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ABSTRACT & COMMENTARY

Which Came First, the Bacteria or the Egg? Gut Microbiota, Diet, and CVD

June 1, 2013
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By Donald J. Brown, ND, Managing Director, Natural Product Research Consultants, Seattle, Washington. Dr. Brown reports he is a retained consultant for Schwabe North America and Linnea SA.

SYNOPSIS: Mouse studies have found that dietary choline is associated with increasing levels of plasma trimethylamine-N-oxide (TMAO) and increasing risk of atherosclerosis — a process that is likely mediated by gut microbiota. Two human clinical trials support these findings. The first demonstrates that phosphatidylcholine (PC) increases plasma levels of TMAO. Following broad-spectrum antibiotic use (and subsequent reduction in gut flora), production of TMAO was reduced after a PC challenge. A large population study demonstrates the connection between TMAO and cardiovascular disease.

SOURCE: Tang WHW, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013;368:1575-1584.

Two prospective clinical studies, which were funded by the National Institutes of Health, were completed at the Lerner Research Institute of the Cleveland Clinic. In the first study, 40 healthy adults who had not taken antibiotics or probiotics in the past month underwent a phosphatidylcholine (PC) challenge during visit 1. For each participant, baseline blood and spot urine samples were obtained after an overnight fast (≥ 12 hours). At baseline, participants were given two large hard-boiled eggs including the yolk (approximately 500 mg of choline each) to be eaten within a 10-minute period together with 250 mg of deuterium-labeled PC (d9-PC), as a tracer. Serial venous blood sampling was performed at 1, 2, 3, 4, 6, and 8 hours after baseline, along with a 24-hour urine collection. These samples were used to measure trimethylamine-N-oxide (TMAO) and d9-TMAO, and choline and betaine levels were also measured in the plasma samples. Time-dependent increases in levels of both TMAO and d9-TMAO, as well other choline metabolites, were detected after the PC challenge. Urine samples also showed the presence of both TMAO and d9-TMAO, and there was a strong correlation between plasma levels of TMAO and absolute urine TMAO level (Spearman’s r = 0.58, P < 0.001).

Six participants then received metronidazole (500 mg twice daily) plus ciprofloxacin (500 mg/day) for 1 week. After receipt of the antibiotics, a repeat PC challenge was performed at visit 2. A third and final PC challenge was performed 1 month or more after the withdrawal of antibiotics (and subsequent “reacquisition of gut flora”), at visit 3. The same plasma and urine measurements were repeated at each visit. Antibiotic use resulted in a near complete suppression of detectable TMAO and d9-TMAO in both plasma and urine at visit 2. In contrast, the time courses of changes in free choline and betaine were not altered by antibiotic use. Following the PC challenge at visit 3, there was a notable time-dependent increase in plasma and urine TMAO and d9-TMAO. (See Table.)

Table 1: Summary of Tang et al Treatments

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Finally, a second study (clinical outcomes) enrolled 4007 adults who were undergoing elective coronary angiography. During a 3-year follow-up, the relationship between fasting plasma levels of TMAO and incidence of major adverse cardiovascular events (death, myocardial infarction [MI], or stroke) was studied. Increased levels of TMAO were associated with an increased risk of major adverse cardiovascular events (hazard ratio for highest vs lowest TMAO quartile, 2.54; 95% confidence interval, 1.96-3.28; P < 0.001). An elevated TMAO level predicted an increased risk of major adverse cardiovascular events after adjustment for traditional risk factors (P < 0.001), as well as in lower-risk groups.

Commentary

Led by Stanley Hazen, PhD, a research group at the Cleveland Clinic Lerner center has been generating quite a bit of press with its reports that first L-carnitine1 and now choline may be converted in humans to TMAO and therefore increase risk of atherosclerosis and major adverse cardiovascular events. The curve ball in their findings is the fact that the production of TMAO from red meat (L-carnitine) or eggs (choline) is largely due to gut microflora.

While we’ve known about TMAO and its precursor trimethylamine (TMA) for decades, the Cleveland Clinic group is the first to definitively demonstrate the gut microbiota connection. Dietary choline and L-carnitine, two TMA-containing compounds, are metabolized by the gut microbiota into TMA. When TMA reaches the liver, oxidizing flavin monooxygenase enzymes convert it to TMAO.2 TMAO is proatherogenic, with one possible mechanism — the suppression of reverse cholesterol transport. A study with mice was the first to demonstrate that oral intake of PC led to an increase in TMAO.3 In animals prone to atherosclerosis, increased TMAO also increased risk of atherosclerosis. Similar to the reviewed human study, use of broad-spectrum antibiotics resulted in reduced TMAO in the plasma and decreased risk of atherosclerosis.

