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Beef, Microbes in the Gut, and Heart Disease

An Expert Interview With Stanley L. Hazen , MD, PhD
Linda Brookes, MSc, Stanley L. Hazen, MD, PhD

Jun 19, 2013

An Expert Interview With Stanley L. Hazen, MD, PhD

About the Interviewee

Stanley L. Hazen, MD, PhD, is Department Chair of Cellular and Molecular Medicine, Section Head of Preventive Cardiology and Rehabilitation, and Vice Chair of Translational Research at the Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio. Dr. Hazen describes the long-term goal of his laboratory as the understanding of the mechanisms through which inflammation contributes to diseases such as atherosclerosis and asthma. Current research programs are focused on the role of myeloperoxidase in promoting oxidant stress in vivo, and its participation in cardiovascular diseases; HDL structure and function; the roles of peroxidases and tobacco exposure in airway remodeling in asthma; and the role of intestinal microbiota in cardiometabolic disease. All research projects rely heavily on chemical and analytical methods to identify specific reactions/products, their mechanisms of formation, and their use as probes to elaborate pathways responsible for disease. Research efforts in each program span from bench to bedside, including basic/genetic, cellular, animal model, and human clinical investigations.
The Studies

Dr. Hazen was lead investigator and senior author of: Koeth RA, Wang Z, Levison BS, et al. Intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013;19:576-585. Tang WHW, Wang Z, Levison BS, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med. 2013;368:1575-1584.

Background to the Interview

Two recently published studies,[1,2] led by Dr. Hazen and colleagues at Cleveland Clinic and supported by both the National Institutes of Health and the Office of Dietary Supplements, have provided evidence suggesting that consuming common foods such as meat, eggs, and dairy products may directly contribute to an increased risk for cardiovascular disease in a way that is not related to the cholesterol or saturated fat content of the food. The researchers showed for the first time that carnitine found in red meat, and choline derived from consuming these foods, is metabolized by gut microbes to trimethylamine (TMA), which in turn is absorbed into the bloodstream and metabolized in the liver to trimethylamine-N-oxide (TMAO), a substance believed to promote atherogenesis. Both studies found an association between high levels of TMAO and increased cardiovascular risk in humans. These studies build on other recently published data from Dr. Hazen and colleagues reporting on the pathway in both humans and mice linking microbiota metabolism of dietary choline and phosphatidylcholine to cardiovascular disease pathogenesis, and demonstrating a direct proatherosclerotic effect of TMAO.[3,4] The first of the more recent studies investigated the effects of dietary L-carnitine, a choline analogue containing a similar TMA structure and found in red meat.[1] Human subjects who consumed L-carnitine as an 8-oz sirloin steak plus 250 mg isotope-labeled L-carnitine showed increased formation of both endogenous and isotopelabeled TMAO. This increase in TMAO was almost completely suppressed when a week-long treatment with antibiotics was given beforehand, indicating that TMAO is produced from dietary L-carnitine via a microbiotadependent mechanism. Omnivores produced more TMAO than vegans or vegetarians following ingestion of Lcarnitine. The presence of specific bacterial taxa in human feces was associated with both plasma TMAO concentration and dietary status. In an independent cohort of 2595 persons undergoing cardiac evaluation, plasma L-carnitine levels predicted increased risks for both prevalent cardiovascular disease and incident major adverse cardiac events (myocardial infarction, stroke or death), but only among persons with concurrent

