• History & Evolution
• Biosynthesis vs. dietary uptake
• Methionine and the microbiome
• Methionine and DNA methylation
• Methionine and cardiovascular disease
• Methionine and neurogenerative disorders
• Methionine and cancer

History & Evolution

1921: discovery (Mueller, J., 1921) | 1928: structure identified (Barger, G., 1928) | 1945-7: first synthesis (Livak, J. et al., 1945).

Methionine’s story begins just over a century ago, when it was discovered in casein by JH Mueller in 1921 (Mueller, J., 1921). In 1928, Barger and Coyne determined the chemical structure of what was until then known as γ-methylthiol-α-aminobutyric acid and suggested a more straightforward name: methionine (Barger, G., 1928). By the 1930s, methionine was recognized as an essential and limiting amino acid involved in protein synthesis, with subsequent research focusing on its vital role in human and animal diets (Rose, W., 1976). In the aftermath of World War II, protein shortages and widespread malnutrition prompted the hunt for a synthetic form of methionine (Livak, J. et al., 1945).

Now, over one million tons of methionine are produced each year from petroleum, with many applications including poultry feed, flavor enhancers, cosmetics, drugs and pesticides (Neubauer, C., 2021).

Methionine is found in humans, animals, plants and bacteria. In humans, methionine has been described as a “double-edged sword” (Navik, U. et al., 2021). It plays a crucial role in cellular, metabolic, epigenetic and genomic processes, influencing a broad range of physiological functions. It has been shown to support immune function, digestion, insulin resistance, to extend lifespan, and to reduce DNA damage and carcinogenesis (Martínez, Y. et al., 2017). However, excessive amounts can be toxic, with adverse effects including increased cholesterol and hyperhomocysteinemia (Navik, U. et al., 2021); (Garlick, P., 2006).

There is a theory that not only is this ancient metabolite essential for life, but – as a self-activating and sulfur containing amino acid – it may have triggered the development of life on Earth itself (Heinen, W., 1996).

Biosynthesis vs. dietary uptake

As an essential amino acid, methionine must be obtained via the diet and gut microbiota (Parkhitko, A. et al., 2019). Primary dietary sources of methionine include eggs, fish, meat, dairy and plant protein sources (Navik, U. et al., 2021) (Rajavel, E., 2020). In bacteria, methionine is synthesized from cysteine and aspartic acid. Plasma methionine concentration in healthy subjects ranges from 13 to 45 µM (Navik, U. et al., 2021).

The Quantitative Metabolomics Database (QMDB), cataloguing the concentrations of over 600 metabolites in healthy individuals reports average levels of 28 µM for women and 30 µM for men.

Methionine is one of four sulfur-containing amino acids, and, along with cysteine, one of two incorporated into proteins (Brosnan, T., 2006). Daily dietary requirements for methionine are usually taken together with cysteine, and the total sulfur amino acid requirement (TSAA) is 13-15 mg/kg/d for adults (Rajavel, E., 2020).

Methionine metabolism involves three key stages (Parkhitko, A. et al., 2019); (Lauinger, L., 2021):
– the methionine cycle,
– the transsulfuration/transmethylation pathway
– the methionine salvage cycle

In the methionine cycle, methionine and adenosine triphosphate (ATP) are converted into the methyl donor S-adenosylmethionine (SAM) by the enzyme methionine adenosyltransferase. After transferring the methyl group, SAM is converted to S-adenosylhomocysteine (SAH) and hydrolyzed into adenosine and homocysteine. This homocysteine can be remethylated to methionine by the methionine synthase enzyme (with vitamin B12 as a coenzyme), which completes the cycle, or diverted to the transssulfuration pathway to form cysteine (Rajavel, E., 2020); (Parkhitko, A. et al., 2019). In the salvage cycle, methionine is regenerated from decarboxylated SAM, following a series of enzymatic steps involving polyamine synthesis.

Methionine is a precursor to other molecules including antioxidants such as glutathione and cystathionine, and amino acids including cysteine, carnitine, taurine and creatine (Navik, U. et al., 2021).

Methionine and the microbiome

Gut microbiota play a major role in methionine metabolism, with around 20% of dietary methionine metabolized in the gut (Wu, X. et al., 2022); (Bauchart C. et al., 2009). In turn, methionine metabolism influences gut health, as evidenced by the interactions between methionine levels, microbial composition and disease states.

A multi-omics study comparing vegan and omnivore diets found that methionine positively correlates with Bacteroides, Blautia, Dorea, Lachnoclostridium and Fusicatenibacter, with all except Bacteroides being more abundant in omnivores (Prochazkova, M. et al., 2022). Another study found that restricting sulfur amino acids like methionine increases the abundance of Firmicutes, Clostridaceae and Turicibacteraceae, while decreasing Verrucomicrobia, compared to a low-calorie diet (Nichenametla, S. et al., 2021). These findings show how methionine intake influences the diversity and abundance of bacterial species in the gut.

Animal studies have shown that restricting dietary methionine, e.g. through a plant-based diet, may improve gut health. Methionine restriction in mice was found to increase short chain fatty acid (SCFA)-producing bacteria such as Bifidobacterium, Lactobacillus, Bacteroides, Roseburia, Coprococcus, and Ruminococcus, and increase inflammation-inhibiting bacteria like Oscillospira and Corynebacterium (Yang, Y. et al., 2019). In contrast, the high fat diet reduced SCFA production and induced gut dysbiosis.

Methionine appears to play a role in several inflammatory diseases associated with disrupted gut microbiota (Wu, X. et al., 2022). For example, a study in patients with inflammatory bowel disease (IBD) showed reduced levels of methionine, serine and sarcosine, along with fewer SCFA-producing gut bacteria (Borren, N. et al., 2021). Another study found changes in methionine and homoserine levels in patients with Parkinson’s disease (PD) (Hertel, J. et al., 2019). PD patients showed an increase in Akkermansia muciniphila, which is involved in sulfur metabolism. This contributed more than 70% of potential methionine production, suggesting a clear link between gut microbiota and the methionine cycle (Wu, X. et al., 2022).

Methionine and DNA methylation

Methionine is essential for DNA methylation, a key epigenetic mechanism affecting gene expression related to growth and development (Lauinger, L., 2021). In this process, DNA methyltransferases transfer a methyl group from SAM to the C-5 position of the pyrimidine ring of cytosine (Jin, B. et al., 2011). Changes in dietary methionine change the ratio of SAM to SAH (the “methylation index”), which influences methylation capacity. This can be measured using immunoassays to detect levels of SAM and SAH in blood and tissue (Hao, X. et al., 2016).

Dysregulation of DNA methylation is associated with some cancers (Bauchart C. et al., 2009). Studies suggest that methionine supplementation can affect DNA methylation, with both positive and negative effects depending on the gene region affected (Waterland, R., 2006).

More research is needed to fully understand methionine’s role in DNA methylation. Metabolomics offers a useful tool with which to understand epigenomics and DNA methylation in more detail, especially when combined with other omics. For example, a 2020 study combined metabolomics, proteomics and genomics to show that activated T cells use methionine to synthesize SAM, identifying methionine as a key nutritional factor in shaping T helper cell activity through histone methylation (Roy, D. et al., 2020). The findings suggest restricting dietary methionine could be a viable intervention in the treatment of autoimmune conditions such as multiple sclerosis.

Layering insights from different omics analyses in this way gives a fuller picture of the interactions between metabolites, proteins, genes and environmental factors that drive disease processes.

Methionine and cardiovascular disease

Too much methionine may increase the risk of cardiovascular disease (CVD). Methionine is involved in DNA synthesis, lipid regulation, oxidative defense and cellular regulation – processes all thought to play a role in CVD and other diseases (Aissa, A. et al., 2017). Impaired DNA methylation may also be a factor, as this increases circulating LDL cholesterol and lowers HDL cholesterol, both of which are risk factors for CVD (Calderón, A. et al., 2020); (Blachier, F. et al., 2020).

Another key mechanism is linked to methionine’s downstream metabolite homocysteine, which is known to be vasotoxic (Troen, A. et al., 2003). In excess, homocysteine can cause hyperhomocysteinemia, which is a well-established risk factor for CVD. A longitudinal population-based study found that a lower concentration of methionine, higher concentration of homocysteine and lower ratio of methionine to homocysteine were associated with an increased risk of CVD (Calderón, A. et al., 2020).

Excess methionine may also damage endothelial cells and increase plasma lipid levels, which both contribute to atherosclerosis. High levels of dietary methionine have been found to increase the risk of acute coronary events in middle-aged men (Virtanen, J. et al., 2006).

By contrast, methionine restriction appears to delay development of CVD and associated risk factors such as obesity (Zhang, Y. et al., 2022).

Methionine and neurogenerative disorders

Excess methionine is linked to neurodevelopmental disorders, like autism and schizophrenia, and neurodegenerative disorders such as Alzheimer’s disease (AD) and PD (Alachkar, A. et al., 2022); (Hertel, J. et al., 2019). A 2023 study involving animal and human AD phenotypes found that methionine intake is associated with mild cognitive impairment, and methionine restriction improves cognitive function (Xi, Y. et al., 2022).

More research is needed to understand the mechanisms, but again, methionine’s role in DNA methylation and homocysteine production may be relevant. A population-based study found links between methionine, homocysteine and dementia development, suggesting that a higher methionine to homocysteine ratio may reduce brain atrophy and lower the risk of dementia (Hooshmand, B. et al., 2019).

