February 2020 Issue

Choline and CVD
By Carrie Dennett, MPH, RDN, CD
Today’s Dietitian
Vol. 22, No. 2, P. 36

Is there truth to the controversial claim that this essential nutrient puts heart health at risk?

Choline is an essential nutrient that plays a critical role in several biological processes, but one byproduct of its metabolism is a molecule that’s been receiving much negative attention—trimethylamine N-oxide (TMAO). Since TMAO was first reported as having a link to CVD in 2011, it’s been the focus of hundreds of research studies. However, it remains unclear whether TMAO is a causal link between diet and CVD, or whether it’s simply a possible marker of underlying disease.

Because the precursors of TMAO synthesis, including choline, have many health benefits, suggestions that people should limit dietary sources of choline, including eggs and meat, to prevent CVD may be premature. It seems that every day, dietitians learn more about the complex interconnections between nutrition, genes, and the gut microbiota in affecting health. This is one of those cases.

Choline and Its Health Benefits
Despite the liver’s ability to synthesize choline, people need dietary choline as well, so the Institute of Medicine recognized choline as an essential nutrient in 1998.1 There’s no Dietary Reference Intake for choline, but the daily Adequate Intake (AI) for adults is 425 mg for women aged 19 and older, 450 mg during pregnancy, and 550 mg for men and lactating women.2 Recommendations are higher during pregnancy and lactation because choline is important for normal fetal and early childhood development, including reducing the risk of several birth defects linked to choline deficiency. Low maternal choline intake and plasma choline levels are associated with higher risk of neural tube defects, even when intake of dietary folate or supplemental folic acid is adequate.3,4 In addition, several randomized controlled trials have shown a beneficial effect of maternal choline supplementation on the brain health of offspring.1

Animal foods, especially eggs, beef, chicken, fish, and milk are the richest sources of choline. What about plant-based eaters? Cruciferous vegetables, certain beans, nuts, seeds, and whole grains also contribute choline to the diet.1,2 In spite of that variety, it’s estimated that only 11% of Americans reach the AI.5 That’s concerning, because choline plays a role in the epigenetic regulation of gene expression,6,7 is a precursor for synthesis of the neurotransmitter acetylcholine, and is a component of the major phospholipids in cell membranes.1,3 Since choline is essential for transporting lipids from the liver, choline deficiency can lead to nonalcoholic fatty liver disease (NAFLD), which may be reversible with choline repletion through supplementation.3,7 Although most women of childbearing age are resistant to NAFLD because of their high estrogen levels, at least 40% have a polymorphism that removes this protection, so adequate consumption of dietary choline is important.8

So where does TMAO enter the picture? When humans ingest foods containing choline or carnitine, an amino acid derivative, it provides certain gut microbes with the materials to produce trimethylamine (TMA), which then enters the portal circulation and travels to the liver, where enzymes oxidize TMA to TMAO.9 Most TMAO is excreted in the urine within 24 hours, about one-half of it unchanged, and about one-half being converted to TMA then back to TMAO in the liver.10

About one-third of choline in food is free choline, which gut microbes prefer. Most dietary choline is in the form of the phospholipid phosphatidylcholine, which isn’t an efficient TMA precursor.10 Eggs are rich in choline, while beef is rich in both choline and carnitine.11 However, most of the choline in eggs isn’t free choline but is bound up as part of lecithin, and lecithin also is a weak precursor for TMA.10 Notably, fish and seafood contain preformed TMAO, which protects enzymes in the fish from salt water.11

TMAO: Marker, Causal Factor, or Something Else?
Elevated TMAO levels are associated with CVD—especially atherosclerosis—and other poor health outcomes, including the development or progression of hypertension, diabetes, and renal failure, but is TMAO a causative factor or simply a marker of disease?12 The origins of this question come from a 2011 study from the Cleveland Clinic, which found an association between elevated fasting plasma levels of TMAO and choline and several types of CVD. The associations held even after adjustment for traditional cardiac risk factors and medications.13

While other studies have found associations between CVD and choline, TMAO, or both, some have not. For example, large population studies in Japan, the Netherlands, and the United States, including the Atherosclerosis Risk in Communities study, found no link between dietary choline intake and CVD. Unfortunately, these studies didn’t also measure circulating TMAO levels.14 A 2017 systematic review and meta-analysis in the Journal of the American Heart Association did find that high TMAO levels were a much stronger predictor of cardiovascular events than were elevated levels of its nutrient precursors, including choline.15 Proposed mechanisms include effects on cholesterol, the hormone angiotensin II—which can raise blood pressure—and increased platelet clumping, possibly leading to blood clots.16,17

“In my view, I don’t think they have very good evidence that TMAO is a causative element in the disease process,” says Marie Caudill, PhD, RD, a professor and researcher in the division of nutritional sciences at Cornell University in Ithaca, New York. “We’re at association at this point. It’s so murky. Association isn’t causation, but we seem to go down that road anyway.”