high TMAO concentrations, suggesting that TMAO is the primary driver of association of L-carnitine with cardiovascular risk. The researchers also investigated chronic dietary L-carnitine supplementation in mice, which altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increased atherosclerosis, but not when the intestinal microbiota was concurrently suppressed. In mice with an intact intestinal microbiota, dietary supplementation with TMAO, or either L-carnitine or choline, reduced in vivo reverse cholesterol transport, suggesting that generation of TMAO by gut microbiota impairs reverse cholesterol transport. Finally, the investigators described additional studies that revealed that TMAO supplementation altered cholesterol and sterol metabolism in multiple compartments (artery wall, liver, intestines), so the mechanisms through which TMAO enhances atherosclerosis appear to be mediated via changes in cholesterol and bile acid metabolism. Dr. Hazen and coauthors commented on the health-related implications of these findings, notably that these studies may help explain the well-known epidemiologic association between dietary red meat ingestion and atherosclerosis and the need to investigate the safety of chronic consumption of over-the-counter L-carnitine dietary supplements. The second study investigated the relationship between intestinal microbiota-dependent metabolism of dietary phosphatidylcholine, the major dietary source of choline, with TMAO levels and adverse cardiovascular events in humans.[2] After a phosphatidylcholine challenge involving ingestion of 2 hard-boiled eggs and deuterium [d9]-labeled phosphatidylcholine in 40 healthy persons, time-dependent increases were seen in both TMAO and d9-TMAO. Levels of TMAO and d9-TMAO were undetectable after another challenge following suppression of intestinal microbiota with oral broad-spectrum antibiotics for 1 week, then reappeared after withdrawal of the antibiotics. These studies were thus consistent with gut microbes playing a role in TMAO formation from ingestion of phosphatidylcholine, which is abundant in egg yolk. The authors also described a separate clinical study[2] in which 4007 sequential consenting patients (mean age, 63 years; two thirds men) undergoing elective coronary angiography were examined. Increased plasma levels of TMAO were associated with a significantly increased risk for a major adverse cardiovascular event (hazard ratio, 2.54 for highest vs lowest TMAO quartile; P < .001) over 3 years of follow-up. This increased risk persisted after adjustment for traditional risk factors. These results supported the previous findings suggesting that pathways dependent on the gut microbiota might contribute to the pathophysiology of cardiovascular disease, concluded Dr. Hazen and his colleagues. Among other implications, these findings suggest that excessive consumption of phosphatidylcholine/choline-containing foods should be avoided. In an editorial in the New England Journal of Medicine, [5] Joseph Loscalzo, MD, PhD (Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts), said that the studies "point to a truly novel and potentially modifiable risk factor for atherothrombotic vascular disease." However, "much remains to be done to determine the precise role of TMAO in atherothrombogenesis -- whether it has a direct effect, acts as an epiphenomenal biomarker, or is a precursor to a more direct effector." Dr. Hazen spoke to Medscape about the implications of both studies.

The Role of Microbes in the Gut and Compounds in Red Meat

Medscape: Both of your recent studies have been viewed as having implications for nutrition, especially as related to newly identified adverse effects associated with the consumption of L-carnitine (in meat) and dietary phosphatidylcholine (in meat, eggs, dairy products). Dr. Hazen: That was just the media hype; we never really pushed that point at all. We have been focusing on the biochemical determinants of atherosclerosis and following where the chemistry and biology lead us, and it happened that some compounds involved are part of the foods we eat on a regular basis. I have been trying to avoid making too many statements about nutritional recommendations, other than saying that I think we should be following what is recommended by the American Heart Association.[6] We also referred to a recent study in which people who consumed a Mediterranean diet, specifically avoiding red meat, showed a 30% reduction in cardiovascular events over almost 5 years.[7] Medscape: Nonetheless, the compounds that were the focus of your studies are usually referred to as "essential" nutrients, or as you referred to choline, "semi-essential," and people take them as supplements even if they don't have a deficiency.

