Jacob Schor ND, FABNO

www.DenverNaturopathicOncology.com

May 17, 2019

A paper published in February 2019 has me rethinking my opinion about eggs and leading me down a rabbit hole of recent research trying to understand how we might better prevent cardiovascular disease.  Eggs were originally singled out years ago as a prime suspect in causing heart disease because they contain large amounts of dietary cholesterol.  But then they were cleared of blame as researchers repeatedly failed to find a positive association between eggs eaten and disease incidence. Now a new study by Victor Zhong et al has again cast suspicion on eggs.  

I originally posted an article to our website suggesting that eggs posed little risk of cardiovascular disease (CVD) in April 2005, over a dozen years ago:  http://www.denvernaturopathic.com/news/eggs.html

I wrote a second article that was published in the Natural Medicine Journal in July 2018 reviewing the research by Qin et al, which actually concluded that egg consumption was associated with a significantly lower risk of cardiovascular disease. [1]

https://www.naturalmedicinejournal.com/journal/2018-07/egg-consumption-may-lower-risk-cardiovascular-disease

This new Zhong study contradicts the earlier information that we thought had answered this question with a degree of certainty.  In the Zhong study, data was pooled from half a dozen prospective cohort studies done in the United States with a median follow up of 17.5 years. Taken together, there were 29,615 adults followed in their research.Each addition of 300 mg of dietary cholesterol eaten per day, that’s about two eggs worth, significantly raised CVD risk by 17%. This same amount of cholesterol increased risk of all-cause mortality by 18%.[2]  This is not what we were expecting to read.

What should we do with this information?  We could just ignore it and assume it’s wrong.  Observational studies such as those used to source these data are prone to error. The data on egg consumption was only gathered at the start of the studies by having participants fill out questionnaires. Such data are less accurate than most researchers like, and inaccurate starting data could have led to inaccurate outcome results.  That would be the easy way to deal with ideas we don’t like.  Another approach is to reconsider our starting assumptions and see if they still seem reasonable.  

The initial idea that dietary cholesterol consumption was linked with heart disease risk came out of Framingham Study.  Eggs are an excellent source of dietary cholesterol and so were early on identified as suspects that could increase risk of cholesterol deposits leading to plaque formation.  Eggs are also an excellent source of dietary choline, in particular phosphatidyl choline but no one was thinking about choline back then.  The role phosphatidyl choline and dietary choline play in cardiovascular disease has only recently begun to be appreciated. 

In 2009 two Swedish scientists, de Faire and Frostegård, reported that they had studied the immunoglobulins that target phosphatidylcholine and their association with CVD.  Such immunoglobulins are protective for individuals with high blood pressure reducing  their risk of heart disease.  At the time, these Swedes were experimenting with what we might describe as phosphatidylcholine vaccines to prevent or treat atherosclerotic plaques.  [3]  By 2010 deFaire’s research was suggesting that measuring these anti-PC immunoglobulins might be predictive of risk; people in the lowest quartile of anti-PC antibodies were at nearly double the risk for heart disease of those in the highest quartile.  Did I get that right?  The antibodies are good because they lower PC.[4]  As a side note, low levels of PC antibodies also increase risk of stroke.[5]

Skip ahead a handful of years and the mechanism of action to explain phosphatidylcholine’s role in increasing CVD is clearly established. The explanation is that bacteria in the large bowel convert phosphatidylcholine (and also the choline containing compounds betaine and l-carnitine) into trimethylamine (TMA), which is then oxidized in the liver to trimethylamine n-oxide (TMAO).  This chemical TMAO has a pronounced atherosclerotic effect. [6 ][7]  The degree phosphatidylcholine (and also betaine and l-carnitine) are converted  to TMA and eventually TMAO varies with the types of bacteria living in the colon.

Eggs are our highest dietary source of choline, about 126 mg/egg.  Other foods also high in choline include peanuts, beets, meat and fish.[8]  Few of us would group these together as heart risky foods and we could imagine that studies that  examined dietary data for disease associations would have easily overlooked choline intake and failed to associate levels with risk.  

