAO has anti-inflammatory effects and improve blood-brain barrier integrity.[22]

More recently, researchers found that plasma levels of choline, but not TMAO, were associated with poor cognition, brain atrophy and other markers of cerebral small vessel disease.[23] Upon closer inspection, the team discovered that only individuals with impaired kidney function showed a link between elevated TMAO levels and an increased risk of dementia.

To further muddy the waters, some research has demonstrated a neuroprotective effect of TMAO.

Keep in mind that TMAO precursors (including choline and betaine) are considered beneficial, if not essential, for the healthy development, maintenance and function of the brain. A 2023 meta-analysis and systematic review including 15 studies with 8357 subjects found that Alpha-GPC did improve neurological function and functional recovery as well as reduced dependency in stroke patients.[24] Interestingly, CDP-Choline (citicoline) did not improve the neurological or functional outcomes in acute stroke patients.

Lastly, a 2023 review published in the International Journal of Molecular Science concluded that “there is no fully conclusive evidence that TMAO is a causal factor of ASCVD [atherosclerotic cardiovascular disease], and, especially whether TMAO induces or just is a marker of hypertension and renal dysfunction requires further study.”[25]

But, things get a bit more complex still…

Choline Sources Impact TMAO Production

As you know, dietary choices have a tremendous impact on how our bodies look, feel, and perform. Research shows that not only is getting adequate daily choline vital, but the source of choline may also play a role, particularly with regard to TMAO production.

A small randomized cross-over study in six healthy men supplied an equivalent amount of choline (550mg/day) from:

• Choline citrate
• Choline bitrate
• Alpha-GPC
• Egg-phosphatidylcholine (PC)

Egg-PC did not rapidly increase TMAO levels whereas the conventional choline supplements did.[26]

It’s worth mentioning that another popular choline supplement, CDP-Choline (citicoline), was not included in this study. Compared to other choline supplements, CDP-Choline is less prone to conversion to TMA and TMAO.[27]

Something else to keep in mind are an alarming pair of recent findings regarding Alpha GPC supplementation. The first study tested the effects of Alpha-GPC on mice as well as human coronary artery endothelial cells and concluded: 

“...our results show GPC promotes atherosclerosis through multiple mechanisms and that caution should be applied when using GPC as a nutritional supplement.”[28]

The researchers then put forth several ways in which Alpha-GPC may promote atherosclerosis:

• Alpha-GPC serves as a substrate for the production of TMAO.
• Alpha-GPC supplementation can shift the gut microbial community by decreasing the relative abundance of short-chain fatty acid (SCFA)-producing bacteria, leading to reduced SCFA production and elevated levels of pro-inflammatory cytokines and chemokines.
• Alpha-GPC can activate MAPK and NFκB signaling in artery endothelial cells, further contributing to the inflammatory dependencies of atherosclerosis

The other key study published in JAMA Network Open, is a 10-year cohort study, including over 12,000,000 men and women aged 50+. It concluded that Alpha-GPC supplementation was significantly associated with a 10-year incident stroke risk in a dose-responsive manner. To be more precise, individuals supplementing with α-GPC vs not supplementing with it had a 46% higher risk of stroke.[29]

The bottom line here is that if you’re being mindful of TMAO levels, then you may want to limit certain choline supplements and look to whole food sources to satisfy your daily choline requirements.

Looking For a Choline-Free Pre Workout?

For the reasons discussed above (and more), Advanced Molecular Labs Pre Workout and its nootropic/productivity supplements (Dopa Rush Cocktail, Dopa Rush Shots, etc.) do NOT contain Alpha GPC (or other choline donors), opting instead for ingredients that support dopamine -- a key neurotransmitter involved in motivation, mood, decision-making, motor control and physical performance.

We’ve addressed the many key roles and benefits of supporting dopamine previously in the AML Articles Section, and we’ll keep doing so as new dopamine research is published.

Most recently, a study in PLoS Biology found that dopamine has “mentalizing abilities” and altering dopamine levels directly affects an individual’s ability to mentalize.[30] Mentalizing is the ability to attribute/understand thoughts, feelings, or intentions in others as well as yourself.

