Let me tell a recent interest, that is a chemical that I had not heard about, and certainly not thought about until I chanced upon a recent paper, well, series of papers written by researchers working at the Stanley Hazen Lab at the Cleveland Clinic. I have to pause a moment to dredge the name from memory. Phenylacetylglutamine. It doesn’t roll off the tongue, at least not with practice. I abbreviate it as PAG. Hazen and his colleagues often abbreviate it as PAGln. I find that confusing, unclear if the fourth letter is 1 or l.) 

PAG is a metabolite produced by intestinal bacteria from dietary phenylalanine.

Well specifically from unabsorbed phenylalanine that remains in the intestines and is fermented by gut microbes to produce phenylpyruvic acid and subsequently phenylacetic acid. The main sources of the dietary phenylalanine are protein-rich foods, such as meat, milk, and eggs.¹ Aspartame, the artificial sweetener, also contains phenylalanine but at this point I haven’t found a citation that clarifies how much of an impact on PAG levels aspartame use has.

PAG is important because a few years back, researchers In Hazen’s lab realized blood levels were closely correlated with risk for cardiovascular disease (CVD). Prior to that the main interest in PAG was as a measure of kidney function. Healthy kidneys were able to filter and excrete PAG out of the body. People with chronic kidney disease (CKD) could not. Back in 2016, Ruben Poesen et al. reported that higher levels of PAG were associated with higher CVD risk in patients with CKD. Poesen had monitored PAG levels in patients with CKD, (n=488). PAG levels were associated with higher mortality (HR per 1-SD increase, 1.77; P=0.003) and greater risk of cardiovascular disease (HR 1.79; P<0.001).²

Hazen’s group has published a series of papers on PAG’s effect on CVD. In their first report published in 2020, Ina Nemet et al. described how they discovered PAG’s importance. They utilized a technique called untargeted metabolomics in which the total metabolomes of control and test groups are analyzed to identify differences between their metabolite profiles. Metabolome means the total number of metabolites present within an organism, cell, or tissue. We’ve entered an era where the metabolites produced by both the patient and the multitudes of other organisms they host in their bodies are considered relevant to health. Such metabolomic differences between individuals are thought relevant to specific biological conditions. Nemet’s group was interested in the metabolome differences that correlate with CVD. People with type-2-diabetes were selected for this initial study, because diabetics have high, but unexplained risks for CVD even when their blood sugar levels are kept under tight control. Nemet realized there was some other factor at work aside from high blood sugars, that put these people at risk for heart problems. 

Untargeted metabolomic analysis was performed on blood samples from a “Discovery Cohort” of diabetics (n=1,162). The metabolites most associated with their eventual major adverse cardiac events over a three year follow up were identified. The top five suspect compounds most associated with events included several we’re familiar with, including trimethylamine-N-oxide (TMAO). Among the unknowns though was this stuff, phenylacetylglutamine (PAG). It became a top suspect when hazard ratios were calculated as the study participants with high levels had twice to three times the risk of having a major adverse cardiac event during the study.(HR 95%: Confidence interval for incident (3 year) risk of MACE (major adverse cardiac event³) of 2.69 (1.61–4.52); P<0.0001).

Once PAG was singled out and identified, the Hazen lab undertook to determine what and 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 commentator summed up those results writing, “Platelets get gutted by PAG.”⁴ 

Injection of PAG into mice sped up clotting in the carotid artery after an injury, an effect that mimics a coronary event. PAG acts by binding to adrenergic receptors. Those are the cell surface glycoproteins that recognize and selectively bind the catecholamines, that is norepinephrine and epinephrine, or what most people still call adrenaline, after they are released during a fight or flight response.

