January 28, 2014

Intestinal Fermentation of L-carnitine in Red Meat Promotes Atherosclerosis

Six-part study determines a connection between L-carnitine and heart disease.
This publication pulled together data from several studies, both mouse and human, to assess the role of trimethylamine-N-oxide (TMAO) on atherosclerosis. TMAO is a product of carnitine metabolism by microbiota in the gut.


Koeth RA, Wang Z, Levison BS, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nature Med. 2013;19(5):576-585. 

Design and Key Findings

This publication pulled together data from several studies, both mouse and human, to assess the role of trimethylamine-N-oxide (TMAO) on atherosclerosis. TMAO is a product of carnitine metabolism by microbiota in the gut. This publication contained 6 sections:

1. Metabolomic studies

Plasma samples were collected from GeneBank (a large clinical repository of patients undergoing elective diagnostic cardiac evaluation), screened, and compared to age- and gender-matched controls. Although plasma L-carnitine did not meet the P-value cutoff for association with cardiovascular disease (CVD) using strict criteria, evaluation with less stringent criteria identified L-carnitine as a compound that was associated with CVD.

2. Confirming gut microbiota involvement

Omnivorous subjects completed an L-carnitine challenge by eating a large amount of L-carnitine (an 8-oz sirloin steak with estimated 180 mg L-carnitine) as well as a capsule containing 250 mg of heavy isotope-labeled L-carnitine (d3-carnitine). d3-carnitine and d3-TMAO were measured in plasma and urine and demonstrated a postprandial increase in d3-carnitine and d3-TMAO in plasma as well as a increase in d3-TMAO in 24-hour urine.
To confirm the potential contribution of gut microbiota in the formation of TMAO from dietary L-carnitine, 5 volunteers were placed on oral broad-spectrum antibiotics for 1 week, and then repeated the L-carnitine challenge described above. There was virtually no detectable native or d3-TMAO in either plasma or urine after a week-long treatment with antibiotics. This data confirms that TMAO production from dietary L-carnitine in humans is dependent on intestinal microbiota. 

3. Omnivores vs. vegans

A post-hoc nutritional survey completed by volunteers suggested that dietary habits may influence the ability to generate TMAO from L-carnitine in the diet. To test this, the L-carnitine challenge described above was conducted on 2 additional groups: volunteers who were vegan >5 years or vegetarians (n=23, and yes, they did eat the steak!). While omnivores showed increases in TMAO and d3-TMAO postprandially, vegans showed nominal plasma and urine TMAO at baseline and had virtually no capacity to generate TMAO or d3-TMAO in response to the L-carnitine challenge. Vegans also had a lower baseline plasma level of L-carnitine compared to omnivorous subjects.

4. Gut microbes and TMAO production

Dietary habits can influence gut microbial taxonomy.1 To determine microbiota composition, fecal samples underwent gene sequencing in both vegans/vegetarians (n=23) and omnivores (n=30). Analysis of proportions of specific taxa revealed significant correlation between taxonomy and plasma TMAO concentrations (P=0.03), but not with plasma carnitine (P=0.77). It was noted that individuals with a high proportion of genus Prevotella had a higher plasma TMAO concentration than those with a high proportion of Bacteroides genus (P<0.05). Specific genera of bacteria were also associated with both vegan and vegetarian dietary habits compared to omnivorous diets.

5. Of mice...

Finally, in mice, it was observed that TMAO production in response to L-carnitine could be induced with microbial colonization in germ-free mice or with chronic dietary L-carnitine intake. Germ-free mice underwent an L-carnitine challenge, which demonstrated no detectable plasma d3-TMAO production after ingestion of d3-carnitine. After several weeks in conventional cages, allowing recolonization of intestinal flora, the same mice acquired the ability to produce d3-TMAO. In a different study, mice fed a carnitine-supplemented chow increased their carnitine-to-TMAO conversion 10-fold compared to a control group. Bacterial taxonomy was completed on the mice and it was observed that as in humans, there were genera associated with increased conversion of carnitine to TMAO, but these genera were not shared with those in humans, as mouse flora and human flora do not have much overlap.

6. Plasma L-carnitine and CVD

In a large, independent cohort (n=2,595) of stable subjects undergoing elective cardiac evaluation, there were significant, dose-dependent associations between plasma carnitine concentration and risk of coronary artery disease (CAD) (P<0.05), peripheral artery disease (P<0.05), and overall CVD (P<0.05). These associations remained significant when adjusting for traditional cardiovascular risk factors (P<0.05). When TMAO concentration and other comorbidities were adjusted for, the association was not present, suggesting that TMAO concentration, rather than carnitine, is the true driver of the association of carnitine with cardiovascular risk.
Further mouse studies examined the mechanisms by which TMAO promotes atherosclerosis. It was found that the association may occur via several mechanisms, including TMAO inhibition of reverse cholesterol transport, lowering total bile acid pool, and/or increasing macrophage SRA and CD36 surface expression and foam cell formation.2