The obvious assumption would be that cutting back on dietary choline and other substrates for TMAO would lower CVD risk.  Given that choline is an essential nutrient we have to wonder whether reducing dietary intake would be worth the risk of deprivation and possible deficiency. More important it is unclear whether choline restriction will even have the desired effect reducing TMAO levels.

There is another source of phosphatidylcholine that gives me pause as I read these studies. Many of the liposomal encapsulated supplements use phosphatidylcholine to form the micellular nano-particles.  My favorite brand of curcumin certainly does, and I’ve taken this myself, not to mention sold a fair bit to patients.  These liposomal capsules deliver daily levels of phosphatidylcholine that are no different from what a person obtains from eating a dozen eggs a week.   We frequently have suggested l-carnitine and beet powder to patients with heart problems.  Could we have done a disservice to patients by inadvertently increasing their risk for CVD?  

So far, the research hasn’t supported such concerns; choline depletion doesn’t appear to lower TMAO levels.  Lemos et al in their 2018 study found that dietary choline has little impact; TMAO levels remained the same in study participants who ate 3 eggs, or took 400 mg of choline a day, compared to people who received no choline. [9]A 2017 meta-analysis that looked at dietary choline and CVD did not find any association with disease or mortality. The authors had identified six studies, comprising 18,076 incident CVD events, 5343 CVD deaths, and 184,010 total participants. Incident CVD was not associated with choline or betaine intake.  A separate 2017 trial found that eating 3 eggs a day, while it did increase plasma choline, still did not affect TMAO levels. [10]  Thus, while the idea that eating eggs or swallowing those liposomal phosphatidylcholine containing supplements might increase TMAO seems logical, the evidence suggests we need not worry, at least for the moment. [11]   It’s all about the colonic bacteria and whether they convert the choline to TMA or not.

 Various diet, nutrient and drug approaches to block the bacterial fermentation processes that produce TMA and then TMAO are being considered. [12][13]  Interventions as simple as caloric restriction in combination with exercise reduce TMAO production. Reducing visceral adiposity also reduces production. [14]A day long water fast drops TMAO levels by almost half. [15]Reducing red meat consumption lowers TMAO production, though this may be the result of reduced l-carnitine, another precursor to bacterial production, or just as likely, it shifts bacterial populations. [16]  Two contradictory papers published in Spring 2019 informed us that adherence to a Mediterranean style diet may [17]  or may not lower TMAO. [18] 

 The bottom line is that TMAO concentrations seem to be little changed by what someone eats. [19]  Egg, fish or meat consumption did not affect TMAO in one study, though dairy still may increase it.  [20]

At the same time, nothing in the current literature suggests that we should discount the phosphatidylcholine-TMAO connection to heart disease.  In fact, each new publication makes the theory all the more compelling. 

A 2018 meta-analysis reported a significant association between TMAO levels and cardiac events and overall mortality. Higher circulating TMAO was associated with a 23% higher risk of CVEs [cardiac vascular events] … and a 55% higher risk of all-cause mortality …”[21]  

 A January 2019 Chinese study comparing urinary TMAO levels reported patients in the upper quartile were nearly twice as likely to suffer from coronary heart disease than patients in the lower quartile.  Diabetic patients whose TMAO was above the mean group were 6-fold more likely to suffer from coronary heart disease than those below the mean.  [22]

TMAO impacts a range of other conditions including cancer and diabetes. [23]  The consensus is that if this pathway can be modulated, we could see wide ranging benefits. Modulation of the pathway should be possible through shifting the gut biome.

Granted that the impact of the gut microbiome is the fad of the moment and is now getting blamed for all things that might ail humans.  In the case of CVD, this is possibly true; the biome research published in the last few years is compelling.  