The 2011 Cleveland Clinic study also added choline or TMAO to the diets of a species of mice that were at elevated risk of developing atherosclerosis, and found that the mice in fact developed atherosclerosis.13 However, this species of mice lacks the gene responsible for the protein that drives reverse cholesterol transport, Caudill says. Reverse cholesterol transport is a process that removes excess cholesterol from the tissues and delivers it to the liver, where it can be excreted or recycled. “They’re looking at an animal model that doesn’t express all the genes humans do,” she says. “When you add that gene back in, TMAO has no adverse effect.” she says.

One headline-grabbing detail of some TMAO studies is that vegans and vegetarians produce far less TMAO after consuming its precursor nutrients than do omnivores.18 For example, a 2017 study published in Circulation found that oral choline supplementation of approximately 450 mg choline per day for two months—an amount far above the AI when added to an omnivore diet—led to an increase in fasting blood TMAO levels and a corresponding increase in clumping of blood platelets. These increases were more pronounced in omnivores than they were in vegans and vegetarians and were attenuated by aspirin therapy, possibly because aspirin alters the gut microbe community and interferes with TMAO production.19 What’s notable is that the percentage of platelet clumping varied widely within each diet group after supplementation, suggesting that a third factor led to differences between individuals.

In an editorial in AME Medical Journal, Caudill stressed that supplemental choline was a more potent substrate for TMA/TMAO than the phosphatidylcholine from food. Furthermore, the study couldn’t measure hard disease endpoints.20 “The problem with surrogate markers is they don’t predict death,” Caudill says. “At the end of the day, what’s important is, does this reduce risk of disease or death?”

A Function of Kidney Function?
Blood TMAO level depends on several factors, including diet, the composition and function of the gut microbiota, permeability of the gut-blood barrier—which controls bacterial metabolites’ access to the bloodstream—genetic regulation of liver enzymes, and how fast we can excrete TMAO in our urine.16

Lowered excretion due to impaired kidney function may have played a role in the results of a 2013 study that fed 40 healthy adults two large hard-boiled eggs, which contained about 250 mg choline, along with 250 mg supplemental choline that was marked so it could be tracked in the body after ingestion. Blood and urine levels of both total TMAO and marked TMAO rose after consumption, although after eight hours total TMAO levels were lower than they were before the challenge. Interestingly, after administering antibiotics and repeating the challenge, TMAO levels didn’t rise, which speaks to the role of the gut microbiota in TMAO production. Also notable? While average TMAO levels were associated with cardiovascular events, they were inversely associated with glomerular filtration rate (GFR). In other words, people with impaired kidney function had trouble clearing TMAO from their systems.21

Caudill says this is a critical detail in TMAO association studies, because most people older than age 55 to 60 are going to have some subclinical kidney impairment. “If your kidneys are slightly compromised, that’s going to lead to an elevation in TMAO.”12 Kidney function aside, another potential wrinkle in association studies is that some research suggests the wrong player in the choline-TMA-TMAO cycle is being targeted. A 2019 study assessed TMA and TMAO levels in both healthy adults and cardiac patients—unusual, since the studies that have found associations between TMAO and increased cardiovascular risk generally haven’t measured TMA levels. They found that the cardiac patients had TMA levels twice as high as levels in the healthy subjects, and that those levels were inversely correlated with estimated GFR, again suggesting that impaired kidney function plays a role. Interestingly, despite the significantly higher TMA levels, the cardiac patients only had slightly higher TMAO levels that didn’t reach statistical significance. The authors also found that TMA, but not TMAO, had biological effects that could harm the circulatory system—specifically, damaging the cells responsible for contracting the heart—and that treatment with TMAO actually protected these cells from damage.22 Moreover, many studies have found other protective functions of TMAO, including keeping cells from losing volume under stress.16,17,23

Genetic and Microbial Influences
Despite many researchers finding associations between TMAO levels and CVD risk and hypothesizing about potential causal mechanisms, no actual mechanisms have been determined. Scientists haven’t yet identified a dedicated TMAO receptor on cells, and little is known about which specific gut microbe species are the primary producers of TMA. So far, researchers only know which broader bacteria groups are associated with higher or lower circulating TMAO levels.14

“We differ in our gut microbiota,” Caudill says. “Those who are sick have a different gut microbiota than those who are well,” she says. “Those who have colon cancer may have a completely different gut microbe population.”