Practice Implications

To begin, it’s important to highlight a notable limitation of this study, which is that most of the research uses a mouse model, and the mice used were a specific strain with a higher risk of atherosclerosis. This mouse data may not correlate with human data. Additionally, the authors themselves point out that the original human data did not pick up a statistically significant association between L-carnitine and atherosclerosis. It was only after “less stringent” criteria were applied that this association was observed. Drawing clinical conclusions based on this less stringent criteria does not necessarily reflect best practice.
This carnitine-TMAO connection suggests another mechanism by which diets high in red meat may lead to cardiovascular disease.
While a quick glance over the article title (or a read of the associated New York Times article)3 may lead to the assumption that L-carnitine is the culprit behind atherosclerosis, a more careful reading clarifies the author’s intent. It is not to vilify this compound or an omnivorous diet, but rather to identify the significance of the gut microbiome as a clear etiological factor for cardiovascular disease.
But the true question is, how does this study impact our current clinical approach to cardiovascular disease and prescriptions of L-carnitine?
Carnitine is an endogenously synthesized compound made from the amino acids lysine and methionine that controls transport of acyl and acetyl groups across the mitochondrial membrane. Carnitine is required in order to obtain energy from fatty acids (via B-oxidation and the citric acid cycle). Since the cells of the heart have a tremendous amount of mitochondria, they have a fairly high requisite for carnitine.
L-carnitine exerts antioxidant effects, such as in the epididymis where it prevents lipid oxidation and protects sperm from reactive oxygen species. Its effects have been studied for attention deficit and hyperactivity disorder (ADHD), chronic fatigue syndrome, infertility, erectile dysfunction, chronic obstructive pulmonary disease (COPD), diabetes, and many other conditions. The most significant consideration is that L-carnitine is frequently recommended to support cardiovascular health, and several studies support such use. Studies using 1 gram of L-carnitine 2 to 3 times daily showed an improvement in heart function and decreased symptoms of angina.4,5 A double-blind trial showed that patients with congestive heart failure taking 500 mg 2 to 3 times daily of propional-L-carnitine experienced a 26% increase in exercise capacity after 6 months.6,7 Other cardiac benefits have been noted with the use of L-carnitine for congestive heart failure.8 Even in patients without congestive heart failure, benefit of carnitine supplementation has been noted. Clinical trials have reported that 4–6 grams of L-carnitine daily improves the chance of surviving a heart attack.9,10 Both IV and oral dosing of L-carnitine have been shown to decrease infarct size and decrease the number of nonfatal heart attacks compared to placebo.11 A recently published systematic review and meta-analysis of 13 controlled trials compared the effect of L-carnitine versus placebo found that L-carnitine decreased all-cause mortality by 27%, reduced ventricular arrhythmia by 65%, and reduced the development of angina by 40% in the setting of acute myocardial infarction. In the same study, there was no reduction in the development of heart failure or reinfarction.12
While robust research demonstrates a positive effect of L-carnitine on cardiovascular disease, this study suggests that carnitine may have an unwanted effect increasing atherosclerosis via TMAO in individuals with specific strains of intestinal flora.
The effect of TMAO is not unique to carnitine. A similar mechanism has been described with lecithin and phosphatidyl choline (PC) in an article published in Nature in 2011. The article reported a high correlation with plasma PC and 2 of its metabolites, TMAO and betaine, with cardiovascular disease in human subjects. Researchers also observed an increase in foam cell formation and aortic atherosclerosis in mice that were fed PC. Again, these observations were dependent upon specific gut flora.13 An April 2013 article in the New England Journal of Medicine reported similar results in humans, where intestinal microbiota-dependant metabolism was associated with adverse cardiovascular events after dietary ingestion of lecithin/PC.14 Again, these are nutritional supplements that people have taken to support cardiovascular health.
This carnitine-TMAO connection suggests another mechanism by which diets high in red meat may lead to cardiovascular disease. There is a longstanding assumption that the primary mechanism by which red meat leads to CVD is the associated increased intake of saturated-fat and cholesterol. This dogma was questioned in a 2010 meta-analysis of prospective cohort studies that found no association between saturated fat intake and CVD.15 Could it be that the Mediterranean diet’s benefit comes from low intake of carnitine? Or even more interesting, could some of the benefits of the Mediterranean diet and other naturopathic therapies be that they promote particular strains of gastrointestinal flora?
A growing body of data links several diseases to the predominant strains of intestinal microflora. In the March 2013 issue of Science, a study in mice showed that early-life microbial exposures influence sex hormone levels and modify risk of autoimmunity.16 Immature female mice that underwent fecal transfer from adult males exhibited elevated testosterone, decreased islet cell inflammation and autoantibody production, and overall, experienced robust protection against type 1 diabetes. Studies in humans have also demonstrated that intestinal flora can influence disease risk, such as type 1 diabetes, or even traits such as leanness versus obesity.17 The microbiome also influences asthma and allergic disease.18
This study proposes a link between microbiome and cardiovascular health, an exciting step forward in understanding another factor in the complex etiology of chronic disease. These provocative results warrant further investigation on the safety of L-carnitine use and may be the tip of the iceberg of information that changes our clinical practice in the future. But these preliminary findings alone do not suggest that we should avoid dietary or supplemental sources of carnitine, especially in light of the body of evidence demonstrating L-carnitine’s protective effects. 
For those at risk of or with current atherosclerosis, it may become prudent to address gastrointestinal microbiota as part of an overall treatment approach, especially in patients who are resistant to a dietary shift towards vegetarianism. It would be exciting to also see further research on supplements such as probiotics and their impact on TMAO production after exposure to carnitine, phosphatidyl choline, and other implicated nutrients. 
Given the growing body of evidence that gut microbiota play a role in overall health, we can eagerly await emerging research to demonstrate how this observation can be used clinically. Perhaps in the future, enterotyping of one’s microbiome will be a useful tool—one more step toward personalized medicine. 

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