Specific families of bacteria are now associated with both higher heart disease risk and other families of bacteria are associated with lower risk.  Some of the data are troubling as they don’t support our routine generalizations of which bacteria are good or bad; some of our probiotic favorites such as Lactobacilliand Bifidobacteriaare associated with increased heart disease risk. [24]  [and while you might think so, that was not a typo.]. Obscure bacteria such as

Bacteroides vulgatusand Bacteroides doreiare associated with lower risk. These are not sold at your local health food store.  Cardiac patients who consume ‘resistant starches’ do however increase levels.  [25] [26]

Fecal transplantation in mice can transfer atherosclerotic propensities.[27]  Antibiotic treatments have then countered these tendencies.  Recent research has sought to identify bacteria species that might serve as probiotics to lower conversion of choline to trimethylamine n-oxide. [28][29]

[****I don’t know where to put this but it’s interesting:  There is a food eaten in Greenland that is made by fermentation of shark meat.  This Greenland Shark’s flesh is toxic because it contains exceptionally high TMAO levels (and also triethylamine (TMA) that occur naturally in the animal.  Yet “hákarl” the traditional fermented shark meat preparation eaten in Greenland is apparently safe for humans; the fermentation process reduces the toxic levels of these constituents rendering the shark meat safe.[30]  Scientific details aside, I suspect this food is still an acquired taste.]

The Cleveland Heart Lab now tests for trimethylamine n-oxide (TMAO). [31]  ARUP Laboratories offers testing for phosphatidylcholine antibodies.  [32]  With easy testing we see a growing list of studies that test various interventions that impact TMAO.

Nora Kalagi and colleagues in Australia conducted a systematic review of these trials, which was published in May 2019. “A spectrum of antibiotics and other therapeutic strategies have been employed to test their potential to modulate TMAO concentrations, assuming the gut microbiome to be the key source of TMAO.” Their paper provides the most comprehensive list of potential interventions seen to date.  [33]

What’s fascinating is that much of the findings contradict our starting assumptions of what is good and what is bad for heart disease. 

Three studies have assessed the effect of taking metformin on TMAO levels and reported inconsistent results.  TMAO levels increased in two studies, Huo in 2009 and Cadedduet al in 2013. [34][35] Yet metformin had no effect for Velebova et al’s study in 2016. [36]

Because they shut down bacterial conversion, antibiotics certainly lower TMAO, at least in the short term.  In one study, TMAO levels increased in forty healthy subjects who ate two hard boiled eggs per day but when the subjects were pretreated for one week with a combination of metronidazole and ciprofloxacin, TMAO was undetectable when the same egg challenge was repeated.  [37]  There’s a U.S. patent for using enteric coated aspirin accompanied by a claim that it significantly reduces TMAO levels in study participants also eating 2 hard boiled eggs per day. [38]  In these few studies, the common denominator is that eating hard boiled eggs is a bad idea.

A January 2019 publication in Nutrients reported that a grape pomace, which supplies resveratrol, lowered TMAO levels in humans by about 10% compared to placebo.[39]

Another study published in the same issue reported that a fermented apple pomace reduced TMAO by 63%. [40]

Oral berberine, long employed to shift gut biota, reduces TMAO production in mice [41]  and also increases Akkermansia levels [42].  Specific strains of lactobacilli plantarumhave been reported to reduce TMAO production also in mice.  [43]  Treating aging mice with antibiotics lowered their gut microbiome populations, reduced TMAO levels and improved endothelial function. [44]    Inulin has been tried but failed to reduce TMAO levels in “… sedentary, overweight/obese adults at risk for T2DM” [fat human pre-diabetics]. [45]  Eating a Mediterranean Diet also failed to change TMAO production [46]

If dietary intake of choline has less impact on TMAO production than what types of bacteria are fermenting the choline, then perhaps worrying about dietary choline is wasted effort.  

What one eats today may have little impact on TMAO production compared to what you were eating yesterday and the weeks leading up to the ‘study.’   Long term dietary patterns may shift the gut biome. For example, a study published this past January fed radioactive tagged l-carnitine, (l-carnitine is also linked to increased TMAO levels via bacterial fermentation as choline is) to 32 vegetarians and 40 omnivores.  “The transformation into atherosclerotic compounds occurred in both groups but occurred to a “markedly lower extent, in vegans/vegetarians.”[47]  

So back to eggs.  The truth is I don’t know if we should worry about them or not.  In a trial of fifty people by Missimer et al published in 2018, eating two eggs per day was more effective than oatmeal at lowering CVD markers and made no difference in TMAO levels. [48]

Even if TMAO is ultimately proven a significant problem, to date we aren’t seeing strong evidence that getting excessive dietary choline makes a difference.  If I had to make a guess, my prediction is that certain people because of their gut biome have a greater tendency to produce TMAO and for them, the unlucky among us, choline and phosphatidylcholine consumption might play an undesirable role.  How should we identify them? Perhaps by testing.  We could use TMAO serum levels to screen and identify patients who might benefit from choline restriction and then watch individually if restriction lowers their TMAO.  We also might try ‘rearranging’ their gut biome and see if that has an effect.  Should we worry about eggs, liposomal supplements, or for that matter, l-carnitine or even beets?  Perhaps, but at this point there are plenty of better things that deserve our attention.