Elevated PAG was clearly associated with a higher risk of heart failure, and led to worse outcomes for patients with heart failure.  They also showed that the gut microbial PAG signaling pathway was linked to numerous heart failure-related features and cardiovascular disease risks.⁵

Another important paper, this one published in 2023 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 through this modulation of adrenergic receptor signaling. Data was gathered from two human cohorts to seek an association between plasma PAG and heart failure. A U.S. cohort (n=3256) and a European cohort (n=829) were both watched. PAG levels were dose-dependently associated with heart failure presence and severity in a manner that was 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.”⁶

In August 2024 a new paper, this one by Saha et al., but again from the Stanley Hazan Lab, was published that adds to our understanding. The earlier reports about how PAG binds to specific adrenergic receptors have been expanded upon. 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 cAMP by cells expressing β2AR receptors but not β1AR receptors. “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.”⁷

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.⁸

Drugs known as beta-blockers have been used for years to reduce risk of adverse heart problems. Around thirty million US adults are taking beta-blockers.

I need to back up and review some of these names of receptor sites and what they signify before moving forward here. Recall the metaphor that receptors are like locks and require the right chemical key, to be activated to trigger some signal within the cell.

The beta-blocker drugs attach to the receptor sites but don’t activate the receptor, and don’t trigger a signal. They prevent the actual hormones from being able to trigger a response. Chemicals that block a receptor site and block action are called antagonists. They are like leaving a broken key in a lock. No one else can open it.

Adrenergic receptors get their name because they are triggered by adrenaline (well epinephrine these days). Adrenaline activates all of the adrenergic receptors kind of like a master key opens all the locks. There are three subtypes to beta-receptors:

Beta-1 receptors are mainly heart and kidneys and when activated increase heart rate, heart pumping force and get the kidneys to release renin from the kidneys.

Beta-2 receptors are found mainly in smooth muscle and when activated relax the smooth muscles in your respiratory system (making it easier to breathe… ie think asthma), relax smooth muscles in the vascular system, lowering blood pressure, in the liver convert glycogen to glucose, and in the heart increase the pumping force.

Beta-3 receptors are located mostly in fat cells and the bladder and when triggered cause fat cells to break down, relax the bladder to increase capacity and cause tremors in the nervous system.⁹

Beta Blockers can pose a special challenge to diabetics as they may block the rapid heart rate and other signals that may warn them of hypoglycemia.¹⁰

If PAG triggers the adrenergic receptors in the heart to chronically stimulate a low-level version of the body’s fight or flight response, this explains how beta-blockers protect against CVD. Repeated PAG stimulation of these receptors leads to chronic damage to the heart and accelerates development of heart failure. Beta-blockers block the beta-2 adrenergic receptors preventing adrenaline/epinephrine from binding to the receptor sites. Beta-blockers also appear to block PAG and neutralize the harmful effects of PAG. Saha et al identified additional binding sites on the receptor that may allow blocking of PAG activation while allowing normal activation by epinephrine. This may allow development of drugs that are more nuanced than the 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.”¹¹

If you’ve never taken a beta-blocker or haven’t heard what the drugs feel like from patients, you might not understand why people dislike taking these drugs. When adrenergic receptors are blocked, nothing is exciting. I recall one patient describing it as ‘life loses its excitement: every experience is decaffeinated.” 

One may find this information interesting but consider it to be relevant to drug development and not naturopathic medicine. The challenge in following mainstream research is often in seeing how it might be translated into naturopathic practice. How do we translate new knowledge about drugs, or in this case, potential drugs, into what we consider natural medicine?

The idea that potential new drugs might be developed to block these alternate receptors and provide CVD protection while still allowing the experience of enthusiasm in life will strike these patients as welcome if not exciting news, even if they might not be able to currently show it.

The obvious implication of these developments is that we now have a compelling explanation and justification for dietary interventions to lower phenylalanine consumption, by reducing animal products, in particular red meat. 

We should pause though to consider another recent study, Heianza et al, published in July 2024 before ruminating further. This study confirmed two things; first, that people with higher levels of PAG in their blood were at greater risk of coronary heart disease (CHD) and second, that a plant-based diet vs. a high meat diet might lower the risk of heart disease. Recall that meat and animal protein are the primary source of phenylalanine, the precursor amino acid that is metabolized into PAG via gut fermentation.

This Heianza study looked at data from two groups of women. One group [1520 women (760 cases and 760 controls)] were part of the Nurses’ Health Study (NHS) and the second group were enrolled in the Women’s Lifestyle Validation Study (n=725).