Dr. Hazen: Words like "essential" are used very loosely. Carnitine is not essential. We make all the carnitine we need from other nutrients found in foods, vegan and omnivore alike. Choline is considered "semi-essential" from a nutritional standpoint because our bodies can make most of the choline needed, but we do need to consume some in our diets or we develop a deficiency state -- which is very rare on a Western diet. Of course, choline is a major component of phosphatidylcholine, the building blocks of our cell membranes, so we could not live without it. However, this does not mean that we have to consume it in a capsule, enema, or in any other form as a supplement. Medscape: Both of your studies were focused on the catabolism of choline to TMA by gut microbes and then hepatic conversion of TMA to TMAO. You have published other detailed studies on this pathway[3,4]; could you explain what changes your research has brought about in the way of thinking about it? Dr. Hazen: First, choline, carnitine, and phosphatidylcholine, which are all dietary nutrients, all contain a similar TMA structure. TMA is generated through cleavage of that structure by gut bacteria. TMA is actually a gas, both at body and room temperature. It smells like rotting fish, and people who have a defect in the hepatic enzymes that convert TMA to TMAO have a genetic disorder, trimethylaminuria, also called fish malodor syndrome. Up until the time of our studies, TMAO in humans was thought to be simply a nitrogenous waste product excreted in the urine. It is a very stable, small molecule and dialysis specialists have long used TMAO measurement as a way of assessing the efficacy of dialysis membranes. That is also one of the reasons why we think that this pathway is going to be particularly important, not just in everyone, but especially in people with chronic kidney disease and end-stage renal disease. As the excretion of TMAO becomes worse, it starts to accumulate, and we think that this may account in part for enhanced cardiovascular risk in people with chronic kidney disease and end-stage renal disease. It is known that traditional risk factors do not adequately capture that enhanced risk in renal patients, but is not exactly known what the pathways involved in cardiac risks in these patients are. So, it is a logical leap that TMAO is involved. Medscape: Could you explain more about what you believe to be the link between TMAO and increased cardiovascular risk in patients? Dr. Hazen: It is important to stress that this increased risk associated with TMAO level is not an alternative to cholesterol; it is in addition to cholesterol. It appears that TMAO is mechanistically supporting enhanced atherosclerosis by changing cholesterol and sterol metabolism in multiple different compartments in the body. In the artery wall, TMAO influences the macrophage foam cell, which is the major cellular hallmark of an atherosclerotic plaque. In the presence of TMAO there are changes in expression levels of multiple different genes, including ones that have been linked to enhanced cholesterol deposition, such as scavenger receptors. In the liver we showed that bile acid synthesis is inhibited because the major enzymes involved, Cyp7a1 and Cyp27a1, are both suppressed. In addition to reduction in bile acid pool size, the composition of the bile acids also changes, and multiple transporters involved in moving bile acids across the different membranes in the hepatocyte are reduced. The same process takes place in the enterocyte in the intestines; we see changes in cholesterol and sterol metabolism. So, cholesterol is necessary to promote atherosclerosis; it is the major lipid that deposits in the artery wall. I would compare it to the electricity needed to turn on a light bulb: TMAO functions like a dimmer switch; if you turn a dimmer switch on all the way up (TMAO increased) with a certain amount of electricity (cholesterol), you will get a lot of light (plaque). If you turn the dimmer switch down (TMAO decreases), for the same amount of electricity (cholesterol) there will be less light (atherosclerotic plaque). So, the net effect of the TMAO is enhancing the potential for cholesterol to get deposited in cells of the artery wall and reducing the elimination of sterols from the body by the pathway called reverse cholesterol transport. Medscape: So, you are saying that TMAO is a causal factor, not just a marker like high-sensitivity Creactive protein? Dr. Hazen: Yes. The reason why I think it is causal is because if you feed it to animals, they get accelerated atherosclerosis. The point that may be a little confusing is, how can it be linked to cholesterol metabolism and not change LDL- or HDL cholesterol levels? HDL- and LDL cholesterol are just surrogates for pathways -- that is, for moving cholesterol; they are not measures of function. Each is a snapshot of how much cholesterol there