As noted above, a longitudinal metabolomics study investigated links between PD, sulfur metabolism and the gut microbiome (Hertel, J. et al., 2019). This showed differences between methionine and cysteine production via cystathionine in PD patients and healthy subjects. Using multiomics, the researchers were able to identify patterns in microbial-host sulfur metabolism that may contribute to PD severity.

Methionine and cancer

Methionine restriction is also an “exciting potential tool in the treatment of cancer” (Wanders, D. et al., 2020). Methionine inhibits cancer cell proliferation and growth in several types of cancer, without damaging healthy cells (in the presence of homocysteine). It also appears to improve efficacy of chemotherapy and radiation therapy in animal models. A 2020 review summarized evidence showing that methionine restriction could inhibit cancer development and/or progression in prostate cancer, breast cancer and colorectal cancer (Wanders, D. et al., 2020). Possible mechanisms vary by cancer type. It may be that methionine restriction inhibits polyamine biosynthesis, induces apoptosis, alters DNA methylation and glutathione formation, and reduces activity of thymidylate synthase, the enzyme that converts dUMP to dTMP.

 
More about the Metabolite of the Month
Reference ranges of methionine in healthy humans
Quantify the impact of methionine

References

Aissa, A. et al.: Methionine-supplemented diet affects the expression of cardiovascular disease-related genes and increases inflammatory cytokines in mice heart. (2017). J Toxicol Environ Health A | DOI: 10.1080/15287394.2017.1357366

Alachkar, A. et al.: L-methionine enhances neuroinflammation and impairs neurogenesis: implication for alzheimer’s disease. (2022). Journal of Neuroimmunology | DOI: 10.1016/j.jneuroim.2022.577843

Barger, G. et al.: The amino-acid methionine; constitution and synthesis. (1928). Biochem J. | DOI: 10.1042/bj0221417

Bauchart, C. et al.: Intestinal metabolism of sulfur amino acids. (2009). Research Reviews | DOI: 10.1017/S0954422409990138

Blachier, F. et al.: Sulfur-containing amino Acids and lipid metabolism. (2020). The Journal of Nutrition | DOI: 10.1093/jn/nxaa243

Borren, N. et al.: Alterations in fecal microbiomes and serum metabolomes of fatigued patients with quiescent inflammatory bowel diseases. (2021). Clin Gastroenterol Hepatol | DOI: 10.1016/j.cgh.2020.03.013

Brosnan, T. et al.: The sulfur-containing amino acids: an overview. (2006). J Nutr | DOI: 10.1093/jn/136.6.1636S

Calderón, A. et al.: Association of homocysteine, methionine, and mthfr 677c>t polymorphism with rate of cardiovascular multimorbidity development in older adults in sweden. (2020). JAMA Netw Open | DOI: 10.1001/jamanetworkopen.2020.5316

Garlick, P. et al.: Toxicity of methionine in humans. (2006). The Journal of Nutrition | DOI: 10.1093/jn/136.6.1722S

Hao, X. et al.: Immunoassay of S-adenosylmethionine and S-adenosylhomocysteine: the methylation index as a biomarker for disease and health status. (2016). BMC Res Notes | DOI: 10.1186/s13104-016-2296-8

Heinen, W. et al.: Organic sulfur compounds resulting from the interaction of iron sulfide, hydrogen sulfide and carbon dioxide in an anaerobic aqueous environment. (1996). Orig Life Evol Biosph | DOI: 10.1007/BF01809852

Hertel, J. et al.: Integrated analyses of microbiome and longitudinal metabolome data reveal microbial-host interactions on sulfur metabolism in parkinson’s disease. (2019). Cell Rep | DOI: 10.1016/j.celrep.2019.10.035

Hooshmand, B. et al.: Association of methionine to homocysteine status with brain magnetic resonance imaging measures and risk of dementia. (2019). JAMA Psychiatry | DOI: 10.1001/jamapsychiatry.2019.1694

Jin, B. et al.: DNA methylation. (2011). Genes Cancer | DOI: 10.1177/1947601910393957

Kaiser, P. et al.: Methionine dependence of cancer. (2020). Biomolecules | DOI: 10.3390/biom10040568

Lauinger, L. et al.: Sensing and signaling of methionine metabolism. (2021). Metabolites | DOI: 10.3390/metabo11020083

Livak, J. et al.: Synthesis of dl-methionine. (1945). J Am Chem Soc | DOI: 10.1021/ja01228a050

Martínez, Y. et al.: The role of methionine on metabolism, oxidative stress, and diseases. (2017). Amino Acids | DOI: 10.1007/s00726-017-2494-2

Mueller, J. et al.: Growth-determining substances in bacteriological culture media. (1921). Proceedings of the Society for Experimental Biology and Medicine | DOI: 10.3181/00379727-18-113

Navik, U. et al.: Methionine as a double-edged sword in health and disease: Current perspective and future challenges. (2021). Ageing Research Reviews | DOI: 10.1016/j.arr.2021.101500

Neubauer, C. et al.: A planetary health perspective on synthetic methionine. (2021). The Lancet Planetary Health | DOI: 10.1016/S2542-5196(21)00138-8

Nichenametla, S. et al.: Differential Effects of Sulfur Amino Acid-Restricted and Low-Calorie Diets on Gut Microbiome Profile and Bile Acid Composition in Male C57BL6/J Mice. (2021). The Journals of Gerontology: Series A | DOI: 10.1093/gerona/glaa270

Parkhitko, A. et al.: Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species. (2019). Aging Cell | DOI: 10.1111/acel.13034

Prochazkova, M. et al.: Vegan diet Is associated with favorable effects on the metabolic performance of intestinal microbiota: A cross-sectional multi-omics study. Front. (2022). Nutr | DOI: 10.3389/fnut.2021.783302

Rajavel, E. et al.: Methionine Nutrition and Metabolism: Insights from Animal Studies to Inform Human Nutrition. (2020). The Journal of Nutrition | DOI: 10.1093/jn/nxaa155

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Roy, D. et al.: Methionine metabolism shapes t helper cell responses through regulation of epigenetic reprogramming. (2020). Cell Metab | DOI: 10.1016/j.cmet.2020.01.006

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• History & Evolution
• Biosynthesis vs. dietary uptake
• Indoxyl sulfate and the microbiome
• Indoxyl sulfate and nephrology
• Indoxyl sulfate and cardiovascular disease
• Indoxyl sulfate and bone disease
• Indoxyl sulfate and neurological disease

History & Evolution

1911: discovery of indoxyl sulfate (Obermayer, F. and Popper, H., 1911) | 1936: confirmation of liver’s role in indoxyl sulfate production (Houssay, B., 1936) | 1950s: discovery of role in kidney disease (Leong, S. and Sirich, T., 2016).

In 1911, Obermayer and Popper discovered high concentrations of a metabolite, then called “indican,” in patients with kidney disease (Obermayer, F. and Popper, H., 1911). Indoxyl sulfate, as it later came to be known, was initially studied as a “putrefaction” product of intestinal microbial metabolism. In the 1950s, researchers explored whether urinary excretion of indoxyl sulfate might indicate various diseases, eventually focusing on its role in kidney disease. This was a major leap forwards in understanding uremia, which had puzzled scientists for decades (Leong, S. and Sirich, T., 2016).

As a key uremic toxin, accumulation of indoxyl sulfate affects the kidneys, cardiovascular system and bones (Colombo, G. et al., 2022). Indoxyl sulfate has been linked to cognitive impairment, oxidative stress and blood-brain barrier permeability, and is also thought to play a role in remote sensing and signaling in renal and extra-renal tissues (Lowenstein, J. and Nigam, S., 2021). Recent research has focused on IS as a potential therapeutic target in chronic kidney disease and cardiovascular disease, especially when these conditions are comorbid.

Indoxyl sulfate is a precursor of indigo, which has been used as a dye for thousands of years. This connection is illustrated vividly in “purple urine bag syndrome,” where constipation or urinary tract infections cause an accumulation of bacteria in urine collection bags, leading to the conversion of indoxyl sulfate into indigo and indirubin, and resulting in purple urine (Tan, C. et al., 2008).

Biosynthesis vs. dietary uptake

Indoxyl sulfate is produced from precursors synthesized via the tryptophan-indole pathway in the gut (Banoglu, E. et al., 2001). In the first step, L-tryptophan, an amino acid obtained from dietary protein, is metabolized by tryptophanase-expressing bacteria in the intestine to produce indole. Indole is absorbed into the bloodstream and is converted to indoxyl in the liver via hydroxylation by cytochrome enzymes including CYP2E1. Sulfotransferase enzymes in the liver complete the process by converting indoxyl into indoxyl sulfate.

Research suggests that increasing dietary tryptophan (typically found in protein-rich foods) may increase indoxyl sulfate production (Lauriola, M. et al., 2023; Leong, S. and Sirich, T., 2016). I ndividuals following high-protein diets show higher levels of indoxyl sulfate in both plasma and urine compared to those on low-protein or vegetarian diets (Patel, K. et al., 2012). Conversely, very low protein diets have been shown to reduce indoxyl sulfate levels (Marzocco, S. et al., 2013). A study of 56 hemodialysis patients found that increasing dietary fiber also reduced indoxyl sulfate levels (Sirich, T. et al., 2014).

In healthy individuals, the indoxyl sulfate concentrations range is10-130 mg/day, clearing rapidly through the kidneys (Patel, K. et al., 2012). Kidney dysfunction inhibits clearing, causing indoxyl sulfate to accumulate, which contributes to renal and cardiovascular toxicity as discussed below (Hung, S. et al., 2017). The full extent of IS’ role in healthy phenotypes remains to be discovered.