Sure enough, 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/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 (PDI) 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. In other words, what we have long assumed, that a high animal protein diet isn’t good for your heart and that eating a plant diet is.¹²

So, what’s all of this tell us? Well first the obvious: a plant-based diet might protect from heart disease. It suggests that those eating an animal based diet might be protected to some degree from the effects of elevated PAG.

As naturopathic doctors, we often work with patients who follow ketogenic diets. These patients commonly report having more energy, stamina, and a feeling of being alive. Given what we now know from the Hazen and the Heianza studies we might hypothesize that keto diets supply more phenylalanine, which is fermented and converted to PAG and this might explain their subjective experience from the keto diet. These patients may be enjoying the excitement of having a steady low-level adrenergic stimulation. No wonder they seem almost addicted to the diet. I have not come across any confirmation to this idea in the scientific literature. I recall that half a dozen years back when writing for the Natural Medicine Journal, I reviewed research by Christine Olson on using the ketogenic diet to reduce seizures in children (https://www.naturalmedicinejournal.com/journal/ketogenic-diet-improves-seizures).

At the time there were some mouse studies suggesting that the keto diet increased GABA levels in the brain.¹³ It might be that keto diet adherents are experiencing two distinct effects, an adrenergic effect accompanied by increased GABA action. No wonder they come across so strongly in favor of their diet when proselytizing to non-adherents.

Will following a ketogenic raise PAG levels and over the long term increase cardiac problems? We don’t have data yet as to say. We can say that a plant-based diet will probably lower heart disease risk.

This brings up a problem. At this point testing PAG levels is an esoteric business and I have not found a commercial laboratory that offers testing. I assume someone will fill the need soon enough.

The idea from the Hazen Lab that a drug might be developed that specifically binds to and blocks the PAG binding site is intriguing. That’s because, often as not, evolution has gifted us with a plant derived chemical that might have a parallel action, that is some botanical might do the same thing and produce a similar result as the pharmaceutical Hazen hopes to develop. No one yet appears to be hunting for such a phytochemical, but when they do start looking, it might be worth examining cacao’s constituents. There is something about chocolate that interrupts the normal age-related progression of CVD. Perhaps that action is related to acting as a PAG agonist?

I don’t know how much phenylalanine is required to raise PAG to risky levels. So we can’t guess how much aspartame, the non-sugar low calorie sweetener, will supply too much phenylalanine and result in increased PAG.¹⁴ No studies have linked aspartame consumption with elevated PAG levels. Two reports make one suspicious though: a 2022 paper by Debras et al., which tracked a large French cohort (n=103 388), collecting data totaling 904,206 person years, and reported that, “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 Second is a 2021 paper that reported patients who have suffered ischemic strokes have high PAG levels.¹⁶ Few of us encourage patients to consume artificial sweeteners but perhaps we should make more of an effort for patients at risk of heart disease or stroke.

As mentioned, 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 to assess kidney function and adjust phenylbutyric acid or glycerol phenylbutyrate dosing.^17,18 That isn’t what we need. Hazen’s lab uses stable-isotope-dilution LC/MS/MS to quantify PAG levels in their research studies. Similar methodology is also used to quantify TNMO levels.¹⁹ An enterprising lab with such equipment might offer to test both metabolites at the same time. 

Hardly anyone is talking about phenylalkylglutamine yet, but there is good reason to think it will become a big thing. It might behoove us to not just learn to spell the name correctly but also to practice saying it out loud. That way we’ll sound like we know what we’re talking about.

