Intestinal Flora Again Implicated in CAD; but This Time, Also CV Events

April 25, 2013

CLEVELAND, OH A major dietary source of choline provided in abundance by egg yolks and meat can, after conversion by intestinal bacteria, raise plasma levels of trimethylamine-N-oxide (TMAO), suggests a report published this week[1]. It also supports earlier evidence that circulating TMAO is proatherogenic and may have potential as a biomarker of later cardiovascular risk.

The new study, actually a pair of prospective clinical studies, complement each other by establishing dietary phosphatidylcholine metabolism by gut flora as a source of circulating TMAO and TMAO levels as predictors of death, MI, and stroke "independent of traditional risk factors, even in low-risk cohorts," according to the authors, led by Dr WH Wilson Tang (Cleveland Clinic, OH).

Their work is detailed in the April 25, 2013 issue of the New England Journal of Medicine. If it sounds familiar, it may be because the same research team published strikingly similar findings two weeks ago in the journal Nature Medicine[2], but naming dietary carnitine--also prevalent in red meat--as an ultimate source of TMAO released by intestinal microbiotia.

Our gut microbes are the biggest endocrine organ in our body. They can make biologically active compounds, and they contribute to disease processes.

The popular press seemed to enjoy itself in covering the study and exploded with stories, of varying accuracy, on how carnitine--also an additive to commercial energy drinks and diet supplements--may help drive the heart-disease risk associated with diets rich in meat.

(Later the same week, heartwire and others reported a meta-analysis[3] suggesting, notably to a befuddled public, that L-carnitine given as a drug in acute MI could limit infarct size and improve clinical outcomes--proving as little else could that the overall story is more complicated than it seems.)

Dr Stanley L Hazen (Cleveland Clinic, OH), senior author of both reports linking food components to CV risk, told heartwire that it was an accident they appeared in the literature within two weeks of each other; with the manuscripts completed and submitted to different journals some time ago, they do not specifically cross-reference each other despite their close, complementary relationship.

More important, he said, their message isn't about L-carnitine or phosphatidylcholine. "It's all about the gut flora and TMAO, and TMAO is about cholesterol." The metabolite is thought to promote atherosclerosis, among other effects, "by changing cholesterol metabolism in the artery wall and other compartments like the liver and the intestines."

Taken further, the message is that "our gut microbes are the biggest endocrine organ in our body. They can make biologically active compounds, and they contribute to disease processes."

"A Truly Novel and Potentially Modifiable Risk Factor"

Hazen said the line of research began with observations by many in the field that the cardiovascular risk associated with diets rich in meat in epidemiologic studies far outstrips that expected from their cholesterol and saturated-fat content. So, the reasoning went, something else about meat seems to be adding to the increased risk.

The current study extends research in mice Hazen and his colleagues reported in 2011[4], which demonstrated a gut-flora–mediated relationship between dietary phosphatidylcholine, circulating levels of three metabolites (choline, TMAO, and betaine), and mechanisms behind arterial cholesterol buildup.

It to a large extent replicated those animal models clinically, with a focus on TMAO, in the test cohort of 40 healthy volunteers. Then TMAO emerged as an independent predictor of CV events in a separate cohort of >4000 patients undergoing elective angiography.

That the findings of the animal study also seem to apply to humans, Hazen said, suggests that TMAO might serve as a marker of CV risk and possibly a treatment target. And insofar that it points to a role for the metabolic effects of intestinal flora in atherogenesis, "it opens up new vistas for potential therapeutic approaches to go after for the treatment of heart disease."

In an accompanying editorial[5], Dr Joseph Loscalzo (Brigham and Women’s Hospital and Harvard Medical, Boston, MA) agreed, writing, "These studies in humans are consistent with the initial preclinical observations and point to a truly novel and potentially modifiable risk factor for atherothrombotic vascular disease."

"Unambiguous" Evidence

Forty healthy volunteers--no chronic illnesses (especially no heart, lung, or hematologic disease), no active infections, and no recent antibiotic therapy--were given a phosphatidylcholine challenge in which they consumed "two large hard-boiled eggs" and a capsule of deuterium-labeled phosphatidylcholine. Six of the participants repeated the challenge after a course of broad-spectrum antibiotics, which suppressed gut flora, and then again a month later after the gut flora had an opportunity to repopulate. Plasma and urinary choline metabolites, as products of ingested phosphatidylcholine, were measured after each challenge.