Indoxyl sulfate and the microbiome

More than 85 species of bacteria produce indole through the action of tryptophanase on tryptophan (Melander, R. et al., 2014). Tryptophanase has been found in bacteria including Lactobacillus, Bifidobacterium longum, Bacteroides fragilis, Parabacteroides distasonis, Clostridium bartlettii and E. hallii (Zhang, L. and Davies, S., 2016). Even more species respond to the presence of indole, despite lacking tryptophanase and thus not producing indole themselves.



The role of the liver in indoxyl sulfate production was known as far back as 1936, when animal studies revealed that indoxyl sulfate could be produced even in the absence of a digestive tract, as long as the liver was intact (Houssay, B., 1936). More recently, metabolomics has confirmed the involvement of the gut microbiome in indoxyl sulfate production: studies comparing indoxyl sulfate levels in conventional and germ-free rats, and in hemodialysis patients with and without colons, highlight the significant role of colon bacteria (Leong, S. and Sirich, T., 2016).

Indoxyl sulfate and nephrology

As a uremic toxin, indoxyl sulfate is strongly implicated in the progression of chronic kidney disease (CKD) (Caggiano, G. et al., 2023). As renal function declines, indoxyl sulfate concentrations rise, making it a useful biomarker of CKD. Both indoxyl sulfate and p-cresol sulfate, another protein-bound uremic toxin, are linked to symptoms of renal disease such as inflammation and fibrosis (Caggiano, G. et al., 2023). Indoxyl sulfate levels increase progressively with each stage of CKD (Clark, W. et al., 2019). Patients with hospital-acquired acute kidney injury have also been found to have higher serum levels of indoxyl sulfate (Menez, S. et al., 2019). Hemodialysis is limited in its ability to remove plasma protein bond, and dialysis patients with end-stage renal disease have been found to have indoxyl sulfate levels more than 20 times higher than those of healthy subjects (Wang, Y. et al., 2020).

Emerging evidence suggests that interventions that reduce indoxyl sulfate levels may slow CKD progression, though results remain inconclusive (Wang, Y. et al., 2020). Generally, there appears to be a good indication that administering prebiotics, probiotics and synbiotics can reduce indoxyl sulfate in patients with CKD and renal failure, although further research is needed (Lim, Y. et al., 2021).

Administration of AST-120 seems to be particularly promising for CKD (Lim, Y. et al., 2021). This treatment works by adsorbing indole and p-cresol to minimize downstream indoxyl sulfate production. AST-120 has been shown to slow the decline in glomerular filtration rate of CKD patients in some clinical studies, although randomized controlled trials have been inconclusive. More research is needed to determine whether gut microbiome modulation is a viable strategy in kidney disease.

Indoxyl sulfate and cardiovascular disease

Cardiovascular disease is higher among patients with CKD than the general population. While this relationship is not entirely understood, uremic toxins are thought to play a role, acting as vascular toxins as they accumulate in the blood (Barreto, F. et al., 2009; Zhao, Y. and Wang, Z., 2020). IS disrupts endothelial integrity and is linked to pro-oxidant, pro-inflammatory and prothrombotic processes, contributing to atherosclerosis, aortic calcification and vascular disease (Lano, G. et al., 2020). Indoxyl sulfateis also an agonist for the aryl hydrocarbon receptor (AhR) on vascular smooth muscle cells, further promoting the formation of atherosclerotic lesions and thrombosis (Leong, S. and Sirich, T., 2016). These mechanisms highlight indoxyl sulfate as a key player in the progression of cardiovascular complications in CKD.



Metabolomics studies have provided further insight into the role of indoxyl sulfate in cardiovascular health. For example, research shows that elevated indoxyl sulfate in plasma predicts cardiovascular events in patients with congestive heart failure and hypertrophic cardiomyopathy (Imazu, M. et al., 2020). Another study showed an association between plasma indoxyl sulfate levels and arterial stiffness in patients with type 2 diabetes (Katakami, N. et al., 2020). Diabetes can disrupt gut microbial composition and metabolism, which may contribute to increased indoxyl sulfate levels.

Indoxyl sulfate and bone disease

Patients with CKD often develop mineral bone disorders (CKD-MBD). Research suggests a link between this disorder and IS, though the precise mechanism is unclear (Liu, W. et al., 2018). One possibility is that the accumulation of uremic toxins weakens bone quality and quantity, potentially leading to uremic osteoporosis. Bone responses to parathyroid hormone (PTH) are also progressively lower in CKD patients, with studies suggesting a connection between PTH resistance, low bone turnover and increased indoxyl sulfate levels. Furthermore, indoxyl sulfate acts on AhR and activates signaling pathways that can induce ferroptosis and disrupt osteoblast differentiation (Chen, H. et al., 2024). Uremic toxin adsorbents may be a potential therapeutic strategy to improve bone health in patients with renal disease.

Indoxyl sulfate and neurological disease

A relatively new area of interest is the link between indoxyl sulfate and neurology, particularly its involvement in Alzheimer’s disease, Parkinson’s disease, anxiety and other neurological disorders. In CKD patients, accumulation of uremic toxins can lead to cerebrovascular lesions, increasing the risk of cognitive disorders and dementia (Sankowski, B. et al., 2020).

Using metabolomics, Sankowski et al. demonstrated that patients with Parkinson’s disease had higher levels of indoxyl sulfate and p-cresol sulfate in cerebrospinal fluid compared to controls, and higher levels of another uremic toxin, trimethylamine N-oxide (TMAO), in plasma (Sankowski, B. et al., 2020).

Another metabolomics study quantified molecular markers in whole blood of dementia patients, and found elevated levels of indoxyl sulfate compared to healthy controls, again suggesting that indoxyl sulfate may be neurotoxic (Teruya, T. et al., 2021).

Indoxyl sulfate levels also positively correlate with severity of anxiety, according to an exploratory metabolomics study (Brydges, C. et al., 2021). While modulating indole levels did not seem to affect treatment outcomes, the fact that a gut microbiome-derived metabolite influenced neural processing provides further evidence of a link between the gut microbiome and neuropsychiatric disorders.

 
Quantify the impact of indoxyl sulfate
Reference ranges of indoxyl sulfate in healthy humans
Learn more about the Metabolite of the Month

References

• History & Evolution
• Biosynthesis vs. dietary uptake
• Kynurenic acid and the microbiome
• Kynurenic acid receptors
• Kynurenic acid and neurology
• Kynurenic acid and cancer
• Kynurenic acid and metabolomic diseases

History & Evolution

1853: discovery (Liebig et al. 1853) | 1904: first synthesized from tryptophan (Ellinger et al. 1904) | 1980s: mode of action discovered

Kynurenic acid (KYNA) was first discovered in 1853 by German chemist Justus von Liebig in dog urine, which inspired its name (Liebig 1853). Fifty years later, another German chemist, Ellinger, identified it as one of the first metabolites isolated from tryptophan, again from dog urine (Ellinger 1904). Initially considered little more than a by-product of the more interesting kynurenine, KYNA caught the attention of researchers investigating the brain and central nervous system (CNS) in the 1980s and 90s, when it was identified as an antagonist of ionotropic glutamate receptors (Turski et al. 2013).

KYNA is found in several mammalian organs and tissues, including the brain, retina, liver, kidneys, intestines, cardiac muscles and bodily fluids, including breast milk (Turska et al. 2022). It is also found in plants, although its role in plant physiology remains largely unexplored (Wróbel-Kwiatkowska et al. 2024).

KYNA is associated with a wealth of health benefits, including antioxidant, anti-inflammatory and neuroprotective properties. Its well-established role in neurotransmission systems links it to several neurological and cognitive conditions, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), schizophrenia and mood disorders (Zhen et al. 2022). More recent evidence points to a role in energy homeostasis and in the immune and digestive systems, and it is also emerging as a potential biomarker of metabolic disease and endocrine disease (Zhen et al. 2022).

Biosynthesis vs. dietary uptake

KYNA is primarily synthesized intracellularly via the kynurenine pathway. While this process occurs endogenously, it relies on tryptophan, an exogenous essential amino acid that must be obtained through diet (Friedman et al. 2018). Dietary sources of tryptophan include seeds, soybeans, dairy, meat, fish, eggs and legumes. KYNA itself is usually found in quite low concentrations in food, with the exception of honey, broccoli and potatoes (Turski et al. 2009).

The kynurenine pathway metabolizes more than 90% of dietary tryptophan in the liver to form kynurenine, KYNA, quinolinic acid (QUIN) and other metabolites (Hou et al. 2023, Stone et al. 2013). In humans, kynurenine is primarily synthesized endogenously from tryptophan. This reaction is catalyzed by the enzyme tryptophan 2,3-dioxygenase (TDO) in the liver and, to a lesser extent, by indoleamine 2,3-dioxygenase (IDO) in other organs including immune cells (Savitz et al. 2020). Alternative mechanisms that may influence KYNA production in the brain involve D-amino acids, D-amino acid oxidase, and the effects of free radicals (Ramos-Chávez et al. 2018).

KYNA is synthesized in the gut microbiome, absorbed through the lumen of the gastrointestinal (GI) tract and transported to the liver, kidneys and other organs via the bloodstream (Turska et al. 2022). Concentration levels in digestive fluids increase steadily following ingestion, with blood levels peaking at around 15-30 minutes following ingestion (Sadok et al. 2023). Any that is not absorbed is excreted in urine.