Beyond its cognitive and behavioral impacts, dopamine also plays a key role in the regulation of muscle tone, visual processing, and protein synthesis.[31]

Takeaway

TMAO is a by-product generated by our gut bacteria from certain nutrients, including choline, and carnitine. How much TMAO your gut produces depends on several factors, including diet, age, gender, gut microbiome composition, kidney function, and activity of the liver enzyme flavin monooxygenase.[32]

Based on the current body of evidence, choline obtained from whole foods appears to have less of an impact on raising TMAO levels compared to choline supplements.

If using Alpha GPC or other choline supplements, then you may want to consider supplementing with Berberine, which has been found in some research to attenuate choline-induced atherosclerosis by modifying the gut microbiome such that less TMAO is produced.[33]

We’d also encourage you to adopt the Mediterranean diet, which has been shown in research to have little impact on TMAO concentrations in healthy adults and continues to rank #1 as the best diet for diabetes, weight loss, joint support, and overall health, according to the U.S. News & World Report.[34,35]

So, is choline bad for the Heart & Brain?

Researchers continue to investigate those very issues, and rest assured, we’ll keep you posted of all the latest findings!

^{© Published by Advanced Research Media, Inc. 2024
© Reprinted with permission from Advanced Research Media, Inc.}

References

1. Wang Z., Tang W.H., Buffa J.A., Fu X., Britt E.B., Koeth R.A., Levison B.S., Fan Y., Wu Y., Hazen S.L. Prognostic value of choline and betaine depends on intestinal microbiota-generated metabolite trimethylamine-N-oxide. Eur. Heart J. 2014;35:904–910. doi: 10.1093/eurheartj/ehu002.
2. Tang W.H., Wang Z., Fan Y., Levison B., Hazen J.E., Donahue L.M., Wu Y., Hazen S.L. Prognostic value of elevated levels of intestinal microbe-generated metabolite trimethylamine-N-oxide in patients with heart failure: Refining the gut hypothesis. J. Am. Coll. Cardiol. 2014;64:1908–1914. doi: 10.1016/j.jacc.2014.02.617.
3. Zhu W, Gregory JC, Org E, et al. Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk. Cell. 2016;165(1):111–124. doi:10.1016/j.cell.2016.02.011
4. Senthong V, Wang Z, Li XS, et al. Intestinal Microbiota-Generated Metabolite Trimethylamine-N-Oxide and 5-Year Mortality Risk in Stable Coronary Artery Disease: The Contributory Role of Intestinal Microbiota in a COURAGE-Like Patient Cohort. J Am Heart Assoc. 2016;5(6):e002816. Published 2016 Jun 10. doi:10.1161/JAHA.115.002816
5. Wang Z, Tang WHW, O’Connell T, Garcia E, Jeyarajah EJ, Li XS, Jia X, Weeks TL, Hazen SL. Circulating trimethylamine-N-oxide levels following fish or seafood consumption. Eur J Nutr. 2022 Aug;61(5):2357-2364. doi: 10.1007/s00394-022-02803-4. Epub 2022 Feb 3. PMID: 35113194; PMCID: PMC9283263.
6. Huc T, Drapala A, Gawrys M, Konop M, Bielinska K, Zaorska E, Samborowska E, et al. Chronic, Low-Dose TMAO Treatment Reduces Diastolic Dysfunction and Heart Fibrosis in Hypertensive Rats. American Journal of Physiology-Heart and Circulatory Physiology, 2018; DOI: 10.1152/ajpheart.00536.2018
7. Mueller D.M., Allenspach M., Othman A., Saely C.H., Muendlein A., Vonbank A., Drexel H., Von E.A. Plasma levels of trimethylamine-N-oxide are confounded by impaired kidney function and poor metabolic control. Atherosclerosis. 2015;243:638– doi: 10.1016/j.atherosclerosis.2015.10.091.