Measurement of deuterium levels by mass spectrometry showed that "a very large proportion" of urinary TMAO derived from the labeled ingested phosphatidylcholine, Hazen said. "It's unambiguous that phosphatidylcholine gets converted into TMAO." Moreover, urinary TMAO levels were closely correlated with plasma levels.

"Plasma levels of TMAO were markedly suppressed after the administration of antibiotics and then reappeared after withdrawal of antibiotics," the group writes. But postprandial plasma levels of the other two phosphatidylcholine products, free choline and betaine, were unaffected by antibiotic intake. That TMAO increased after the third phosphatidylcholine challenge pointed to resurgence of the intestinal flora.

Risk Doubled in "Low-Risk" Groups

The group also followed 4007 primarily male patients undergoing elective angiography for evaluation of possible coronary disease; none had experienced acute coronary syndromes. Those with baseline TMAO levels in the highest quartile, compared with the lowest quartile, showed an unadjusted hazard ratio (HR) for death, MI, or stroke of 2.54 (95% CI 1.96–3.28, p<0.001) after three years.

The HR remained significantly increased at 1.88 (95% CI 1.44–2.44, p<0.001) after adjustment for age, sex, smoking status, systolic blood pressure, LDL- and HDL-cholesterol levels, diabetes status, and C-reactive protein levels. It was still significant, 1.43 (95% CI 1.05–1.94, p=0.02), after further adjustment for myeloperoxidase levels, glomerular filtration rate, total white-cell count, body-mass index, and use of aspirin, statins, ACE inhibitors, angiotensin-receptor blockers, and beta-blockers.

Breaking the composite end point into components, the high-TMAO-level group showed an HR of 3.37 (95% CI 2.39–4.75, p<0.001) for death and 2.13 (95% CI 1.48–3.05, p<0.001) for nonfatal MI or stroke.

"Basically, none of the risk factors for atherosclerosis or CAD [we looked at] attenuated the intensity of this [TMAO] signal--it stays really robust." And, Hazen said, that applies to virtually all of the subgroups examined, including some that would traditionally be classified as lower risk. Those included those aged <65, without CAD or CAD equivalents, with baseline LDL <70 mg/dL, normal levels of other lipid markers and blood pressure, nonsmokers, and those managed in a primary-prevention setting.

"Even among subjects who were later categorized as not having significant CAD, because they had <50% [coronary] stenoses, a high TMAO doubled their risk for heart attack, stroke, or death, and it was significant even after adjustments," Hazen said.

In his editorial, Loscalzo writes that the current findings "raise the distinct possibility that atherothrombogenesis can be modulated or attenuated by a variety of novel strategies. These include, but are not limited to, modifying the diet to limit the intake of choline-rich food; altering gut microbiota with the use of probiotic approaches to limit synthesis of trimethylamine . . . or suppressing the synthesis of TMAO pharmacologically."

Supported by grants from the National Institutes of Health (NIH) and its Office of Dietary Supplements. The clinical study GeneBank was supported by grants from the NIH and a Cleveland Clinic/Case Western Reserve University Clinical and Translational Science Award. Hazen was supported by a gift from the Leonard Krieger Fund. Mass spectrometry instrumentation used was housed within the Cleveland Clinic Mass Spectrometry Facility with partial support through a Center of Innovation by AB Sciex. Tang reports consulting for Medtronic and St Jude Medical and receiving grants from Abbott Laboratories, St Jude Medical, Medtronic, and FoldRX. Hazen reports consulting for Abbott, Cleveland Heart Lab, Esperion, Lilly, Liposcience, Merck, and Pfizer and receiving grants from the NIH, Liposcience, Esperion, and the Leducq Fondation; and royalties from Abbott, Cleveland Heart Lab, Esperion, Frantz Biomarkers, Liposcience, and Siemens. Disclosures for the coauthors are listed at Loscalzo had no disclosures.


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