Kynurenic acid and the microbiome

In the GI tract, tryptophan metabolism is modulated by gut microbiota including the bacterial phyla Actinobacteria, Bacteroides, Firmicutes, Fusobacteria, and Proteobacteria (Dehhaghi et al. 2020). Gut microbiota can influence the kynurenine pathway by regulating IDO-1 activity or tryptophan availability (Dehhaghi et al. 2019). For example, administering Bifidobacterium infantis to germ-free mice has been shown to increase KYNA levels (Desbonnet et al. 2008). Similarly, IDO and TDO have been found to reduce the kynurenine-to-tryptophan ratio in germ-free animals, but when those animals were exposed to microbiota, the enzyme activity normalized (Cryan et al. 2012).

The production of kynurenine metabolites is known to affect host physiology, with multiple studies demonstrating how manipulating microbial populations influences the kynurenine pathway and neural, endocrine and immune pathways (Cryan et al. 2012) (Dehhaghi et al. 2019). Differences in gut microbiota of healthy individuals and those with clinical conditions have been observed in diseases associated with the kynurenine pathway, such as anxiety, depression, schizophrenia, AD, PD, multiple sclerosis, ASD and irritable bowel syndrome (Purton et al. 2021).

There is still much to learn about the role of specific bacteria in producing KYNA and related therapeutic possibilities (Dehhaghi et al. 2019). A 2021 systematic review by Purton et al. showed that probiotics could modulate metabolite activity in the kynurenine pathway. However, evidence regarding the effect of prebiotics on this pathway remains limited (Purton et al. 2021).

Find out more about neurometabolomics, an evolving approach to better understand the role of KYNA and other metabolites in brain processes and function.

Kynurenic acid receptors

KYNA is a ligand to several receptors, acting as a signaling molecule for multiple physiological processes:

• It is an antagonist of the three ionotropic glutamate receptors N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainite, which mediate its effects in the brain (Schwarcz, R. et al. 2012). This makes it a useful test for glutamate involvement in synaptic transmission, for example in cases of stroke (Stone et al. 2000).
• KYNA is also thought to be an antagonist of the alpha7 nicotinic acetylcholine receptor (α7nAChR) (Albuquerque et al. 2013). However, this hypothesis is controversial: the results of studies showing that KYNA blocks NMDA receptors but not nicotinic receptors have not been consistently reproducible, while investigations using KYNA analogs may be vulnerable to misinterpretation (Stone et al. 2020). Given the role of α7nAChR in CNS disorders, there’s a strong case for more research to better understand if and how KYNA may block its action.
• KYNA has been more assuredly identified as an agonist of the G-protein coupled receptor (GPCR) GPR35, which is primarily present in the GI tract (Turski et al. 2013). Research in this area has led to the suggestion that KYNA could have a mediating effect on GI disorders, including ulcers, colitis and colon obstruction. KYNA’s role in modulating GPR35 also links it to immune system regulation, as does its role as an agonist of the aryl hydrocarbon receptor (AhR) (Tsuji et al. 2023). In 2020, researchers using DCyFIR, a CRISPR-based GPCR screening platform for ligand and drug discovery, found that KYNA activates hydroxycarboxylic acid receptor (HCAR3) in addition to GPR35 (Kapolka et al. 2020).

Because of its varied signaling effects on the immune system, inflammation, neuropathology, cancer and gut homeostasis, KYNA has been described as a “double-edged sword” (Wirthgen et al. 2017). Its anti-inflammatory and immunosuppressive functions are beneficial in some respects, but elevated levels of circulating KYNA are also associated with several diseases, as discussed below.

Interestingly, there appears to be a sex-specific association between circulating KYNA and immune response: a study using untargeted metabolomics found that male patients with COVID-19 had higher levels of KYNA than females, correlating with age, inflammation and disease severity (Cai et al. 2021). This may explain a potential link between KYNA levels and the differing outcomes in men and women with COVID-19.

Kynurenic acid and neurology

KYNA levels are altered in various mental disorders, with decreased levels observed in patients with affective psychosis, chronic schizophrenia, AD, cluster headaches, and chronic migraines (Wirthgen et al. 2017). Patients with PD show increased ratios of KYNA and KYNA/kynurenine, along with lower ratios of QUIN and QUIN/KYNA (Chang et al. 2018). Elevated KYNA in the cerebrospinal fluid of schizophrenia patients and its correlation with inflammatory biomarkers in AD suggest a link between KYNA and neuroinflammation (Wennström et al. 2014).
These findings highlight KYNA’s role in the pathophysiology of neurodegeneration and mental disorders, potentially through mechanisms involving oxidative stress, glutamatergic excitotoxicity, and inflammation. The participation of gut microbiota associated with the alternative routes to KYNA production involving D-amino acids and free radicals may also be relevant here (Ramos-Chávez et al. 2018).

The white-paper, Complex chronic diseases have a common origin, published by biocrates in 2023, includes a discussion of the relationship between KYNA, NDMA receptors and major depressive disorder (MDD). While KYNA inhibits the NMDA receptor and is considered neuroprotective, its fellow kynurenine metabolite quinolinic acid (QUIN) has the opposite effect and is considered neurotoxic. The QUIN/KYNA ratio is increased in the serum of patients with MDD) compared to healthy subjects, and correlates with the duration of remission, suggesting a role for these metabolites in disease progression (Savitz et al. 2015).

A metabolomics study by Erabi et al. found that KYNA was decreased in MDD, and lower levels showed a better therapeutic response to escitalopram (Erabi et al. 2020). This suggests KYNA is a potential overlapping biomarker in diagnosis and predicting therapeutic response.

KYNA does not effectively cross the blood-brain barrier, therefore limiting the potential efficacy of oral administration of KYNA in mitigating neurological and psychological diseases (Turska et al. 2022).

Kynurenic acid and cancer

Kynurenic acid’s effects in the periphery are not as well understood as its role in the brain. Still, there is a growing – if ambiguous – body of evidence confirming the presence of KYNA in several types of cancer in tumor tissue from patients with colon adenocarcinoma, glioblastoma, renal cell carcinoma (RCC) and oral squamous cell carcinoma (SCC) (Walczak et al. 2020). Elevated levels of KYNA have been found in the serum of patients with colon adenocarcinoma and lung cancer, and have been associated with invasiveness of lung cancer. In other cases, such as glioblastoma, cervical and prostate cancer, KYNA levels have been found to be lower than in healthy controls.

The precise mechanisms remain unclear. One possibility is that KYNA’s action on GPR35 initiates carcinogenesis and influences cell proliferation, survival and metastasis (Basson et al. 2023). KYNA may also interfere with nicotinamide adenine dinucleotide (NAD+) pathways, which is central to cancer biology. And as noted, KYNA activates the AhR pathway, which is also commonly activated in cancer patients, though data suggests this can be both pro- and anti-metastatic (Basson et al. 2023).

Multiple clinical trials are investigating the kynurenine pathway as a potential target for cancer therapies. More research is needed to understand where KYNA fits in the puzzle.

Kynurenic acid and metabolic diseases

Metabolomic and epidemiological research has shown that KYNA may play a role in diabetes and other metabolic diseases, and may protect against obesity and nonalcoholic fatty liver disease (Zhen et al. 2022). Deficiency in dietary KYNA is associated with adipose tissue dysfunction, with links to insulin sensitivity, energy homeostasis and lipid and carbohydrate metabolism (Tomaszewska et al. 2019). In contrast, KYNA synthesis may be influenced by physical exercise, potentially resulting in increased thermogenesis and helping to limit weight gain, insulin resistance and inflammation (Zhen et al. 2022).
Metabolomics research is increasing our understanding of the role of KYNA in metabolic disease and in turn revealing potential therapeutic targets. For example, a recent metabolomics study using an animal model found that KYNA production increases following hepatic ischemia-reperfusion injury, which diverts resources from NAD synthesis (Xu. et al. 2022). Augmenting NAD levels were found to mitigate oxidative stress, inflammation and cell death in the liver.

References

Using mass spectrometry in metabolic profiling

Metabolomics relies on mass spectrometry (MS), a prevalent methodology known for its high sensitivity, reproducibility, and versatility. MS is a quantitative and qualitative analytical technique that uses the mass-to-charge (m/z) ratio of previously ionized molecules to provide insights into biochemical pathways, metabolic fluxes, and the physiological status of organisms. Due to the diverse physical and chemical properties of compounds such as lipids, amino acids, and organic acids, as well as their widespread and fluctuating distribution across biological matrices, different MS-based technologies are used (Ren et al. 2018; Zhang et al. 2015).

Mass spectrometry, which consists of three main components: an ion source, a mass analyzer, and a detector. Ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) ionize molecules from solutions/solid-state, mixtures, while electron ionization (EI) is used to ionize molecules from the gas phase (after gas-chromatography (GC) separation).

Mass analyzers like time-of-flight (TOF), quadrupole, and ion trap separate ions based on their m/z ratios. Detectors such as electron multipliers and photomultiplier tubes then amplify ion signals for analysis. Liquid chromatography-mass spectrometry (LC-MS) combines liquid chromatography separation with MS detection, suitable for complex mixtures, whereas GC-MS offers high resolution for volatile compounds but requires derivatization for non-volatile ones. Each platform has advantages and limitations, guiding their selection for metabolomics studies based on the nature of the analytes and analytical requirements (Ahmad et al. 2020, Hoffmann et al. 2007).

Sample preparation

Effective metabolomic analysis calls for meticulous sample preparation to extract metabolites from the biological matrices, enhance detectability, and mitigate potential interference in subsequent mass spectrometry (MS) analysis.

If you’re unsure about the optimal matrix for your study, consider referring to ‘Which sample matrix should I use for my metabolomics study?’.