In an ideal world, researchers will identify a strain of bacteria that lowers TMAO production and then really clever individuals will realize that these same bacteria are already present in some desirable food stuff, for example in blue cheese, and our problems will be solved.  Add that to my list of ideal scenarios in the ideal world of my imagination.


[1]Qin C, Lv J, Guo Y, et al; The China Kadoorie Biobank Collaborative Group. Associations of egg consumption with cardiovascular disease in a cohort study of 0.5 million Chinese adults [published online ahead of print May 21, 2018]. Heart.

[2]Zhong VW1, Van Horn L1, Cornelis MC1, et al.  Associations of Dietary Cholesterol or Egg Consumption With Incident Cardiovascular Disease and Mortality. JAMA. 2019 Mar 19;321(11):1081-1095. 

[3]de Faire U1, Frostegård J. Natural antibodies against phosphorylcholine in cardiovascular disease. Ann N Y Acad Sci. 2009 Sep;1173:292-300. 

[4]de Faire U, Su J, Hua X, Frostegård A, et al. Low levels of IgM antibodies to phosphorylcholine predict cardiovascular disease in 60-year old men: effects on uptake of oxidized LDL in macrophages as a potential mechanism. J Autoimmun. 2010 Mar;34(2):73-9. 

[5]Sjöberg BG, Su J, Dahlbom I, et al. Low levels of IgM antibodies against phosphorylcholine-A potential risk marker for ischemic stroke in men. Atherosclerosis. 2009 Apr;203(2):528-32. 

[6]Senthong V, Li XS, Hudec T, et al. Plasma trimethylamine n-oxide, a gut microbe-generated phosphatidylcholine metabolite, is associated with atherosclerotic burden. J. Am. Coll. Cardiol. 2016;67:2620–2628.

[7]Wang Z, Klipfell E, Bennett BJ, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011 Apr 7;472(7341):57-63. 

[8]https://lpi.oregonstate.edu/mic/other-nutrients/choline

[9]Lemos B1, Medina-Vera I, Malysheva OV, Caudill MA, Fernandez ML. Effects of Egg Consumption and Choline Supplementation on Plasma Choline and Trimethylamine-N-Oxide in a Young Population. J Am Coll Nutr. 2018 May 15:1-8. 

[10]DiMarco DM, Missimer A, Murillo AG, et al. Intake of up to 3 Eggs/Day Increases HDL Cholesterol and Plasma Choline While Plasma Trimethylamine-N-oxide is Unchanged in a Healthy Population. Lipids. 2017 Mar;52(3):255-263. 

[11]Meyer KA, Shea JW. Dietary Choline and Betaine and Risk of CVD: A Systematic Review and Meta-Analysis of Prospective Studies. Nutrients. 2017 Jul 7;9(7). pii: E711. 

[12]Kalagi NA, Abbott KA, Alburikan KA, et al. Modulation of Circulating Trimethylamine N-Oxide Concentrations by Dietary Supplements and Pharmacological Agents: A Systematic Review. Adv Nutr. 2019 May 10. pii: nmz012. 

[13]Kuka J, Liepinsh E, Makrecka-Kuka M, et al. Suppression of intestinal microbiota-dependent production of pro-atherogenic trimethylamine N-oxide by shifting L-carnitine microbial degradation. Life Sci. 2014 Nov 11;117(2):84-92. 

[14]Erickson ML, Malin SK, Wang Z, Brown JM, Hazen SL, Kirwan JP. Effects of Lifestyle Intervention on Plasma Trimethylamine N-Oxide in Obese Adults. Nutrients. 2019 Jan 16;11(1). pii: E179. 

