Reference
Heianza Y, Tiwari S, Wang X, et al. Gut-microbiota-related metabolite phenylacetylglutamine and risk of incident coronary heart disease among women. J Clin Endocrinol Metab. 2024:dgae525.
Study Objective
To investigate whether higher plasma levels of phenylacetylglutamine (PAG) are associated with a greater risk of incident coronary heart disease (CHD) and test whether adherence to a plant-based diet modifies the association
Key Takeaway
Higher PAG was associated with higher risk of CHD, particularly in women with animal-rich diets and fewer plant foods. Adherence to plant-based diets might reduce the association between this microbial metabolite and CHD risk.
Design
This paper describes 2 independent, prospective, observational case-control studies.
In the first analysis, investigators examined associations between plasma PAG in a nested case-control study using data from the Nurses’ Health Study (NHS). In a separate analysis, investigators used data from the Women’s Lifestyle Validation Study (WLVS) to examine the relationship between dietary intake and PAG.
Participants
Participants in these studies were either members of the NHS or a subgroup of that study, the WLVS.
Investigators gathered the NHS data from 1,520 women (760 cases and 760 controls) and analyzed the relationship between PAG levels and incident CHD over a period of 11 to 16 years.
The NHS is a prospective cohort study of American, female registered nurses (N=121,700), aged 30 to 55 years when enrolled in 1976. Information on demographics, lifestyle factors, medical history, and disease status was collected through a self-administered questionnaire in 1976. The data has been updated every 2 years since then through follow-up questionnaires. Researchers acquired blood samples from 32,826 women from 1989 to 1990 and 18,743 women from 2000 to 2002 and began a case-control study looking at incidence of CHD compared to controls without disease over 11 to 16 years of follow-up.
In total, there were 1,524 participants in this NHS section of the study (762 CHD cases and 762 matched controls), but 4 were excluded from the analysis. Blood samples had been stored in liquid-nitrogen freezers at -130 degrees Celsius or colder until analysis.
The WLVS is an observational study begun in 1989 among a subset of participants from the NHS cohorts. This group was originally used to investigate the validity of self-reported diet and lifestyle. During a 15-month period between 2010 and 2012, along with giving extensive anthropometric data that included records on diet and physical activity, these women provided 2 blood samples. These data and blood samples were adequate to compare PAG levels and dietary intakes of 725 women.
Study Parameters Assessed
Investigators assessed the levels of phenylacetylglutamine, a gut microbiome metabolite that has been associated with CHD disease, in all participants. They tracked nonfatal myocardial infarctions through medical records and deaths through next-of-kin reports or searches of the National Death Index. Investigators tracked fatal CHD through medical records or autopsy reports.
The WLVS portion of the study considered adherence to dietary patterns, using a validated plant-based dietary index (PDI) that characterizes habitual animal and plant food intakes.
Members of the WLVS kept 7-day dietary records, and their blood samples were tested for plasma levels of insulin, proinsulin, C-peptide, and lipids (total cholesterol, high-density lipoprotein [HDL] cholesterol, and triglycerides) in addition to PAG levels. Demographic data were collected and confirmed.
Primary Outcome
Whether PAG is associated with CHD risk and whether a plant-based diet lowers risk
Key Findings
Higher PAG levels were associated with a greater risk of CHD (P<0.05 for dose-response relationship).
Higher PAG levels were also associated with greater red and processed meat intake and lower vegetable intake (P<0.05 for all).
A significant interaction was found between PAG and adherence to plant-based diet index and CHD (P interaction=0.008); higher PAG levels were associated with an increased risk of CHD (relative risk per 1 SD: 1.22 [95% CI: 1.05, 1.41]) among women with low PDI but not among those with high PDI.
Transparency
This study was sponsored by multiple government, educational, and nonprofit groups, including the National Institutes of Health (NIH), the National Heart, Lung, and Blood Institute, the National Institute of Diabetes and Digestive and Kidney Diseases, and the National Institute of General Medical Sciences, and Tulane University. One of the researchers was a postdoctoral fellow supported by grants from the Japan Society for the Promotion of Science and the American Heart Association. None of these grants suggest a conflict of interest.
Practice Implications & Limitations
The simple take-home from this and other recent papers on phenylacetylglutamine is that this metabolite of the gut biome appears to cause cardiac damage and increase the risk for adverse cardiac events. Diets that supply the gut microbiome with an abundance of phenylalanine increase production of PAG, and we now understand that the link between specific diets and cardiovascular disease (CVD) is via PAG production, and that PAG binds to specific adrenergic receptors, stimulating excitatory responses that lead to damage.
Given how few of us are familiar with this novel metabolite phenylacetylglutamine, let us step back to consider some of the background information we may not have previously turned our attention to.