While preparation techniques play a crucial role in improving the detection and stability of metabolites, they also introduce complexities and variability into the sample preparation workflow. Sample clean-up procedures are necessary to eliminate unwanted matrix components, ameliorate matrix effects and enhance the sensitivity and selectivity of MS detection (Gong et al. 2017). Despite efforts to optimize sample preparation protocols, striking a balance between maximizing metabolite detection and minimizing sample variability and matrix effects remains challenging (Fan et al. 2012).

Extraction and analysis

Metabolomics analysis relies on various extraction techniques, each tailored to specific metabolite proprieties and sample matrices. Liquid-liquid extraction (LLE), solid-phase extraction (SPE), and protein precipitation are a few examples, each offering distinct advantages and applicability across different sample types (Nováková et al. 2024).

LLE involves partitioning analytes between two immiscible liquid phases, making it ideal for extracting non-polar and moderately polar metabolites. SPE uses solid-phase sorbents to selectively retain analytes, making it applicable to a wide range of sample types with high selectivity (Schomakers et al. 2022, Chetwynd et al. 2015). By contrast, protein precipitation removes proteins using organic solvents or acids, providing a rapid and effective approach for high-throughput analyses.

Derivatization methods enhance metabolite detection and stability, particularly those with low volatility or poor ionization efficiency, but as with preparation techniques, they can complicate the sample preparation workflow (Skov et al 2015).

Data analysis and interpretation

After data preprocessing, which involves peak picking, retention time alignment, and normalization, researchers strive to mitigate systematic variations that may be lurking in the raw data. To facilitate the identification of differences between experimental conditions, the data interpretation phase uses statistical methods, which may include the following (Lamichhane et al. 2018, Cambiaghi et al. 2017): 

• t-tests
• analysis of variance (ANOVA)
• linear mixed models
• multivariate analyses such as principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA)
• metabolite set analysis such as the hypergeometric test and Fisher Exact test
• data fusion techniques such as joint and individual explained analysis (JIVE) and deeply integrated single-cell omics (DISCO).

A few common challenges to look out for at this stage are handling missing values, managing high data dimensionality, and controlling false discovery rates. Researchers address these through various strategies such as imputation techniques, dimensionality reduction methods, and stringent statistical corrections. Quality control measures and validation strategies are also imperative for ensuring the robustness and reproducibility of results (Perez de Souza et al. 2024, Anwardeen et al. 2023). Multivariate data analysis techniques help find patterns and correlations within metabolomics datasets. Limonciel, 2023, introduces the “STORY Principle”, a systematic framework for interpreting metabolomics data and guiding researchers through each step.

Applications of MS-based metabolomics in recent research publications

Metabolomics can be applied in diagnostics, prognostics, biomarker discovery, treatment monitoring, and understanding disease mechanisms across various fields such as cardiometabolic diseases, diabetology, oncology, and toxicology.

The study of cardiometabolic diseases, including diabetes, is an especially important application of MS in metabolomics research. Huang et al. 2024, investigated the protective effect of Liu-Wei-Di-Huang-Wan (a traditional Chinese formulation composed of six commonly used Chinese herbs) in mice with type 2 diabetes mellitus. They identified 30 endogenous metabolites including 3-hydroxybutyric acid, citric acid, hexadecanoic acid, and octadecanoic acid. They found that treatment with Liu-Wei-Di-Huang-Wan improved metabolic patterns in diabetic kidney disease mice, comparable to gliquidone, by regulating metabolites associated with key metabolic pathways. In another study, Matter-Rieck et al. 2024, showcase the potential role of omentin in regulating systemic metabolism, particularly lipid metabolism, particularly lipid metabolism, and influencing insulin sensitivity, further exploring its involvement in type 2 diabetes. Omentin levels were positively associated with lipids such as phosphatidylcholines (PCs) and acylcarnitines, and negatively associated with certain amino acids.

Epidemiology and genetics offer insights into disease etiology and progression. Rosario et al. 2023, identified significant metabolic and transcriptional dysregulation associated with gastrointestinal adenocarcinomas. The findings reveal alterations in pathways such as steroid metabolism and tryptophan/kynurenine metabolism, with sex-specific differences. Investigating gut microbiota stability, Frost et al. 2021, found that instability, characterized by an increase in potential pathogens like Enterobacteriaceae and a decrease in beneficial bacteria such as Bifidobacteria, was significantly associated with metabolic disorders including diabetes mellitus and fatty liver disease.

Metabolomics is also an important investigative tool in oncology, presenting the metabolic signatures linked to cancer development and treatment response. Zhang N et al. 2024, used LC-MS and GC-MS analysis of plasma metabolites to study biomarkers for neoadjuvant therapy efficacy in HER2 + breast cancer. Significant differences were found between responders and non-responders, with 100 metabolites identified and enriched in 40 pathways. Area under the curve (AUC) values for discriminating groups exceeded 0.910, and 18 metabolites showed potential for monitoring efficacy. In another study, Decker et al. 2024, described connections between cholesterol precursors, oxysterols, and diverse factors in women diagnosed with breast cancer. Their research shows the linkage of cholesterol precursors to metabolic factors like body mass index (BMI), cardiovascular disease (CVD) and oxysterols, particularly those generated via reactive oxygen species (ROS), and key characteristics of breast cancer tumors, including obesity.

Metabolic profiling is increasingly recognized in cardiology studies. A study by Kretzschmar et al. 2024, analyzed plasma metabolites in patients with acute decompensated heart failure (ADHF) and chronic heart failure (CHF). They found distinct metabolic signatures associated with each phenotype, including potential novel biomarkers for heart failure such as 1-methyl histidine and 3-indolepropionic acid. Another interesting study by Andrews et al. 2024, highlights significant changes in lipid metabolism in mice, specifically focusing on alterations in acylcarnitines, amino acids, bile acids, ceramides, sphingomyelins, and triacylglycerols. These findings pinpoint the mechanisms underlying hepatic steatosis and atherosclerosis development in mice fed the hyperhomocysteinemic diet.

Finally, in neuroscience, Ge et al. 2024, conducted a metabolomic analysis from 48 patients with disorders of consciousness (DoC), showing distinct metabolic profiles linked to etiology, consciousness levels, and prognosis. Their findings highlight the critical role of phospholipid metabolism and identify potential biomarkers for improved diagnosis and treatment of DoC.

As mass spectrometry evolves, we’ll see more opportunities to apply the methodology within metabolomics studies. If you are considering using this technique in your own study, feel free to contact the biocrates team with any questions.

References

Andrews, S.G. et al.: Diet-Induced Severe Hyperhomocysteinemia Promotes Atherosclerosis Progression and Dysregulates the Plasma Metabolome in Apolipoprotein-E-Deficient Mice (2024) Nutrients | DOI: 10.3390/nu16030330

Anwardeen, N.R.et al.: Statistical methods and resources for biomarker discovery using metabolomics (2023) BMC Bioinformatics 24 | DOI:10.1186/s12859-023-05383-0

Cambiaghi A et al.: Analysis of metabolomic data: tools, current strategies and future challenges for omics data integration (2017) Briefings in Bioinformatics | DOI: 10.1093/bib/bbw031

Causon J.: Review of sample preparation strategies for MS-based metabolomic studies in industrial biotechnology (2016) Analytica Chimica Acta | DOI: 10.1016/j.aca.2016.07.033

Chetwynd J. et al.: Solid-Phase Extraction and Nanoflow Liquid Chromatography-Nanoelectrospray Ionization Mass Spectrometry for Improved Global Urine Metabolomics (2014) Analytical Chemistry | DOI: doi.org/10.1021/ac503769q

Decker, N.S et al.: Associations between lifestyle, health, and clinical characteristics and circulating oxysterols and cholesterol precursors in women diagnosed with breast cancer: a cross-sectional study. (2024) Sci Rep 14 | DOI: 10.1038/s41598-024-55316-x

Fan, T.WM.: Considerations of Sample Preparation for Metabolomics Investigation. (2012) The Handbook of Metabolomics. Methods in Pharmacology and Toxicology | DOI: 10.1007/978-1-61779-618-0_2

Frost F. et al.: Long-term instability of the intestinal microbiome is associated with metabolic liver disease, low microbiota diversity, diabetes mellitus and impaired exocrine pancreatic function (2021) Gut | DOI: 10.1136/gutjnl-2020-322753

Ge Q, et al.: Serum metabolism alteration behind different etiology, diagnosis, and prognosis of disorders of consciousness (2024) Chin Neurosurg J. | DOI: 10.1186/s41016-024-00365-4

Gong Z-G. et al.: The Recent Developments in Sample Preparation for Mass Spectrometry-Based Metabolomics (2017) Critical Reviews in Analytical Chemistry, 47(4), 325–331 | DOI: 10.1080/10408347.2017.1289836

Hoffmann E. et al.: Mass Spectrometry: Principles and Applications (2007) Hoboken, NJ: John Wiley & Sons. | ISBN: 978-0-470-03310-4

Huang JH. et al.: A GC-MS-Based Metabolomics Investigation of the Protective Effect of Liu-Wei-Di-Huang-Wan in Type 2 Diabetes Mellitus Mice (2020) Int J Anal Chem. Aug | DOI: 10.1155/2020/1306439

Javed A et al.: Basic Principles and Fundamental Aspects of Mass Spectrometry (2022) Mass Spectrometry in Food Analysis | DOI: 10.1201/9781003091226-2

Kretzschmar T. et al.: Metabolic Profiling Identifies 1-MetHis and 3-IPA as Potential Diagnostic Biomarkers for Patients With Acute and Chronic Heart Failure With Reduced Ejection Fraction. (2024) Circ Heart Fail | DOI: 10.1161/CIRCHEARTFAILURE.123.010813