is at a certain point. HDL tends to move cholesterol from the periphery centrally, into the liver, and out of the body, and LDL delivers cholesterol to the peripheral tissues. They do not tell you anything about flux. Experts in the HDL community have been saying for a long time that HDL cholesterol is a surrogate and not as good as HDL function. Technically speaking, that is true also for LDL. In fact, none of the things that we have mechanistically seen TMAO doing should influence LDL cholesterol, and we do not see any relationship between TMAO and LDL or HDL. Medscape: These experiments were in mice; how relevant are the results to humans? Dr. Hazen: There are certainly many differences between animals and humans. We proved mechanistically in mice that TMAO directly enhances atherosclerosis in atherosclerosis-prone mouse models. We also showed that you can reduce reverse cholesterol transport with either TMAO or the foods containing the precursors, choline or L-carnitine, which both reduce reverse cholesterol transport, but only in the presence of intact gut flora. As soon as you suppress the gut flora, you no longer reduce it. However, in humans we do not yet have data connecting TMAO to reverse cholesterol transport, because no one yet knows how to measure it in humans. What we do see in humans is a very strong correlation between circulating levels of TMAO and both prevalent cardiovascular disease and future risk for heart attack, stroke, death, need for heart surgery -- all the adverse cardiovascular events. Medscape: So, you showed that this association has no direct relation to cholesterol levels? Dr. Hazen: We showed that TMAO predicts cardiovascular outcomes regardless of cholesterol levels. When we analyzed the data above and below an LDL cholesterol level of 100 mg/dL or 70 mg/dL, as well as above and below HDL 45 mg/dL, apoB above and below 80 mg/dL, or apoA1 above and below 100 mg/dL, the relationship between TMAO and cardiovascular risk did not change.[2] It is remarkable how robust the prognostic value of this TMAO is, regardless of all the different risk factor tests. Medscape: Was it also predictive in different subgroups? Dr. Hazen: The association was apparent in persons with or without cardiovascular disease, as well as within diabetic and nondiabetic persons alike, or those with vs without hypertension and smokers or nonsmokers -whichever way we looked at it.[2]

The Role of TMAO in Cardiovascular Residual Risk

Medscape: Could this represent the residual risk for cardiovascular disease identified in patients with low LDL cholesterol levels on statin therapy?[8] Recent studies that focused on reducing this risk by increasing low levels of HDL cholesterol do not appear to have been successful.[9,10] Could lowering TMAO be the way to reduce that residual risk? Dr. Hazen: Perhaps. In the New England Journal of Medicine paper[2] we reported that, statistically, inclusion of TMAO as a covariate resulted in a net 8.6% reclassification for improvement in risk estimation over traditional risk factors (net reclassification improvement, a significant P < .001). I hesitate to call it "residual risk"; it is independent of LDL- and HDL cholesterol levels. Once we can find people at risk, the next step is whether we can inhibit or treat that risk. Obviously these studies need to be done in the future, both attacking this pathway and seeing whether it represents a new therapeutic angle for lowering cardiovascular risk. It does not even have to be drugs, because it seems that nutrition and diet approaches can make a difference. However, we will also need to determine whether, by identifying someone who is at risk, in the same way as with high-sensitivity C-reactive protein, we can identify a population that has a higher risk and then get them to more aggressive goals -- for example, by intensifying statin therapy. Would these people benefit from more aggressive preventive efforts? Medscape: Would you risk creating a "worried well" population of patients with no symptoms but raised TMAO? Dr. Hazen: In our cohort of over 1000 primary prevention patients, we found that TMAO predicted future risk quite well. It is almost a CAD risk equivalent, a twofold increase, which is equivalent to the risk of having diabetes in our cohort. And those were people who just had a coronary angiogram and were told that there is less than 50% stenosis in all of their major vessels and they did not have anything to worry about. So, more