The group from the Cleveland Clinic also found similar results with L-carnitine in both mice and humans.1 Chronic dietary L-carnitine supplementation in mice altered cecal microbial composition, enhanced synthesis of TMA and TMAO, and increased atherosclerosis. This did not occur if gut microflora was eliminated using antibiotics. A very interesting finding was the difference in TMAO production in human omnivores or vegans/vegetarians following consumption of a sirloin steak (approximately 180 mg of L-carnitine) and 250 mg of a radiolabeled L-carnitine. The increase in plasma TMAO was notable in the omnivores (n = 5) but was not seen in a vegan (> 5 years) subject. In a larger sample size of omnivores (n = 51) and vegans/vegetarians (n = 23), it was observed that omnivores had higher plasma levels of TMAO compared to omnivores, and the findings with the six participants reported above were also repeated following the L-carnitine challenge. Plasma L-carnitine levels in subjects undergoing cardiac evaluation (n = 2595) predicted increased risks for both prevalent cardiovascular disease and incident major adverse cardiac events (MI, stroke, or death), but only among those with concurrently high levels of TMAO.

So, other than bad news for those of you on the Paleo diet or regularly consuming steak and eggs for breakfast, what’s the takeaway here? The practical lesson is that the maintenance of a high-fat diet (particularly from animal sources) increases the risk of cardiovascular disease (due partly to dysbiosis?) and that a vegan/vegetarian diet is lower in choline4 and L-carnitine and higher in fiber and prebiotics that are likely to contribute to a healthier microbiota and all the associated digestive, immune, and cardiovascular benefits. It will be interesting to see what the ramifications of these findings are for high-dose L-carnitine therapy as well as energy drinks that are high in L-carnitine. The same holds true for phosphatidlycholine and choline-containing supplements (such as some multivitamins for both adults and children). While the press is quoting Dr. Hazen about the potential hazards of these supplements, I would like to see more specific tests to determine if the amount in supplements is in fact contributing to higher TMAO levels — particularly multivitamins that contain such a low amount of choline.

Reductionist and commercial thinking are probably racing ahead to routine plasma TMAO testing. Low-dose antibiotics are already used to reduce TMA production in persons with trimethyluria. Will we see targeted, non-systemic antibiotic therapy if the microbiota that are responsible for generating TMA from choline and L-carnitine are discovered? Lower your TMAO but create dysbiosis anyway?

Finally, is there a probiotic strategy on the horizon? In a germ-free mouse model carrying a “humanized” microbiome colonized with human infant flora, exposure to Lactobacillus paracasei decreased TMA and TMAO while L. rhamnosus increased TMA and TMAO.5 Developing a probiotic or probiotic combination that could reduce TMAO production in the occasional meat and egg eater might be of interest.

However, all roads lead back to diet. In an interview with the New York Times, Dr. Hazen probably sums it up best when he confesses to previously eating red meat several times a week, about 12 ounces at a time.6 Now he eats it once every 2 weeks and has no more than 4-6 ounces at a time.

References

  1. Koeth RA, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nature Med 2013;doi:10.1038/nm.3145.
  2. Rak K, Rader DJ. The diet-microbe morbid union. Nature 2011;472:40-41.
  3. Wang Z, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 2011;472:57-63.
  4. Stella C, et al. Susceptibility of human metabolic phenotypes to dietary modulations. J Proteome Res 2006;5:2780-8.
  5. Martin FP, et al. Probiotic modulation of symbiotic gut microbial-host metabolic interactions in a humanized microbiome mouse model. Mol Syst Biol 2008;4:doi:10.1038/msb4 100190.
  6. Kolata G. Culprit in heart disease goes beyond meat’s fat. New York Times. April 7, 2013. Available at: www.nytimes.com/2013/04/08/health/study-points-to-new-culprit-in-heart-disease.html?pagewanted=all&_r=0. Accessed May 17, 2013.

Summary Points

  • Human research has provided a link between intake of choline (as well as L-carnitine) and increased levels of plasma TMAO. The link appears to be due to gut microbiota metabolizing TMA, which is later converted in the liver to TMAO.
  • Plasma TMAO levels are directly correlated with major adverse cardiac events.
  • Altering gut flora through the use of antibiotics (in both animals and humans) and a vegan/vegetarian diet appears to decrease levels of TMAO.