Lamichhane S.:Chapter Fourteen – An Overview of Metabolomics Data Analysis: Current Tools and Future Perspectives (2018) Comprehensive Analytical Chemistry | DOI: 10.1016/bs.coac.2018.07.001

Limonciel A. The STORY principle A guide to the biological interpretation of metabolomics (2023) biocrates life sciences gmbh | DOI: 10.1016/bs.coac.2018.07.001

Nováková S. et al.: Comparison of Various Extraction Approaches for Optimized Preparation of Intracellular Metabolites from Human Mesenchymal Stem Cells and Fibroblasts for NMR-Based Study (2024) Metabolites | DOI: 10.3390/metabo14050268

Perez de Souza L. et al.: Computational methods for processing and interpreting mass spectrometry-based metabolomics. (2024) Essays Biochem | DOI: 10.1042/EBC20230019

Ratter-Rieck J.: Omentin associates with serum metabolite profiles indicating lower diabetes risk: KORA F4 Study (2024) BMJ Open Diabetes Research and Care | DOI: 10.1136/bmjdrc-2023-003865

Ren J-L. et al.: Advances in mass spectrometry-based metabolomics for investigation of metabolites (2018) Royal Society of Chemistry | DOI: 10.1039/C8RA01574K

Rosario D. et al.: Systematic analysis of gut microbiome reveals the role of bacterial folate and homocysteine metabolism in Parkinson’s disease (2021) Cell Reports 34 | DOI: 10.1016/j.celrep.2021.108807

Schomakers V. Et al.: Polar metabolomics in human muscle biopsies using a liquid-liquid extraction and full-scan LC-MS (2022) TAR Protocols | DOI: 10.1016/j.xpro.2022.101302

Skov K. et al.: LC–MS analysis of the plasma metabolome—A novel sample preparation strategy (2015) Journal of Chromatography B | DOI: 10.1016/j.jchromb.2014.11.033

Zhang A. et al.: Metabolomics for Biomarker Discovery: Moving to the Clinic (2015) Biomed Res Int. | DOI: 10.1155/2015/354671

Zhang N et al.: Metabolomics assisted by transcriptomics analysis to reveal metabolic characteristics and potential biomarkers associated with treatment response of neoadjuvant therapy with TCbHP regimen in HER2 + breast cancer. (2024) Breast Cancer Res. | DOI: 10.1186/s13058-024-01813

• History & Evolution
• Biosynthesis vs. dietary uptake
• Butyric acid and the microbiome
• Butyric acid as a signaling molecule
• Butyric acid and the immune system
• Butyric acid and the brain
• Butyric acid and cardiometabolic diseases
• Butyric acid and cancer: the “butyrate paradox”

History & Evolution

1814-23: first observed in impure form by Michel Eugène Chevreul (Chevreul 1823) | 1869: first synthesized by Lieben and Rossi (Goldberg 2009)

Butyric acid is a four-carbon straight short chain fatty acid (SCFA) found in the esters of animal fats and plant oils. Its name comes from the Ancient Greek for butter, which is where it was first identified. Butyric acid is responsible for the foul smell found in rancid butter, parmesan cheese, vomit and body odor (Huang et al. 2018). Interestingly, as for isovaleric acid, some esters of butyric acid have a more appealing scent, and are often used in perfumes.

Butyric acid is one of three common SCFAs in the human gut, alongside acetic acid and propionic acid, which together make up 90-95% of the SCFAs in the colon (Ríos-Cavián et al. 2016). It is a major source of energy for the colon and is used in treatments for colorectal cancer, hemoglobinopathies and gastrointestinal diseases (Huang et al. 2018).

In industry, butyric acid has applications in food, textile production, animal feed, and biofuels, often chemically synthesized through the oxidation of propylene-derived butyraldehyde, or through syngas fermentation (Huang et al. 2018).

Biosynthesis vs. dietary uptake

Most SCFAs in the gut come from dietary fibers: because humans lack the enzymes to digest these, they pass through the intestinal tract and are fermented by host bacteria. Butyric acid is a conjugate of butyrate, which is produced through the fermentation of hydrolysis-resistant starches and dietary fiber by anaerobic bacteria in the colon (Wong et al. 2006). Some butyrate is also produced as proteins and peptides are digested in the bowel (Macfarlane et al. 2003).

Diet, composition of the microbiome, and intestinal transit time all influence butyric acid formation, as with the other SCFAs (Morrison et al. 2016). Most of the dietary fiber from which butyric acid is produced comes from plant sources, such as resistant starch, cruciferous vegetables, and foods with a high sulphur content (Rivière et al. 2016). Dietary butyric acid is found in dairy products, red meat, and fermented foods such as sauerkraut. Around 5% of the saturated fat in dairy products comes from butyric acid (Månsson 2008). Butyric acid can also be taken in supplement form.

Butyric acid and the microbiome

SCFAs are a popular research topic in medical biochemistry because of their potential role in gut function, glucose homeostasis, metabolic regulation, and appetite (Blakeney et al. 2019; Vijay et al. 2021). They are also known to influence inflammation and immune response.

In the colon, butyrate is a source of energy for endothelial cells, promotes cell differentiation and apoptosis, and can inhibit colonic acidification (Wong et al. 2006). Some studies suggest that butyrate can suppress colorectal cancer, though results are inconclusive (Silva et al. 2020). Butyric acid has been shown to influence pathogenesis of gastrointestinal disease and gut dysbiosis, and animal studies show that higher concentrations of butyric acid in the colon reduce the severity of inflammation.

Butyric acid as a signaling molecule

SCFAs are known to act as signaling molecules between gut microbiota and host, with receptors in many different cell and tissue types (Morrison et al. 2016). Butyric acid is an endogenous agonist of one of these receptors, hydroxycarboxylic acid receptor 2 (HCA2). HCA2 is a protein receptor that can inhibit the breakdown of fats, giving butyric acid a key role in lipid metabolism. Butyric acid is also an agonist of the peroxisome proliferator-activated receptor (PPAR), a nutrient sensor which helps to stabilize lipid metabolism and inhibit cancer cell proliferation in the colon (Hong et al. 2019).

One notable way in which butyric acid regulates the inflammatory process is by stimulating the production of eicosanoids, which are lipid mediators derived from arachidonic acid (Vinolo et al. 2011). These are also known to regulate other immune processes involved in cancer, asthmas, and arthritis (Harizi et al. 2008).

Butyric acid and the immune system

As noted, butyric acid exerts several effects in the human gut which affect immune processes (Kovarik et al. 2013). Butyric acid is thought to increase acetylation of histone H3, in turn influencing the behavior of regulatory T cells, which can inhibit the immune response (Borycka-Kiciak et al. 2017). Through this mechanism, SCFAs link crosstalk between the human microbiome and immune system, though it is not clear whether this is by increasing tolerance in the microbiome, or by reducing the inflammatory response (Morrison et al. 2016).

Recent research has highlighted the significance of butyric acid in the gut microbiome, particularly its role in maintaining immune function and metabolic balance. For instance, a study investigating age-associated gut dysbiosis in older individuals living with HIV found a notable decrease in butyrogenic potential, correlating with alterations in plasma tryptophan metabolites (Brivio et al. 2023)

A few clinical studies have observed an anti-inflammatory effect from the therapeutic use of SCFAs in cases of inflammatory bowel disease, radiation proctitis, and diabetes. Growing evidence suggests SCFAs support the immune system and metabolism through gut-liver inflammatory pathways (Morrison et al. 2016).

Butyric acid and the brain

In addition to its role in the gastrointestinal tract, butyric acid may also contribute to links between gut dysbiosis and neurological conditions, such as depression, Alzheimer’s disease, Parkinson’s disease, and autism spectrum disorder (Silva et al. 2020).

Studies looking at the use of probiotics to increase butyrate-producing bacteria in the gut suggest butyrates could help reduce anxiety and lower stress (Bourassa et al. 2016). A review by Bourassa et al. proposed possible mechanisms for butyric acid’s neuroprotective effects, including mitochondrial activity, G-protein coupled receptors, histone acetylation, and microbiome homeostasis. A clear line was drawn between the consumption of a high fiber diet, butyrate production, and protection against multiple neurological conditions through these pathways (Bourassa et al. 2016).

Butyric acid and cardiometabolic diseases

The role of SCFAs in lipid and energy metabolism links them to certain metabolic conditions. Butyrate has been shown to protect against diet-induced obesity and insulin resistance, which suggests it may offer potential therapeutic role in obesity-related diseases and diabetes (Lin et al. 2012). Animal studies confirm that butyric acid supplementation can improve insulin sensitivity: in one study, butyric acid caused fat loss and improved insulin tolerance in mice (Heimann et al. 2015, Gao et al. 2009). More research is needed to confirm the effect in humans.

Gut microbiota have a well-established link to coronary artery disease and atherosclerosis. One animal study has shown that butyrate supplementation could reduce atherosclerotic lesions, while another suggested that butyric acid seems to mediate gut microbiota and the circulatory system (Onyszkiewicz  et al. 2019). Some studies have suggested that butyric acid affects arterial blood pressure, with one showing a significant hypotensive effect when butyric acid concentration in the colon was increased (Onyszkiewicz et al. 2019). The precise mechanism is unknown: it may result from bacterial metabolites having a stimulating enterosyne effect on the enteric nervous system, or from metabolite-derived molecules entering the circulatory system and influencing arterial blood pressure through various organs (Onyszkiewicz et al. 2019).