studies need to be done to see who should have their TMAO measured. The present study was not a small number of people, and the data are pretty strong. Medscape: How can TMAO be measured nowadays? Dr. Hazen: TMAO is straightforward to measure. We currently use mass spectrometry, but this is timeconsuming and not something that one can order from the hospital lab. We are currently involved in the development of a test using nuclear magnetic resonance (NMR). It turned out that the hydrogens on TMAO are unique and have a different NMR signature. The Cleveland Clinic has licensed the technology for measuring TMAO to LipoScience (Raleigh, North Carolina).[11,12] LipoScience uses NMR-based technology to measure lipoprotein particle subfractions and does about 2 million lipoprotein assays per year. The same technique that measures LDL and HDL particles can be applied to measure TMAO. The plan is for our clinical assay to be available for measuring TMAO through this platform by the end of summer 2013. Their machine has recently been cleared by the US Food and Drug Administration for availability in hospital labs.[13] It will not be available so quickly for TMAO measurement, but at least TMAO testing will be available for research studies before the end of the year. In the future, just like we do a blood test for cholesterol and triglycerides, we may do a blood test for TMAO for advice on dietary patterns as well as for other bacterial products that we think are biologically linked to glucose metabolism. These are going to be the subjects of future papers. We should be thinking of our intestinal microbial community as the largest endocrine organ in our body. The nutrients you ingest go through the filter of the intestinal microbes, and depending on the nutrient input and microbial composition, you have a different capacity to make different biologically active compounds. Compounds that diffuse in the blood and act at a different site meet all the definitions of a hormone, so this is why the microbial community can be considered like an endocrine organ, in a way. Medscape: How would one lower high levels of TMAO? Would it be by diet, or would it mean treating individualized gut microbiota? Dr. Hazen: Dietary efforts, including reduction in meat consumption, appear to be associated with reduced TMAO levels. As for treating an "individual gut microbe"? There are trillions of microbes, but if you look at the top 95% of them, in terms of broad classifications of family and genus they are pretty much the same in everyone. Proportions may be shifted in someone who eats a more vegetarian diet with more roughage compared with someone who has more carnivorous eating patterns, but the 95% are similar except for some proportional changes. In that 5% is where there is huge variability. If you are talking about a microbe making a product that is biologically active, it need not be abundant; it can be a really small proportion, because it is making something catalytically. It does not have to be one of the major microbe types; it just has to be present in sufficient numbers to produce the compound at a blood level that will allow us to see if it causes whatever phenotype we are looking at. One of the really intriguing findings of our L-carnitine paper was how substantially different the metabolism was of carnitine in vegetarians and vegans compared with omnivores. Chronic exposure to a diet that included Lcarnitine shifted the microbiota composition. It was a subtle shift, but the proportions of the microbes that tracked with TMAO levels in vegan or vegetarian status still accounted for a very small proportion of the microbes in the entire intestines. The vast majority of the microbes are similar among omnivores, vegetarians, and vegans. Medscape: Does that mean that if you eat any meat at all on a regular basis, whether a small amount or large portions, your gut microbiota are similar? Dr. Hazen: We do not know. To be defined as a vegetarian or a vegan in our studies, participants had to claim to have no meat product during the past year. How long you have to change your diet before you see a shift, we do not know. But at least now we can measure blood levels of TMAO, and it is going to be much easier than trying to look at the entire microbiome in the stool. Medscape: In your studies you used antibiotics to suppress the gut microbiota, but presumably treatment with antibiotics to suppress production of TMAO would not be a good idea. Dr. Hazen: Right. In fact, not only would it not be a good idea, it would be futile. What we already saw, at least in mice, is that when we initially did our experiments with only one antibiotic, we completely eliminated TMAO