The first paper suggesting that PAG is associated with increased risk of mortality and heart disease may be the 2016 publication in which Ruben Poesen et al reported that higher levels of PAG were associated with higher risks in patients with chronic kidney disease (CKD). Poesen had monitored PAG levels in 488 patients with CKD, at the time considering it a marker of kidney dysfunction and progressive disease. Risk of mortality (51 events) and CVD (75 events) were correlated with kidney function and PAG levels. PAG levels were associated with mortality (hazard ratio [HR] per 1-SD increase, 1.77; 95% confidence interval [CI], 1.22–2.57; P=0.003) and cardiovascular disease (HR, 1.79; 95% CI, 1.32–2.41; P<0.001).1 As kidney function decreases, PAG levels build up, and many of the early PAG papers are related to CKD.
Our interest in PAG is, in a way, similar to our interest in other previously obscure bacterial metabolites now viewed as harmful; metabolites such as indoxyl sulfate, trimethylamine-N-oxide (TMAO), and lipopolysaccharide (LPS) suddenly seem relevant.2
We have reached a turning point in the understanding of the etiology of cardiovascular disease. It is now accepted that the gut microbiota and its metabolites are significantly related to development of cardiovascular disease. Investigative efforts have shifted to the mechanisms of how these metabolites induce the formation of vulnerable atherosclerotic plaques and whether this knowledge will provide targets for developing future preventive strategies.
The big picture these days of how and why CHD develops is increasingly about the shifts that have occurred in the modern human diet, which now contains greater quantities of red meat and artificial sweeteners rather than grains, vegetables, or even natural sugars. There has been a corresponding increase in conditions such as diabetes, obesity, and cardiovascular disease. Multiple theories have been expounded to explain these increases in disease occurrence.
So many dramatic changes have occurred over the last century, not just in diet but also in lifestyle, technology, and environment, that it is easy to be misled. Many of these changes when quantified over time appear to be statistically associated with increasing disease rates, but these correlations are not necessarily causative. For example, the scarcity of vinyl records might be erroneously considered as a possible contributing factor to increases in metabolic disease, but that is probably not a causative association.
We have reached a turning point in the understanding of the etiology of cardiovascular disease.
Currently solid research suggests that these increases in disease are secondary to shifts in the gut microbiome, a result of the major dietary shifts that have led to exposure to certain bacterial metabolites that lead, in turn, to pathologic changes.3
The PAG metabolite, which this study focused on, is derived from the microbial metabolism of dietary protein, specifically unabsorbed phenylalanine, which is fermented by gut microbes to produce phenylpyruvic acid and subsequently phenylacetic acid. The main sources of dietary phenylalanine are protein-rich foods, such as meat, milk, and eggs.4 Aspartame, the artificial sweetener, also contains phenylalanine.
Phenylacetic acid is converted to PAG in the liver by conjugation with glutamine.5 The key gut microbial pathways responsible for this conversion were identified by Zhu et al in 2023. Zhu and colleagues were also able to demonstrate through metagenomic analyses that these pathways occurred in higher abundance in the gut microbiomes of heart disease patients compared to controls.6
The most important contributions to our understanding of PAG have come from Stanley Hazen’s laboratory at the Cleveland Clinic. This group has published at least 3 major papers on PAG since 2020.
In the team’s first PAG paper, published in 2020, Ina Nemet et al described their initial process of discovery. Using a technique called untargeted metabolomics led them to focus their attention on PAG. They studied patients with type 2 diabetes mellitus (T2DM) in this initial work, in the hope of identifying metabolic pathways linked to CVD. They chose diabetics because they have a strikingly high, but unexplained, risk for CVD. As Nemet points out in the paper, “… the degree of blood-glucose control within T2DM is a poor indicator of incident CVD risks, and numerous anti-diabetes medications lower plasma glucose levels without significantly impacting CVD development.” They realized something beyond the poor regulation of blood sugar levels was contributing to the disease burden in T2DM.
Nemet’s group performed untargeted metabolomic analysis on blood samples from a “discovery cohort” of diabetics (N=1,162) undergoing elective cardiac evaluation, and the group identified the metabolites most associated with major adverse cardiac events during a 3-year follow-up. The top 5 compounds most associated with cardiac events included several already known suspects, including trimethylamine N-oxide (TMAO) and trimethyllysine (TML). Among the unknown compounds was phenylacetylglutamine, which became a top suspect as it had a high hazard ratio (HR, 95% CI for incident [3-year] risk of MACE [major adverse cardiac events]: 2.69 (1.61–4.52); P<0.0001).7
Once they identified PAG, these researchers undertook to determine how it affects the body. In vitro studies showed exposure of human blood samples to PAG “accelerated the rate of collagen-dependent platelet adhesion and spreading observed under physiological shear flow.” A commentary on Nemet’s study succinctly summed up this effect in its simple title: “Platelets get gutted by PAG.”8
Ex vivo studies with mice showed that injection of PAG sped up the rate at which blood clots in the carotid artery after an injury, in effect mimicking the path to a coronary event. It was also evident to these researchers that PAG acted by binding to adrenergic receptors. Those are the cell-surface glycoproteins that recognize and selectively bind the catecholamines norepinephrine and epinephrine after they are released from sympathetic nerve endings and the adrenal medulla.