[15]Washburn RL, Cox JE, Muhlestein JB, et al. Pilot Study of Novel Intermittent Fasting Effects on Metabolomic and Trimethylamine N-oxide Changes During 24-hour Water-Only Fasting in the FEELGOOD Trial. Nutrients. 2019 Jan 23;11(2). pii: E246. 

[16]Wang Z, Bergeron N, Levison BS, et al. Impact of chronic dietary red meat, white meat, or non-meat protein on trimethylamine N-oxide metabolism and renal excretion in healthy men and women. Eur Heart J. 2019 Feb 14;40(7):583-594. 

[17]Barrea L, Annunziata G, Muscogiuri G, et al. Trimethylamine N-oxide, Mediterranean diet, and nutrition in healthy, normal-weight adults: also a matter of sex? Nutrition. 2019 Jun;62:7-17. 

[18]Griffin LE, Djuric Z, Angiletta CJ et al. 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. 

[19]Kühn T, Rohrmann S, Sookthai D, et al. Intra-individual variation of plasma trimethylamine-N-oxide (TMAO), betaine and choline over 1 year. Clin Chem Lab Med. 2017 Feb 1;55(2):261-268. doi: 10.1515/cclm-2016-0374.

[20]Rohrmann S, Linseisen J, Allenspach M, von Eckardstein A, Müller D. Plasma Concentrations of Trimethylamine-N-oxide Are Directly Associated with Dairy Food Consumption and Low-Grade Inflammation in a German Adult Population. J Nutr. 2016 Feb;146(2):283-9. 

[21]Qi J, You T, Li J, Pan T, Xiang L, Han Y, Zhu L Circulating trimethylamine N-oxide and the risk of cardiovascular diseases: a systematic review and meta-analysis of 11 prospective cohort studies. J Cell Mol Med. 2018 Jan;22(1):185-194. 

[22]Yu D, Shu XO, Rivera ES, et al. Urinary Levels of Trimethylamine-N-Oxide and Incident Coronary Heart Disease: A Prospective Investigation Among Urban Chinese Adults. J Am Heart Assoc. 2019 Jan 8;8(1):e010606. 

[23]Oellgaard J, Winther SA, Hansen TS, Rossing P, von Scholten BJ. Trimethylamine N-oxide (TMAO) as a New Potential Therapeutic Target for Insulin Resistance and Cancer. Curr Pharm Des. 2017;23(25):3699-3712. 

[24]Yamashita T, Emoto T, Sasaki N, Hirata KI. Gut Microbiota and Coronary Artery Disease. Int Heart J. 2016 Dec 2;57(6):663-671. 

[25]Yoshida N, Emoto T, Yamashita T, et al. Bacteroides vulgatus and Bacteroides dorei Reduce Gut Microbial Lipopolysaccharide Production and Inhibit Atherosclerosis. Circulation. 2018 Nov 27;138(22):2486-2498. 

[26]Yoshida N, Sasaki K, Sasaki D, et al. Effect of Resistant Starch on the Gut Microbiota and Its Metabolites in Patients with Coronary Artery Disease. J Atheroscler Thromb. 2018 Dec 27. 

[27]Gregory JC, Buffa JA, Org E, et al. Transmission of atherosclerosis susceptibility with gut microbial transplantation. J Biol Chem. 2015 Feb 27;290(9):5647-60. 

[28]Ramezani A, Nolin TD, Barrows IR, et al. Gut Colonization with Methanogenic Archaea Lowers Plasma Trimethylamine N-oxide Concentrations in Apolipoprotein e-/- Mice. Sci Rep. 2018 Oct 3;8(1):14752. 

[29]Qiu L, Tao X, Xiong H, Yu J, Wei H. Lactobacillus plantarum ZDY04 exhibits a strain-specific property of lowering TMAO via the modulation of gut microbiota in mice. Food Funct. 2018 Aug 15;9(8):4299-4309. 

[30]Osimani A, Ferrocino I, Agnolucci M, et al. Unveiling hákarl: A study of the microbiota of the traditional Icelandic fermented fish. Food Microbiol. 2019 Sep;82:560-572. 