levels at the beginning, but when we harvested the aortas just over half a year later, we found that blood TMAO levels were completely back to normal because the intestinal microbes had developed tolerance. So, even if, let us say, a fraction of 1% has a resistance to the antibiotic, in the beginning it looks as though the TMAO is 99% inhibited. But at 20 weeks it is back up to normal, because bacteria have numbers on their side. They have a doubling time in hours, and the very low-abundance resistant forms have a selective advantage because all the susceptible bacteria are being eliminated. None of the clinical trials of chronic antibiotic use in atherosclerosis have been successful to date.[14-16] My suspicion is that if the antibiotic had inhibited TMAO production in the beginning, I see no reason why it would do so after a continuous year of use of the antibiotic, as any resistant microbial form would eventually take over and repopulate the intestines. Medscape: So, that is not something that physicians will be considering in the future, but do you think they can be doing anything else at this stage? Dr. Hazen: Two things. One is that the test for TMAO will be available on a limited basis -- not widely available in every lab -- before the end of the year. Second, these studies basically reinforce a lot of existing knowledge in terms of diet. These kinds of studies do not address how much you eat and how much is linked to heart risk. That is done by epidemiology studies like the Health Professionals Follow-Up Study or the Nurses' Health Study, in which over 100,000 people have been followed for over 20 years with over 1 million patient-years of follow-up and thousands of mortality events. It is that kind of data, which involves very detailed food questionnaires given out every few years over the course of several decades, that allow us to conclude that eating 1 portion of red meat per day accounts for about a 13% increase in risk for total, cardiovascular, and cancer mortality over an average follow-up period of 22 years.[17] From our studies we perhaps now understand at a mechanistic level why red meat is more associated with cardiovascular risk than would be predicted simply by its cholesterol or saturated fat content. If you look at the mortality data from large studies, and then at the cholesterol content of red meat and the saturated fat content, neither is high enough to account for the enhanced mortality risk,[18] and that is why people have argued that there may be another contributor, such as grilling meat, which produces high benzo[a]pyrene concentrations, or that people who eat steak also increase their sodium intake. Medscape: There is also a carcinogenic effect. Dr. Hazen: There is, and whether or not this pathway is linked is something that needs to be determined. Medscape: Can you say how much people should cut back on foods containing L-carnitine or phosphatidylcholine? Dr. Hazen: They should try cutting back on foods that are particularly rich in them, which goes almost hand in hand with cutting back on foods rich in cholesterol and saturated fat. That is because animal cells do not just have free-floating fat and cholesterol; they are bound in membranes inside the cells. The major building block of membranes is lecithin or phosphatidylcholine, so almost every type of food that is high in fat and cholesterol is also high in phosphatidylcholine, the main dietary source of choline. Free choline also exists, but usually it is in the form of phosphatidylcholine, unless you are following a vegetarian or vegan diet, and then you are probably getting more free choline. However, I want to be clear that I am hesitant to say that we are studying nutrition. We are just studying the fundamental biochemical pathways linked to nutrition, and it just happens that we have discovered a link between some of these compounds via the gut flora that make the metabolite. Essentially we first found the link between TMAO and atherosclerosis in subjects. We then reverse-engineered the process and determined that it comes from what we eat. It just so happens that those foods are high in saturated fat and cholesterol. So, our approach is not to say or not to make recommendations on different food choices; instead it is to determine what the pathway is. Personally, I like a good steak, and in the future I want to be able to have a tablet so that I can continue to eat a small portion occasionally. I do not think that a wholly vegan diet is the way to go, because that has other associated risks, such as being high in carbohydrate and low in vitamins B12 and D... Medscape: Where do you see this research heading now? Dr. Hazen: Our data are opening up 2 exciting clinical possibilities. One is the need to identify new pathways for cardiovascular risk, because even lowering the LDL cholesterol below 70 mg/dL is not sufficient. There is still significant residual risk that is not being treated with our current lifestyle changes and our current therapeutic approaches.

The other clinical possibility follows from the major genetic studies that have looked at attributable cardiovascular risk but have not even been able to attribute 10% of the risk. So, if it is not genes, it is environment, and our biggest environmental exposure is what we eat, which is a foreign body being ingested. Two different people can experience the same food differently because they have different gut flora. One person may generate a little more of a compound like TMAO than the other. That concept is a new way of thinking about complex diseases like atherosclerosis and other cardiometabolic diseases. It can also apply to obesity and insulin resistance. Data links intestinal flora involvement in those phenotypes in both mice and humans.[19-21] There have been very exciting data from a gut flora transplant in persons with metabolic syndrome who received either their own fecal samples or those from a lean donor. Persistent changes in insulin sensitivity occurred just by transplanting the intestinal flora from one individual to another.[22] That is a whole new way of thinking, and it might lead to opportunities for new therapeutics. I now think of statins, HMG-CoA reductase inhibitors, lowering LDL cholesterol, as our Homo sapiens enzyme inhibitors. I predict in our future that we will also have in our medicine cabinets drugs that target bacterial enzymes -- not antibiotics, but compounds that just inhibit the microbe enzyme activity so that they continue to live but without being able to generate the metabolite that we are trying to suppress. I think that is going to be a new and exciting kind of therapeutic for heart disease in the future.
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Cite this article: Beef, Microbes in the Gut, and Heart Disease. Medscape. Jun 19, 2013.