8. Lombardo M, Aulisa G, Marcon D, Rizzo G, Tarsisano MG, Di Renzo L, Federici M, Caprio M, De Lorenzo A. Association of Urinary and Plasma Levels of Trimethylamine N-Oxide (TMAO) with Food Nutrients. 2021 Apr 23;13(5):1426. doi: 10.3390/nu13051426. PMID: 33922680; PMCID: PMC8145508.
9. Yang Q, Han H, Sun Z, Liu L, Zheng X, Meng Z, Tao N, Liu J. Association of choline and betaine with the risk of cardiovascular disease and all-cause mortality: Meta-analysis. Eur J Clin Invest. 2023 Oct;53(10):e14041. doi: 10.1111/eci.14041. Epub 2023 Jun 15. PMID: 37318151.
10. Shea JW, Jacobs DR Jr, Howard AG, Lulla A, Lloyd-Jones DM, Murthy VL, Shah RV, Trujillo-Gonzalez I, Gordon-Larsen P, Meyer KA. Choline metabolites and incident cardiovascular disease in a prospective cohort of adults: Coronary Artery Risk Development in Young Adults (CARDIA) Study. Am J Clin Nutr. 2024 Jan;119(1):29-38. doi: 10.1016/j.ajcnut.2023.10.012. Epub 2023 Oct 21. PMID: 37865185; PMCID: PMC10808833.
11. Bao M, Li H, Li J. Circulating trimethylamine-N-oxide is correlated with high coronary artery atherosclerotic burden in individuals with newly diagnosed coronary heart disease. BMC Cardiovasc Disord. 2024 May 21;24(1):265. doi: 10.1186/s12872-024-03937-5. PMID: 38773380; PMCID: PMC11106919.
12. Head SJ, Farooq V, Serruys PW, et al. The SYNTAX score and its clinical implications. Heart 2014;100:169-177.
13. Spasova N, Somleva D, Krastev B, Ilieva R, Borizanova A, Svinarov D, Kinova E, Goudev A. Association of the trimethylamine-N-oxide with cardiovascular risk and vascular alterations in middled-aged patients with risk factors for cardiovascular diseases. Biosci Rep. 2024 Apr 29;44(5):BSR20232090. doi: 10.1042/BSR20232090. PMID: 38669041.
14. Li D, Ke Y, Zhan R, Liu C, Zhao M, Zeng A, Shi X, Ji L, Cheng S, Pan B, Zheng L, Hong H. Trimethylamine-N-oxide promotes brain aging and cognitive impairment in mice. Aging Cell. 2018 Aug;17(4):e12768. doi: 10.1111/acel.12768. Epub 2018 May 10. PMID: 29749694; PMCID: PMC6052480.
15. Tu R, Xia J. Stroke and Vascular Cognitive Impairment: The Role of Intestinal Microbiota Metabolite TMAO. CNS Neurol Disord Drug Targets. 2024;23(1):102-121. doi: 10.2174/1871527322666230203140805. PMID: 36740795.
16. Lizheng Xie, Li Pan, Baiyun Liu, Hongwei Cheng, Xiang Mao. Research progress on the association between trimethylamine/trimethylamine-N-oxide and neurological disorders. Postgraduate Medical Journal. Volume 100, Issue 1183, May 2024, Pages 283-288, https://doi.org/10.1093/postmj/qgad133
17. Dolkar P, Deyang T, Anand N, Rathipriya AG, Hediyal TA, Chandrasekaran V, Krishnamoorthy NK, Gorantia VR, Bishir M, Rashan L, Chang SL, Sakharkar MK, Yang J, Chidambaram SB. Trimethylamine-N-oxide and cerebral stroke risk: A review. Neurobiol Dis. 2024 Mar;192:106423. doi: 10.1016/j.nbd.2024.106423. Epub 2024 Jan 28. PMID: 38286388.
18. Nam HS. Gut microbiota and ischemic stroke: the role of trimethylamine N-oxide. J Stroke. 2019;21(2):151–9.
19. Hu, X., Ren, H. & Cao, Y. The association between trimethylamine N-oxide levels and ischemic stroke occurrence: a meta-analysis and Mendelian randomization study. BMC Neurol 23, 413 (2023). https://doi.org/10.1186/s12883-023-03458-2
20. Wang L, Nan Y, Zhu W, Wang S. Effect of TMAO on the incidence and prognosis of cerebral infarction: a systematic review and meta-analysis. Front Neurol. 2024 Jan 8;14:1287928. doi: 10.3389/fneur.2023.1287928. PMID: 38259655; PMCID: PMC10801906.
21. Meyer KA, Shea JW. Dietary Choline and Betaine and Risk of CVD: A Systematic Review and Meta-Analysis of Prospective Studie Nutrients. 2017 Jul 7;9(7):711. doi: 10.3390/nu9070711. PMID: 28686188; PMCID: PMC5537826.