1.  Note that various forms of phenylalanine are sold as nutritional supplements. It has at times been experimented for treatment of depression. [PRATT RT, GARDINER D, CURZON G, PIERCY MF, CUMINGS JN. PHENYLALANINE TOLERANCE IN ENDOGENOUS DEPRESSION. Br J Psychiatry. 1963 Sep;109:624-8.]
2. Poesen R, Claes K, Evenepoel P, et al. Microbiota-Derived Phenylacetylglutamine Associates with Overall Mortality and Cardiovascular Disease in Patients with CKD. J Am Soc Nephrol. 2016 Nov;27(11):3479-3487. 
3. Sometimes MACE means “major acute coronary event” and not “major adverse coronary event”: Bosco E, Hsueh L, McConeghy KW, Gravenstein S, Saade E. Major adverse cardiovascular event definitions used in observational analysis of administrative databases: a systematic review. BMC Med Res Methodol. 2021 Nov 6;21(1):241.
4. Parra-Izquierdo I, Bradley R, Aslan JE. Platelets get gutted by PAG. Platelets. 2020 Jul 3;31(5):618-620.
5. Nemet I, Saha PP, Gupta N, et al. A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors. Cell. 2020 Mar 5;180(5):862-877.e22
6. Romano KA, Nemet I, Prasad Saha P, et al. Gut Microbiota-Generated Phenylacetylglutamine and Heart Failure. Circ Heart Fail. 2023 Jan;16(1):e009972.
7. EurekAlert AAAS: Gut microbial pathway identified as target for improved heart disease treatment. August 6, 2024. Cleveland Clinic. https://www.eurekalert.org/news-releases/1055118 accessed Aug 27, 2024
8. Saha PP, Gogonea V, Sweet W, et al. Gut microbe-generated phenylacetylglutamine is an endogenous allosteric modulator of β2-adrenergic receptors. Nat Commun. 2024 Aug 6;15(1):6696.
9. https://my.clevelandclinic.org/health/treatments/22318-beta-blockers Accessed 9/1/2024.
10. https://www.mayoclinic.org/diseases-conditions/high-blood-pressure/in-depth/beta-blockers/art-20044522 Accessed 9/1/2024.
11. Science Daily, August 20, 2024. Gut microbial pathway identified as target for improved heart disease treatment. Accessed Aug 28, 2024. https://www.sciencedaily.com/releases/2024/08/240820124432.htm
12. 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 Jul 30:dgae525.
13. Olson CA, Vuong HE, Yano JM, Liang QY, Nusbaum DJ, Hsiao EY. The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet. Cell. 2018 Jul 12;174(2):497.
14. Krishnamoorthy NK, Kalyan M, Hediyal TAet al. Role of the Gut Bacteria-Derived Metabolite Phenylacetylglutamine in Health and Diseases. ACS Omega. 2024 Jan 8;9(3):3164-3172. https://pubs.acs.org/doi/10.1021/acsomega.3c08184
15. Debras C, Chazelas E, Sellem L, et al. Artificial sweeteners and risk of cardiovascular diseases: results from the prospective NutriNet-Santé cohort. BMJ. 2022 Sep 7;378:e071204.
16. Yu F, Li X, Feng X, et al.. Phenylacetylglutamine, a Novel Biomarker in Acute Ischemic Stroke. Front Cardiovasc Med. 2021 Dec 23;8:798765.
17. Brusilow SW. Phenylacetylglutamine may replace urea as a vehicle for waste nitrogen excretion. Pediatr Res. 1991 Feb;29(2):147-50. doi: 10.1203/00006450-199102000-00009. PMID: 2014149.
18. Mokhtarani M, Diaz GA, Lichter-Konecki U, Berry SA, Bartley J, McCandless SE, Smith W, Harding C, Le Mons C, Coakley DF, Lee B, Scharschmidt BF. Urinary phenylacetylglutamine (U-PAGN) concentration as biomarker for adherence in patients with urea cycle disorders (UCD) treated with glycerol phenylbutyrate. Mol Genet Metab Rep. 2015 Sep 27;5:12-14. doi: 10.1016/j.ymgmr.2015.09.003. PMID: 28649536; PMCID: PMC5471406.
19. Tang Y, Zou Y, Cui J, et al. Analysis of two intestinal bacterial metabolites (trimethylamine N-oxide and phenylacetylglutamine) in human serum samples of patients with T2DM and AMI using a liquid chromatography tandem mass spectrometry method. Clin Chim Acta. 2022 Nov 1;536:162-168.