For example, Caudill’s group found that TMAO was elevated in women at higher risk of colon cancer in a study that used a subset of participants from the Women’s Health Initiative, but she says that lowering TMAO in those women wouldn’t lower cancer risk.24 “TMAO might be a great marker for a disrupted gut microbiome, or maybe a marker of greater kidney dysfunction than we thought,” Caudill says. “Then we could start treating the actual problem.”

Determining the exact relationship between diet, gut microbes, and disease endpoints will be challenging, but it’s likely an important step in determining who—if anyone—might benefit from nutritional or pharmaceutical interventions to reduce TMAO production. In rats, a high-salt diet can increase plasma TMAO levels, decrease TMAO excretion in urine—and alter the gut microbiota.25 On the pharmaceutical front, the TMAO synthesis pathway has become one of the first gut microbiota targets for drug intervention to prevent CVD.14

Individual genetic variation also may play a role in elevated TMAO levels by increasing enzymatic conversion of TMA to TMAO in the liver by flavin-containing monooxygenases (FMOs). Humans have five FMO genes—and five FMO enzymes—but it’s the conversion of TMA to TMAO by FMO3 that may be a culprit in atherosclerosis and CVD. Several factors can increase FMO3 gene expression—estrogen and insulin increase it, while testosterone and glucagon repress it. Notably, FMO3 expression is elevated in individuals with type 2 diabetes.17 What’s more, FMO3 may have adverse effects on blood lipids and glucose independently of TMAO formation, although more research is needed.13,26

Public Health Recommendations
Some research studies that have found associations between TMAO levels and CVD have gone so far as to recommend limiting or avoiding choline-rich foods and dietary supplements containing choline. Caudill says this isn’t only premature, but it’s concerning, because following that advice may have unintended consequences. “The demand for choline is so high,” she says, adding that there’s evidence that going above the AI may be beneficial in certain populations, while other populations may need to be cautious about supplementing with both choline and folic acid, which can increase cell division. But this requires a nuanced therapeutic approach, not a sweeping public health recommendation.

“If you want to go that route, you would have to tell people to reduce their intake of fish,” Caudill says. “You may even have to tell people to eliminate fish.”

It’s a conundrum that many fish species contain both TMA and TMAO. Fish intake acutely raises TMAO levels, yet fish is a significant component of the Mediterranean diet and independently has been shown to benefit cardiovascular health.16 A small 2017 study using participants in the larger European Prospective Investigation into Cancer and Nutrition trial to identify blood markers of intake of various foods—for potential use in epidemiologic studies—found that TMAO was a good blood marker of intake of both lean and fatty fish.27 One 2017 study on healthy young men found that blood levels of TMAO were about 50 times higher after consuming fish than they were after consuming eggs or beef.28

Liz Shaw, MS, RD, CLT, CPT, San Diego–based owner of Shaw’s Simple Swaps and a spokesperson for choline, says it’s important for dietitians to explain what TMAO really is, where it’s found, and what its uses are to demystify some of the headline hype. “We know very well within the RD world that science continues to support the importance of consuming seafood at least twice a week in a healthy diet.”

Bottom Line for RDs
If it’s unclear which individuals may benefit from TMAO-lowering strategies, how can dietitians counsel their clients who have concerns?

“I think it’s extremely important to always validate a client’s fears and help them recognize a safe and effective approach to rationally treating those fears,” Shaw says. Shaw adds that she thinks there’s enough evidence to support continuing to include foods that naturally may contain TMAO or potentially produce TMAO. “No one food (or compound) is going to be the trigger; it’s their environment, genetics, lifestyle, and nutrition plan that will have the biggest impact on their total health.”

Clients who regularly eat eggs and meat tend to get enough choline in their diets, but vegans—and vegetarians who avoid eggs—need to be more thoughtful about meeting the AI. Consuming foods such as Brussels sprouts, broccoli, cauliflower, soymilk, wheat germ, peanuts, and beans can help, but supplemental choline may be needed to make up shortfalls.

Shaw says she often recommends helping clients take steps to improve the balance of their gut microbiota through their food choices, rather than cutting out choline-rich foods such as fish or eggs. If kidney function is impaired, or insulin resistance is present, she says appropriate medical and dietary therapies are the logical place to start.

“We have to show that lowering TMAO levels in blood shows benefit,” Caudill says. Then, she says, we might intervene, with drugs that interrupt the choline-TMA-TMAO pathway, by altering the microbiota, or by altering nutrition. “Right now, there is just no good evidence that in humans elevated TMAO is doing harm.”

— Carrie Dennett, MPH, RDN, CD, is the nutrition columnist for The Seattle Times, owner of Nutrition By Carrie, and author of Healthy for Your Life: A Holistic Guide to Optimal Wellness.


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