22. Hoyles L, Pontifex MG, Rodriguez-Ramiro I, Anis-Alavi MA, Jelane KS, Snelling T, Solito E, Fonseca S, Carvalho AL, Carding SR, et al. Regulation of blood-brain barrier integrity by microbiome-associated methylamines and cognition by trimethylamine-n-oxide. Microbiome 2021, 9, 235.
23. Yaqub A, Vojinovic D, Vernooij MW, Slagboom PE, Ghanbari M, Beekman M, van der Grond J, Hankemeier R, van Duijn CM, Ikram MA, Ahamad S. Plasma trimethylamine-n-oxide (TMAO): associations with cognition, neuroimaging, and dementia. Alzheimers Res Ther. 2024 May 20;16(1):113. doi: 10.1186/s13195-024-01480-1. PMID: 38769578; PMCID: PMC11103865.
24. Sagaro GG, Amenta F. Choline-Containing Phospholipids in Stroke Treatment: A Systematic Review and Meta-Analysis. J Clin Med. 2023 Apr 14;12(8):2875. doi: 10.3390/jcm12082875. PMID: 37109211; PMCID: PMC10143951.
25. Canyelles M, Borras C, Rotllan N, Tondo M, Escola-Gil JC, Blanco-Vaca F. Gut Microbiota-Derived TMAO: A Causal Factor Promoting Atherosclerotic Cardiovascular Disease? Int J Mol Sci. 2023 Jan 18;24(3):1940. doi: 10.3390/ijms24031940. PMID: 36768264; PMCID: PMC9916030.
26. Bockmann KA, Franz AR, Minarski M, Shunova A, maiwald CA, Schwarz J, Gross M, Poets CF, Bernhard W. Differential metabolism of choline supplements in adult volunteers. Eur J Nutr. 2022 Feb;61(1):219-230. doi: 10.1007/s00394-021-02637-6. Epub 2021 Jul 21. PMID: 34287673; PMCID: PMC8783899.
27. Synoradzki K, Grieb P. Citicoline: A Superior Form of Choline?. Nutrients. 2019;11(7):1569. Published 2019 Jul 12. doi:10.3390/nu11071569
28. Wang Z, Hazen J, Jia X, et al. The Nutritional Supplement L-Alpha Glycerylphosphorylcholine Promotes Atherosclerosis. Int J Mol Sci. 2021;22(24):13477. Published 2021 Dec 15. doi:10.3390/ijms222413477
29. Lee G., Choi S., Chang J., Choi D., Son J.S., Kim K., Kim S.M., Jeong S., Park S.M. Association of L-alpha Glycerylphosphorylcholine With Subsequent Stroke Risk After 10 Years. JAMA Netw. Open. 2021;4:e2136008. doi: 10.1001/jamanetworkopen.2021.36008
30. Schuster BA, Sowden S, Rybicki AJ, Fraser DS, Press C, Hickman L, Holland P, Cook JL. Disruption of dopamine D2/D3 system function impairs the human ability to understand the mental states of other people. PLoS Biol. 2024 Jun 13;22(6):e3002652. doi: 10.1371/journal.pbio.3002652. PMID: 38870319; PMCID: PMC11175582.
31. Roelands B, Hasegawa H, Watson P, Piacentini MF, Buyse LD, Schutter G, Meeusen RR. The effects of acute dopamine reuptake inhibition on performance. Med Sci Sports Exerc 2008;40:879–85
32. Gatarek P, Kałużna-Czaplińska J. Trimethylamine N-oxide (TMAO) in human health. EXCLI J. 2021 Feb 11;20:301-319. doi: 10.17179/excli2020-3239. PMID: 33746664; PMCID: PMC7975634.
33. Li X, Su C, Jiang Z, et al. Berberine attenuates choline-induced atherosclerosis by inhibiting trimethylamine and trimethylamine-N-oxide production via manipulating the gut microbiome. NPJ Biofilms Microbiomes. 2021;7(1):36. Published 2021 Apr 16. doi:10.1038/s41522-021-00205-8
34. Griffin LE, Djuric Z, Angiletta CJ, Mitchell CM, Baugh ME, Davy KP, Neilson AP. A Mediterranean diet does not alter plasma trimethylamine N-oxide concentrations in healthy adults at risk for colon cancer. Food Funct. 2019 Apr 1;10(4):2138-2147. doi: 10.1039/c9fo00333a. Epub 2019 Apr 2. PMID: 30938383; PMCID: PMC6552673.
35. https://www.usnews.com/info/blogs/press-room/articles/2024-01-03/u-s-news-reveals-the-2024-best-diets