Butyric acid and cancer: the “butyrate paradox”

As noted, butyric acid has been shown in several studies to inhibit the proliferation of cancer cells in the colon, by inducing apoptosis, inhibiting cancer gene expression, inhibiting cancer cell proliferation, and promoting anti-inflammatory processes (Williams et al. 2007). However, other studies challenge the notion of a chemopreventive effect from butyrate, and there is a lack of agreement particularly when comparing in vitro and in vivo studies, referred to as the butyrate paradox (Lupton 2004). It seems likely that butyrate’s chemopreventive effect depends on the amount of butyrate, time of exposure during the tumorigenic process, and type of dietary fat. Our understanding of the underlying molecular mechanisms is likely to grow with the advance of genomic and metabolomic technologies. Because butyric acid is a by-product of fiber fermentation, this could explain why high fiber diets help to protect against colorectal cancer, as well as obesity, stroke, type 2 diabetes and other conditions.

Learn more about the roles of butyric acid and other SCFAs in complex chronic diseases such as cancer, Alzheimer’s disease, depression, inflammatory bowel disease, multiple sclerosis and diabetes in our whitepaper “Complex chronic diseases have a common origin”.

References

• History & Evolution
• Biosynthesis and dietary uptake
• Choline and the gut microbiome
• Choline and cardiovascular diseases
• Choline and neurology
• Choline supplementation and Alzheimer’s disease
• Choline as biomarker
• Potential dietary and microbiome-based interventions with choline
• References

History & Evolution

1849: first isolation | (Zeisel et al., 2012)1862: named after the Greek word for bile | 1930s: first beneficial properties discovered

Choline is an essential nutrient that plays a key role in phospholipid synthesis, with far-reaching effects in human health and disease.

It was first isolated from pig bile in 1849 by Adolph Strecker, who later named it after the Greek word for bile, chole (Zeisel et al., 2012). In these early years, choline was “discovered” multiple times, only for researchers to realize later that they were looking at the same compound: Babo and Hirschbrunn named their new substance “sinkaline” (Griffith et al., 1954), while Oscar Liebreich identified a molecule called “neurine”, a component of what he referred to as “protagon, the mother substance of all” (Zeisel et al., 2012). Investigations into the structure of choline by Adolf von Baeyer in 1867 eventually revealed that choline, sinkaline and neurine were the same molecule (Griffith et al.,1954). (What we now refer to as neurine is a downstream metabolite of choline).

Choline emerged as a potential nutrient in the 1930s, when it was found to prevent fatty liver in dogs and rats (Zeisel et al., 2012). Throughout the 1950s and 60s, researchers began to describe the primary pathways for choline synthesis. By the 1990s, it was established as an essential B vitamin-like nutrient for human health, with food agencies including it in dietary recommendations.

Today, choline’s vital role in cell structure and signaling, neurotransmission, gene expression and lipid metabolism opens fascinating avenues of research spanning a wide range of metabolic processes and pathologies (National Institutes of Health 2022).

Biosynthesis vs. dietary uptake

Humans produce some choline endogenously, but not enough to fulfil its role (Wallace et al. 2018). This means dietary intake must compensate for the deficiency.

De novo synthesis of choline occurs via the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. This process takes place in the liver: a PEMT enzyme methylates phosphatidylethanolamine (PE) through three sequential reactions to form phosphatidylcholine (PC) (Vance et al., 2014). This is the primarily endogenous pathway to generate new choline molecules, though choline can also be obtained through the hydrolyzation of PCs and from the breakdown of phosphocholine to cytidine diphosphate-choline (CDP-choline) (Li et al. 2023).

Dietary choline is found in meat, poultry, fish, dairy products and eggs, as well as cruciferous vegetables and legumes (Fischer et al. 2010). When these foods are consumed, pancreatic and mucosal enzymes facilitate the release of choline, which is absorbed in the small intestine and transported through the portal circulation to the liver. Here, choline is stored along with endogenously derived choline, and further released into the body via the bloodstream and lymphatic circulation.(National Institutes of Health 2022).

Recommended daily intake of choline for men and women is around 550mg and 425mg respectively (Wallace et al. 2018). Age, environment and genetics influence choline requirements (Sanders et al., 2007). Because PEMT enzyme is induced by estrogen, postmenopausal women tend to be more susceptible to choline deficiency than other groups (Fischer et al. 2010).

Choline metabolism produces several important metabolites, thus choline deficiency can affect multiple physiological systems. Acetylcholine, an ester of choline and acetic acid, supports neurotransmission and affects cognitive and motor functions (Leermakers et al., 2015). Betaine, another derivative of choline, is involved in homocysteine synthesis and influences cholesterol levels (Leermakers et al., 2015). Choline is also a precursor of membrane phospholipids such as PC and sphingomyelin, which are critical for cell structure, signaling and transport (Sanders et al., 2007). As a precursor of very-low-density-lipoproteins, PC plays a role in transporting triglycerides from the liver, which may explain links between choline deficiency and hepatosteatosis (Yao et al.,1988; Laura K et al., 2012).

Choline and the gut microbiome

Choline metabolism by gut bacteria in the intestine results in trimethylamine (TMA), which is converted to trimethylamine-N-oxide (TMAO) in the liver (Arias et al., 2020). High levels of TMA can cause trimethylaminuria, characterized by a fish-like body odor (Wallace et al., 2018). There has been speculation that elevated TMAO levels may be linked to atherosclerosis, although the evidence remains inconclusive.

Less than 1% of gut microorganisms contribute to TMA production, but the small amount that do are highly effective (Arias et al., 2020). Several intestinal microbiota are involved in TMA synthesis from choline, including Anaerococcus hydrogenalis, Clostridium asparagiformis, Clostridium hathewayi, Clostridium sporogenes, Desulfovibrio desulfuricans, Escherichia fergusoni, Ed. tarda, Klebsiella pneumoniae, Proteus penneri, and Providencia rettgeri. Levels of TMA and TMAO are particularly influenced by Firmicutes and Proteobacteria populations (Arias et al., 2020).

In mice, low levels of TMA-producing bacteria have been found to significantly reduce choline availability to the host, with more pronounced effects as these bacteria become more abundant (Romano et al., 2015). In humans, dietary choline depletion has been shown to alter Gammaproteobacteria and Erysipelotrichi populations, with a direct effect on levels of liver fat (Spencer et al., 2010).

Choline and cardiovascular disease

There is growing evidence of a link between choline metabolites and cardiovascular disease (CVD) (Tang et al., 2013). However, much of the research in this area relies on study populations with a high prevalence of CVD, which may limit the generalization of the conclusions to other demographics (Shea et al., 2024).

TMAO’s role in CVD is particularly controversial. Some animal studies have shown TMAO to exacerbate atherosclerosis (Romano et al., 2015), but other studies, including observational studies in humans have failed to confirm whether TMAO is “a bystander or a mediator” in CVD (Janeiro et al., 2018; Meyer et al., 2017).

The role of choline itself is similarly blurred. This is perhaps unsurprising given that dietary sources of choline, such as red meat, are correlated with both positive and negative effects on cardiovascular health. A recent investigation using data from the Coronary Artery Risk Development in Young Adults (CARDIA) prospective cohort study found a positive association between plasma choline and CVD, independent of TMAO and betaine (Shea et al., 2024). This study also found that red meat and fried foods were significantly associated with plasma choline concentrations.

Choline’s involvement in homocysteine metabolism may offer another clue about its role in CVD, as elevated homocysteine concentrations are established risk factors for CVD (Ganguly et al., 2015). However, while choline is known to influence homocysteine regulation, there is little evidence that increasing choline consumption offers significant cardiovascular benefits through this mechanism (Meyer et al., 2017).

Choline and neurology

Choline is essential for neurological development and function (Sanders et al., 2007). Its deficiency is associated with apoptosis and neuronal cell death, potentially contributing to neurological disorders.

A recent systematic review and meta-analysis found that higher maternal choline intake was associated with positive cognitive effects and neurodevelopment in children, including memory, attention and visuospatial learning (Obeid et al., 2022).

There may also be a link between choline and cognitive function in older adults. A study of choline intake in adults aged 60 and over found that consuming at least 187.5mg per day reduced the risk of low cognitive performance by around 40% using three different cognition measures (Liu et al., 2021). Improvements tailed off at 399.5mg per day, suggesting a “U-shaped” effect with increasing choline consumption.

Cognitive impairment in older people is often caused by cerebrovascular disease, which itself may be influenced by choline levels. A 2021 study in China found that patients with higher levels of circulating choline and betaine had a reduced risk of cognitive impairment after acute ischemic stroke (Zhong et al., 2021). Earlier research suggests that CDP-choline has a neuroprotective effect in preclinical models of brain ischemia and trauma, and while findings in clinical trials have been inconclusive, there have been some promising results in cases of slower neurodegeneration, such as glaucoma and vascular cognitive impairment, and in Alzheimer’s disease (AD) (Grieb et al., 2014).

Choline supplementation and Alzheimer’s disease

Low choline intake appears to increase the risk of dementia and AD (Yuan et al., 2022). A recent metabolomic analysis found that low circulating choline levels associate with AD progression (Judd et al., 2023). A proteomics analysis of hippocampal tissue in an AD mouse model showed that choline supplementation led to changes in key proteins involved in AD pathology (Dave et al., 2023). It remains unclear whether choline supplementation can reduce neuropathology in advanced AD, but given choline’s crucial role in neurobiology, it may still be a worthwhile preventive strategy (Judd et al., 2023).