Nemet’s data demonstrated that elevated circulating levels of PAG are associated with a higher risk of heart failure and lead to worse outcomes for patients with heart failure. They also showed that the gut microbial PAG signaling pathway was mechanistically linked to numerous heart failure–related features and cardiovascular disease risks.9
A 2023 paper, also from the Hazen lab, by Romano et al, confirmed that PAG is correlated with atherothrombotic heart disease in humans and mechanistically linked to cardiovascular-disease pathogenesis in animal models via modulation of adrenergic receptor signaling. Investigators monitored 2 human cohorts for an association between plasma PAG and heart failure. They watched both a US cohort (n=3,256) and a European cohort (n=829). PAG levels were dose-dependently associated with heart failure presence and severity in a manner independent of traditional risk factors and renal function in both cohorts. At this point, the authors were clear that “Modulating the gut microbiome in general, and PAG production specifically, may represent a potential therapeutic target for modulating HF.”10
In August 2024, Saha et al, again from the Hazan lab, had a new paper published. It provides the most detailed explanation to date of the mechanisms as to how PAG creates a problem. Saha et al, writing in Nature Communications, have fine-tuned and verified their earlier hypothesis about how PAG binds to specific adrenergic receptors. They demonstrated that PAG exposure “interacts with previously undiscovered locations on beta-2 adrenergic receptors on heart cells once it enters the circulation.” Binding onto these receptors quickly increased the production of cyclic adenosine monophosphate (cAMP) by cells expressing beta-2 adrenergic receptors (β2ARs) but not β1ARs. “PAG was shown to interact with beta-2 adrenergic receptors to influence how forcefully the heart muscle cells contract – a process that investigators believe contributes to heart failure.”11 That PAG acts on the β2 receptor and not the β1 receptor is of great interest as it may lead to development of targeted drugs that may neutralize the harmful effects of PAG.12
This, in part, explains the benefit seen with medications called beta-blockers, which target these adrenergic receptors and block the body’s fight-or-flight response. It may be the repeated stimulation of these receptors by PAG leads to chronic damage to the heart and accelerating development of heart failure. Beta-blockers block the β2 adrenergic receptors, preventing adrenaline from binding to the receptor sites. Beta-blockers appear to neutralize the harmful effects of PAG that lead to heart failure, at least in preclinical experiments. In this recent study, Saha et al have identified additional binding sites on the receptor that may allow blocking of PAG activation while allowing normal activation by epinephrine. This may allow the development of drugs that are more nuanced than current beta-blockers. Dr Hazen is quoted in an August 20, 2024, article in Science Daily as saying:
“A beta-blocker that is more targeted in blocking the harmful signaling from the adrenergic receptors, but allowing the healthy signals through, would be an entirely new approach for treating or preventing cardiovascular disease risk. This would have the potential to improve the quality of life for patients who rely on beta-blockers to calm down their body’s stress responses.”13
One may find this information interesting but consider it to be about drug development and not naturopathic medicine. The challenge in following mainstream research is often interpreting how it fits into naturopathic practice. How do we translate new knowledge about drugs or, in this case, potential drugs into what we consider natural medicine?
If you have patients who take beta-blockers, they will confirm that these drugs make it difficult to get excited about much anything. The idea that potential new drugs might block these alternate receptors and provide CVD protection while still allowing the experience of enthusiasm in life will certainly strike these patients as welcome, if not exciting, news even if they might not be able to currently show it.
Before we consider the clinical implications of this study, we still must pause and ask ourselves whether the association seen by Heianza et al in their results is a causative association or just a correlation between 2 phenomena that are dependent on some yet unrevealed factors or the result of unknown confounders? Some of us have mistakenly jumped to hasty conclusions in the past and now, as a result, might take a moment to dampen our enthusiasm by reading through the Bradford-Hill criteria before running after the next exciting idea.
