Commentary on Gut microbiotica

Gut microbiota: an environmental risk factor for cardiovascular disease

The human gut is home to a vast number of commensal microorganisms, the intestinal microbiota, which influence physiology and metabolism within the body. Not only is the gut microbiota important for metabolism of xenobiotics, amino acids and carbohydrates, but its dynamic nature, especially in response to changes in diet, has fuelled research investigating a potential role in susceptibility to cardiovascular disease and obesity. Indeed, recent evidence has implicated the gut microbiota in insulin resistance and non-alcoholic fatty-liver disease.1,2 Increasingly, research suggests that the gut microbiota appear to function at the intersection between host genotype and diet to influence host metabolism.

Studies have adopted a metabolomics approach to identify small molecules that may act as potential triggers for cardiovascular disease. The most recent candidate is trimethylamine-N-oxide (TMAO) which is a metabolite of dietary choline (major sources eggs, liver, beef and pork).3 The gut microbiota play an intermediate role in converting choline, released by hydrolysis of phosphatidylcholine, to trimethylamine; this metabolite then undergoes oxidation by flavin monooxygenase enzymes in the liver to TMAO, which is then released into the circulation. In a mouse model susceptible to atherosclerosis, increased dietary choline not only resulted in increased plasma levels of TMAO, but also to greater development of plaque in the arteries of mice. In contrast, after treatment with broad-spectrum antibiotics, which effectively abolished the intestinal flora, there was no evidence of circulating TMAO in the plasma and no increase in the severity of atherosclerosis with a high-choline diet.

These findings prompt a key question: is an increase in plasma levels of TMAO a risk factor or risk marker for cardiovascular disease?

A recent report4 aimed to investigate this further. The report comprised two prospective clinical studies. In the first study, healthy volunteers who had not received antibiotics or probiotics within the previous month underwent a dietary phosphatidylcholine challenge (ingestion of two hard-boiled eggs, each containing about 250 mg choline, and 250 mg of deuterium-labelled phosphatidylcholine). Repeat challenges were performed after treatment for one week with antibacterials (metronidazole 500 mg twice daily plus ciprofloxacin 500 mg daily), and one month or more after completion of the antibacterial course (after the gut flora had been re-established). TMAO and deuterium-labelled TMAO were detected in the plasma after the baseline dietary phosphatidylcholine challenge. However, suppression of the gut flora effectively abolished these responses. After the gut flora had been re-established one month after completion of antibacterial treatment, the phosphatidylcholine challenge resulted in detectable levels of TMAO and deuterium-labelled TMAO, although there was some intersubject variability in the recovery of these responses.

The second study investigated whether fasting plasma TMAO levels were associated with incident major adverse cardiovascular events (MACE, defined as death, myocardial infarction [MI] or stroke) over 3 years follow-up in 4,007 patients (mean age 63 years, 64% male) who underwent elective coronary angiography. This patient group was characterised by a high prevalence of pre-existing cardiovascular risk factors; 32% had diabetes, 72% had hypertension, 65% were current or ex-smokers, and 42% had a history of MI. Median lipids were 96 mg/dL interquartile range [IQR] 78-117 mg/dL (2.5 [2.0-3.0] mmol/L) for low-density lipoprotein cholesterol, 34 mg/dL IQR 28-41 mg/dL (0.88 [0-72-1.06] mmol/L) for high-density lipoprotein (HDL) cholesterol and 118 mg/dL IQR 85-170 mg/dL (1.33 [0.96-1.92] mmol/L) for triglycerides; 60% of patients were on statins.