[31]http://www.clevelandheartlab.com/blog/horizons-tmao-testing-a-new-way-to-assess-heart-attack-and-stroke-risk/

[32]http://ltd.aruplab.com/Tests/Pub/3001549

[33]Kalagi NA, Abbott KA, Alburikan KA, Alkofide HA, Stojanovski E, Garg ML. Modulation of Circulating Trimethylamine N-Oxide Concentrations by Dietary Supplements and Pharmacological Agents: A Systematic Review. Adv Nutr. 2019 May 10. pii: nmz012. 

[34]Cadeddu C, Deidda M, Nocco S, et al. Effects of metformin treatment on myocardial and endothelial functioning in insulin resistance patients: a metabolomic study. J Diabetes Metab 2013;4:302–4.

[35]Huo T, Cai S, Lu X, Sha Y, Yu M, Li F. Metabonomic study of biochemical changes in the serum of type 2 diabetes mellitus patients after the treatment of metformin hydrochloride. J Pharm Biomed Anal 2009;49(4):976–82.

[36]Velebova K, Hoang T, Veleba J, et al. The effect of metformin on serum levels of trimethylamine-N-oxide in patients with type 2 diabetes/prediabetes and chronic heart failure. In Diabetologia, 2016, 59, SS533–SS533.

[37]Tang WW, Wang Z, Levison BS, et al.  Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013;368(17):1575–84.

[38]Hazen SL, inventor; Cleveland Clinic Foundation, assignee. Treating and preventing disease with TMA and TMAO lowering agents. United States patent application US 14/866, 375. 2016.

http://www.freepatentsonline.com/y2019/0038642.html

[39]Annunziata G, Maisto M, Schisano C, et al. Effects of Grape Pomace Polyphenolic Extract (Taurisolo®) in Reducing TMAOSerum Levels in Humans: Preliminary Results from a Randomized, Placebo-Controlled, Cross-Over Study. Nutrients. 2019 Jan 10;11(1). pii: E139. 

[40]Tenore GC, Caruso D, Buonomo G, et al. ed Annurca Apple Puree as a Functional Food Indicated for the Control of Plasma Lipid and Oxidative Amine Levels: Results from a Randomised Clinical Trial. Nutrients. 2019 Jan 9;11(1). pii: E122. 

[41]Shi Y, Hu J, Geng J, et al.  Berberine treatment reduces atherosclerosis by mediating gut microbiota in apoE-/- mice. Biomed Pharmacother. 2018 Nov;107:1556-1563. 

[42]Zhu L, Zhang D, Zhu H, et al. Berberine treatment increases Akkermansia in the gut and improves high-fat diet-induced atherosclerosis in Apoe-/- mice. Atherosclerosis. 2018 Jan;268:117-126. 

[43]Qiu L, Tao X , Xiong H, Yu J , Wei H . Lactobacillus plantarum ZDY04 exhibits a strain-specific property of lowering TMAO via the modulation of gut microbiota in mice. Food Funct. 2018 Aug 15;9(8):4299-4309. 

[44]Brunt VE, Gioscia-Ryan RA, Richey JJ, et al. Suppression of the gut microbiome ameliorates age-related arterial dysfunction and oxidative stress in mice. J Physiol. 2019 May;597(9):2361-2378. 

[45]Baugh ME, Steele CN, Angiletta CJ, et al. Inulin Supplementation Does Not Reduce Plasma Trimethylamine N-Oxide Concentrations in Individuals at Risk for Type 2 Diabetes. Nutrients. 2018 Jun 20;10(6). pii: E793. 

[46]Griffin LE, Djuric Z, Angiletta CJ, et al. 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. 

[47]Koeth RA, Lam-Galvez BR4, Kirsop J, Wang Z, et al. l-Carnitine in omnivorous diets induces an atherogenic gut microbial pathway in humans. J Clin Invest. 2019 Jan 2;129(1):373-387. 

[48]Missimer A, Fernandez ML, DiMarco DM, et al. Compared to an Oatmeal Breakfast, Two Eggs/Day Increased Plasma Carotenoids and Choline without Increasing Trimethyl Amine N-Oxide Concentrations. J Am Coll Nutr. 2018 Feb;37(2):140-148.