The first question that must be asked is whether the demonstrated relationship between PAG and CHD is one of correlation, causation, or chance? Do elevated levels of PAG cause coronary heart disease or could there be some other relationship occurring? The meaning of these 2 words is specific: Correlation means there is a statistical association between the variables, while causation means that a change in 1 variable causes a change in another variable. In this case, is it the increase in the bacterial metabolite PAG that leads to an increase in CHD or is it the other way around? Could CHD raise PAG levels? Other relationships might also account for this association, including pure chance.
Upon consideration of the depth of research on this relationship and the delineation of underlying mechanisms, it is most likely that the demonstrated association is causative—that is, elevated PAG increases risk of CHD, and PAG levels increase with higher intake of animal protein, which supplies additional dietary phenylalanine.
The evidence suggests the association is strong, consistent, specific, and displays temporality, meaning the relationship is sequential, with PAG exposure preceding development of CHD. We see a biological gradient in which greater exposure equates with greater risk of disease. The explanations for the phenomenon are plausible and coherent. The experiments published to date are compelling.
There are also multiple in vitro and ex vivo experiments in support of this cause-and-effect hypothesis and detailed explanations and proofs of the mechanism by which PAG causes this disease.
However, we should not expect to see confirmation through human randomized, controlled trials because it would be unethical to intentionally give a study participant PAG, as it is assumed to cause harm.
These novel developments in our understanding of heart disease are both new and quickly evolving. The obvious implication is that we now have a compelling explanation and justification for dietary interventions that lower phenylalanine consumption, such as reducing animal products, in particular, red meat.
We can translate certain other implications of this information on PAG into naturopathic practice as well. We often work with patients who follow ketogenic diets. Some of us encourage patients to do so. Often these patients report having more energy, stamina, and a feeling of being alive. If we hypothesize that these keto diets supply more phenylalanine, which is fermented and eventually converted to PAG, we can understand their subjective experience. These patients are enjoying the excitement of steady, low-level adrenergic stimulation. No wonder some advocates seem almost addicted to the diet. Will this PAG stimulation over the long term increase cardiac problems? We don’t have data yet as to whether following a ketogenic diet increases PAG, but we ought to wonder, particularly for patients consuming meats.
The knowledge that a diabetic who tightly controls their blood sugar is not gaining the benefit of a reduction in CVD risk might lead us to question the value of low-carb diets for these patients, especially if such a diet increases PAG levels.
The challenge is that at this point, we don’t really know how much dietary shifts affect PAG levels. We do not have access to testing that will tell us a patient’s PAG level or allow us to track changes over time. Nor do we know how quickly levels will shift with dietary interventions.
The idea from the Hazen lab that a drug might be developed that binds to and blocks the PAG binding site is intriguing. Often as not, evolution has gifted us with a plant-derived chemical that might latch to such a receptor and produce a similar result as the pharmaceutical Hazen is hoping to develop. Although no one appears to be looking for a phytochemical with such an action yet, when they do, I would suggest that they take a hard look at cocoa constituents. We know there is something about chocolate that seems to interrupt the normal age-related progression of CVD. Perhaps that interruption has something to do with blocking this PAG effect?
The nonsugar sweetener aspartame requires a package warning label stating that it contains phenylalanine. This is the amino acid at the head of the metabolic chain that ends in PAG.14 Will consuming aspartame raise PAG levels? At this point, we might suspect so but don’t know. No studies have linked aspartame consumption with elevated PAG levels. Yet a 2022 paper by Debras et al should make us wonder about the possibility. Their study tracked a large French cohort (N=103,388), collecting data totaling 904,206 person years. The team reported that in those people using artificial sweeteners, “Aspartame intake was associated with increased risk of cerebrovascular events (1.17, 1.03 to 1.33, P=0.02; incidence rates 186 and 151 per 100,000 person years in higher and non-consumers, respectively).”15 Also worth mentioning is a 2021 paper that reported higher PAG levels in patients who have suffered ischemic strokes.16 Few naturopaths encourage patients to consume artificial sweeteners, but perhaps we should make more of a fuss for patients at risk for heart disease or stroke.
To my knowledge, there is not yet an easily available way to test patients’ PAG levels. There are 24-hour urine tests that measure urinary PAG levels to assess kidney function and adjust the doses of phenylbutyric acid or glycerol phenylbutyrate when treating patients with urea-cycle disorders.17,18 But that isn’t the information that we are seeking. Hazen’s lab used stable-isotope-dilution LC/MS/MS to quantify PAG levels in their research. Similar methodology is also used to quantify TNMO levels.19 An enterprising lab with such equipment might offer to test both metabolites.
Hardly anyone is talking about phenylacetylglutamine yet, but it’s about to become a big thing. It might behoove us to not just learn to spell the name but also to practice saying that name out loud.
Conflict of Interest Disclosure
The author declares no conflict of interest.