Over 3 years, 513 (13%) patients experienced a MACE. In addition to a higher risk profile (i.e. older age, higher fasting glucose and higher prevalence of diabetes, hypertension and previous MI), this group also had higher baseline plasma levels of TMAO compared with patients who did not experience a MACE (median [IQR]: 5 [3.0-8.8] µM versus 3.5 [2.4-5.9] µM). Patients with TMAO plasma levels in the highest quartile had more than 2-fold increase in the risk of a MACE than those in the lowest quartile (hazard ratio 2.54, 95% CI 1.96-3.28, p<0.001), and this persisted after adjustment for traditional cardiovascular risk factors (hazard ratio 1.88, 95%CI 1.44-2.44, p<0.001). Moreover, the prognostic value of an elevated TMAO plasma concentration was still evident in individuals considered at low risk of cardiovascular events, including those with low-density lipoprotein cholesterol <70 mg/dL (1.8 mmol/L).

What are the take-home messages from these data?

The results of this study add to emerging evidence that the gut microbiota is an important player in atherogenesis. Indeed, metabolism by the intestinal flora has been recently linked with a potential deleterious association between egg yolk consumption (a major dietary source of choline) and the development of atherosclerotic plaque.5 By implication, therefore, limiting the intake of potential sources of choline (such as red meat and eggs) may favourably impact cardiovascular health. Incidentally, as such foods also tend to be high in cholesterol, further benefit on lipids may be accrued.

Ultimately, these data reinforce the importance of guideline recommendations for a Mediterranean style diet, including lean protein (fish, poultry), nuts, pulses, vegetables and fruit, together with regular physical activity, to maintain cardiovascular health. While recent analyses from the PREDI-MED study6 suggest that such an approach may incur additional cost for the individual, there are benefits at the societal level, when the costs associated with the morbidity, disability and mortality of cardiovascular disease are taken into account. Thus, a healthy lifestyle and eating well remains the fundamental tenet underpinning cardiovascular health.

Finally, the study raises the tantalising possibility that targeting the gut microbiota or related metabolic pathways, may offer potential therapeutic benefit. However, enthusiasm for the hypothetical advantages of targeting the gut flora needs to be tempered by the wider implications of bacterial antibiotic resistance.

What does this study show?

  • The gut microbiota play a key role in the metabolism of dietary choline (major sources are in eggs and red meat), ultimately resulting in plasma trimethylamine N-oxide (TMAO).
  • In this study, elevated plasma TMAO concentration was predictive of the risk for incident major adverse cardiovascular events (MACE, defined as death, MI or stroke). Patients with the highest TMAO plasma levels had more than 2-fold increased risk of MACE versus those with the lowest levels.
  • These findings add to emerging evidence implicating the gut microbiota as an environmental risk factor for cardiovascular disease.

Traffic pollution linked with cardiovascular disease, independent of traffic noise

Previous studies have indicated a link between road traffic and cardiovascular disease.7-10 However, whether this association is mainly explained by air pollution or noise due to traffic is not clear. Both factors are believed to cause an imbalance in the autonomic nervous system, via mechanisms involving systemic oxidative stress and inflammation, which may in turn impact vascular function, thrombogenicity and plaque stability. However, recent findings from the German Heinz Nixdorf Recall Study (n=4,814, mean age 60 years), reported at EuroPrevent 2013,11 delineate long-term exposure to fine particle matter (PM) air pollution as a causative factor, independent of traffic noise.

This analysis was based a cohort of 4,238 subjects enrolled in this study. Proximity to roads with high traffic volume was calculated with official street maps; long-term exposure to particle pollutants was assessed with a chemistry transport model and road traffic noise was recorded using validated tests. Subclinical atherosclerosis was evaluated by measurement of vascular vessel calcification in the thoracic aorta by computed tomography imaging.

Both small particulate matter (designated as PM2.5) and proximity to major roads were associated with an increasing level of aortic calcification. For every increase in particle volume up to 2.4 µm (PM2.5), the extent of calcification in the thoracic aorta increased by 20.7%; additionally, for every 100 metre proximity to heavy traffic, the extent of calcification increased by 10%. Further analysis showed that these associations were independent of each other. Thus, these data add to previous findings that exposure to traffic pollution increased coronary atherosclerosis.12

Understanding the mechanisms underlying this association is highly relevant. Indeed, recent experimental findings13 implicate impairment of the potentially atheroprotective activities of HDL particles.

In this study, apolipoprotein E deficient mice were exposed to either filtered air for 2 weeks (control), diesel exhaust (≈250 µg/m3) for 2 weeks, or diesel exhaust for 2 weeks followed by filtered air for one week. In the control group, HDL inhibited LDL-induced monocyte migration in a monocyte chemotaxis assay. However, in the groups exposed to diesel exhaust, HDL induced more monocyte migration than LDL alone, suggestive of pro-inflammatory activity. There was partial recovery of anti-inflammatory activity after exposure to filtered air for one week.

Furthermore, exposure to diesel exhaust was associated with the formation of dysfunctional pro-oxidative HDL, as indicated by decreased paraoxonase activity, increased lipid peroxidation and increased susceptibility to oxidation of apolipoprotein B-containing lipoproteins. Evidence that these changes persisted after exposure to filtered air for one week suggested that there may be persistent changes in the protein and/or lipid components of HDL. However, investigation of this hypothesis is complicated by the heterogeneous nature of HDL particles, as well as the highly dynamic remodelling among HDL particles. Further understanding of the complex biology of HDL particles should provide important insights.

What does this study show?

  • In this experimental model, the anti-oxidant and anti-inflammatory activities of HDL were impaired after short-term exposure to diesel exhaust fine particulate matter.
  • These findings suggest that dysfunctional HDL might be one of the mediators of vascular damage associated with long-term exposure to traffic pollution.


  1. Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science 2005;307:1915–20.
  2. Dumas ME, Barton RH, Toye A et al. Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice. Proc Natl Acad Sci USA 2006;103:12511–6.
  3. Wang Z, Klipfell E, Bennett BJ et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 2011;472:57–63.
  4. Tang WH, Wang Z, Levison BS et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013;368:1575-84.
  5. Spence JD, Jenkins DJA, Davignon J. Egg yolk consumption and carotid plaque. Atherosclerosis 2012;224:469-73.
  6. Bes-Rastrollo M et al. EuroPrevent 2013, 18-20 April 2013, Rome, Italy.
  7. Rosenlund M, Bellander T, Nordquist T, Alfredsson L. Traffic-generated air pollution and myocardial infarction. Epidemiology 2009;20:265-71.
  8. Gan WQ, Tamburic L, Davies HW, Demers PA, Koehoorn M, Brauer M. Changes in residential proximity to road traffic and the risk of death from coronary heart disease. Epidemiology 2010;21:642-9.
  9. Ostro B, Lipsett M, Reynolds P et al. Long-term exposure to constituents of fine particulate air pollution and mortality: results from the California Teachers Study. Environ Health Perspect 2010;118:363-9.
  10. Sørensen M, Andersen ZJ, Nordsborg RB et al. Road traffic noise and incident myocardial infarction: a prospective cohort study. PLOS One 2012; doi: 10.1371/journal.pone.0039283.
  11. Kaelsch H, Hennig F, Moebus S et al. Is urban particulate air pollution or road traffic noise responsible for the association of traffic proximity with subclinical atherosclerosis? Results from the Heinz Nixdorf Recall Study. Presented at EuroPRevent 2013 Poster presentation P307.
  12. Hoffman B, Moebus S, Mohlenkamp S et al; Heinz Nixdorf Recall Study Investigative Group. Residential exposure to traffic is associated with coronary atherosclerosis. Circulation 2007; 116:489-96.
  13. Yin F, Lawal A, Ricks J, Fox JR et al. Diesel Exhaust Induces Systemic Lipid Peroxidation and Development of Dysfunctional Pro-Oxidant and Proinflammatory High-Density Lipoprotein. Arterioscler Thromb Vasc Biol. 2013 Apr 4. [Epub ahead of print].