Discussing the above study from Arizona State University, lead researcher Prof. Ramon Velazquez commented, “It’s a twofold problem. First, people don’t reach the adequate daily intake of choline established by the Institute of Medicine in 1998. Second, there is vast literature showing that the recommended daily intake amounts are not optimal for brain-related functions.” The study found that choline deprivation in both healthy mice and AD-symptomatic transgenic mice altered levels of amyloid-beta protein and tau protein, which are involved in neurofibrillary tangles associated with AD (Judd et al., 2023).

Choline as a biomarker

The above associations suggest that choline may be a potential biomarker of several different diseases. Metabolomic profiling has been used to measure choline and its metabolites in pathologies such as liver dysfunction and AD (Sha et al., 2010; Donatti et al., 2020).

An interesting example of choline’s potential as a biomarker involves the links between smoking, choline metabolism and risk of CVD. Findings suggest that smoking alters the association between plasma choline and risk of acute myocardial infarction (AMI) (Schartum-Hansen et al., 2015).Choline levels also tend to differ in the population based on fitness or smoking status, as can be seen in biocrates’ quantitative metabolomics database (QMDB).

Potential dietary and microbiome-based interventions with choline

Despite the wide availability of choline in food, most people do not meet the daily recommended intake (Arias et al., 2020). As noted above in relation to cognitive function, supplementation may be useful in some cases.

There has been significant research in animal models. A 2023 study found that choline supplementation in pigs enhanced gut microbiome diversity and epithelial activity, which may help to promote ovarian follicular development and ovulation (Zhan et al., 2023). Another study from 2022 investigated the effects of choline supplementation on atherosclerosis in mice. It found that choline promoted beneficial changes in gut microbial composition and function, including increasing the abundance of anti-inflammatory microbiota and gene expression relating to TMA and TMAO degradation, but notably did not aggravate atherosclerosis (Liu, C. et al. 2022).

Further research in human subjects may clarify the efficacy of these and other choline-based dietary and microbiome-related interventions.

References

Arias, N. et al.: The Relationship between Choline Bioavailability from Diet, Intestinal Microbiota Composition, and Its Modulation of Human Diseases. (2020) Nutrients | DOI: doi.org/10.3390/nu12082340.

Dave, N. et al.: Dietary choline intake is necessary to prevent systems-wide organ pathology and reduce Alzheimer’s disease hallmarks. (2023) Aging Cell | DOI: doi.org/10.1111/acel.13775.

Donatti, A. et al.: Circulating Metabolites as Potential Biomarkers for Neurological Disorders—Metabolites in Neurological Disorders. (2020) Metabolites | DOI: doi.org/10.3390/metabo10100389.

Fischer, L. et al.: Dietary choline requirements of women: effects of estrogen and genetic variation. (2010) Am J Clin Nutr. | DOI: doi.org/10.3945/ajcn.2010.30064.

Ganguly, P. et al.: Role of homocysteine in the development of cardiovascular disease. (2015). Nutr J. | DOI: doi.org/10.1186/1475-2891-14-6.

Grieb, P. : Neuroprotective properties of citicoline: facts, doubts and unresolved issues. (2014) CNS Drugs | DOI: doi.org/10.1007/s40263-014-0144-8.

Griffith, W. et al.,: II. Chemistry, Ch. 5. Choline. (1954) In The Vitamins: Chemistry, Physiology, Pathology, by Sebrell Jr. W. and Harris R. New York. Academic Press Inc. sciencedirect.com/book/9781483197043/the-vitamins

Janeiro, M. et al.: Implication of Trimethylamine N-Oxide (TMAO) in Disease: Potential Biomarker or New Therapeutic Target. (2018).Nutrients | DOI: doi.org/10.3390/nu10101398.

Judd, J. et al.: Inflammation and the pathological progression of Alzheimer’s disease are associated with low circulating choline levels. (2023) Acta Neuropathol | DOI: doi.org/10.1007/s00401-023-02616-7.

Laura K. et al.: Phosphatidylcholine biosynthesis and lipoprotein metabolism, Biochimica et Biophysica Acta (BBA). (2012) Molecular and Cell Biology of Lipids | DOI: doi.org/10.1016/j.bbalip.2011.09.009

Leermakers, E. et al.: Effects of choline on health across the life course: a systematic review. (2015) Nutrition Reviews | DOI: doi.org/10.1093/nutrit/nuv010.

Li, J. et al.: Phosphatidylethanolamine N-methyltransferase: from Functions to Diseases. (2023) Aging Dis. | DOI: doi.org/10.14336/AD.2022.1025.

Liu, C. et al.: Choline and butyrate beneficially modulate the gut microbiome without affecting atherosclerosis in APOE*3-Leiden.CETP mice. (2022) Atherosclerosis | DOI: doi.org/10.1016/j.atherosclerosis.2022.10.009.

Liu, L. et al.: Choline Intake Correlates with Cognitive Performance among Elder Adults in the United States. (2021) Behav Neurol. | DOI: doi.org/10.1155/2021/2962245.

Meyer, K. et al.,: Dietary Choline and Betaine and Risk of CVD: A Systematic Review and Meta-Analysis of Prospective Studies. (2017) Nutrients | DOI: doi.org/10.3390/nu9070711.

National Institutes of Health.: Choline: Fact Sheet for Health Professionals. (2022) .Accessed March 2024. https://ods.od.nih.gov/factsheets/Choline-HealthProfessional/.

Obeid, R. et al.: Association between Maternal Choline, Fetal Brain Development, and Child Neurocognition: Systematic Review and Meta-Analysis of Human Studies. (2022).Adv Nutr. | DOI: doi.org/10.1093/advances/nmac082.

Romano, K. et al.: Intestinal Microbiota Composition Modulates Choline Bioavailability from Diet and Accumulation of the Proatherogenic Metabolite Trimethylamine-N-Oxide. (2015) ASM Journals DOI: doi.org/10.1128/mbio.02481-14

Romano, K. et al.: Intestinal Microbiota Composition Modulates Choline Bioavailability from Diet and Accumulation of the Proatherogenic Metabolite Trimethylamine-N-Oxide. (2015) ASM Journals | DOI: doi.org/10.1128/mbio.02481-14.

Sam, C. et al.,: Physiology, Acetylcholine. (2023) In: StatPearls [Internet]. FL: StatPearls Publishing. | ncbi.nlm.nih.gov/books/NBK557825/

Sanders, L. et al.,: Choline: Dietary Requirements and Role in Brain Development. (2007) Nutr Today | DOI: doi.org/10.1097/01.NT.0000286155.55343.fa.

Schartum-Hansen, H. et al.: Plasma choline, smoking, and long-term prognosis in patients with stable angina pectoris. (2015) European Journal of Preventive Cardiology 22 (5): 606–614. | DOI: doi.org/10.1177/2047487314524867.

Sha, W. et al.: Metabolomic profiling can predict which humans will develop liver dysfunction when deprived of dietary choline. (2010) FASEB J. | DOI: doi.org/10.1096/fj.09-154054.

Shea, J. et al.: Choline metabolites and incident cardiovascular disease in a prospective cohort of adults: Coronary Artery Risk Development in Young Adults (CARDIA) Study. (2024) The American Journal of Clinical Nutrition | DOI: doi.org/10.1016/j.ajcnut.2023.10.012.

Spencer, M. et al.: Association between composition of the human gastrointestinal microbiome and development of fatty liver with choline deficiency. (2010) Gastroenterology | DOI: doi.org/10.1053/j.gastro.2010.11.049.

Tang, W. et al.: Intestinal Microbial Metabolism of Phosphatidylcholine and Cardiovascular Risk. (2013) N Engl J Med | DOI: doi.org/10.1056/NEJMoa1109400.

Vance, D.: Phospholipid methylation in mammals: from biochemistry to physiological function. (2014) Biochimica et Biophysica Acta (BBA) – Biomembranes | DOI: doi.org/https://doi.org/10.1016/j.bbamem.2013.10.018.

Wallace, T. et al.: Choline: The Underconsumed and Underappreciated Essential Nutrient. (2018 ) Nutr Today. | DOI: doi.org/10.1097/NT.0000000000000302.

Yao, Z. et al., : The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes. (1988) J Biol Chem. | pubmed.ncbi.nlm.nih.gov/3343237/

Yuan, J. et al.: Is dietary choline intake related to dementia and Alzheimer’s disease risks? Results from the Framingham Heart Study. (2022) Am J Clin Nutr. | DOI: doi.org/10.1093/ajcn/nqac193.

Zeisel, S.: A brief history of choline. (2012) Ann Nutr Metab. | DOI: doi.org/10.1159/000343120.

Zhan, X. et al.: Choline supplementation regulates gut microbiome diversity, gut epithelial activity, and the cytokine gene expression in gilts. (2023) Front. Nutr. | DOI: doi.org/10.3389/fnut.2023.1101519.

Zhong, C. et al.: Choline Pathway Nutrients and Metabolites and Cognitive Impairment After Acute Ischemic Stroke. (2021) | DOI: doi.org/10.1161/STROKEAHA.120.031903.

• History & Evolution
• Biosynthesis vs. dietary uptake
• TMAO and the microbiome
• Is TMAO good or bad?
• TMAO and cardiovascular diseases
• TMAO and diabetes
• TMAO and chronic kidney disease (CKD)
• TMAO and trimethylaminuria (TMAU)

History & Evolution

Trimethylamine N-oxide (TMAO) is an amine oxide and osmolyte commonly found in marine crustaceans and marine fish, protecting them against water pressure, temperature, salinity, and urea (Velasquez et al. 2016).

TMAO concentration appears to increase in fish tissues as they inhabit greater depths in the ocean, allowing them to withstand increasing hydrostatic pressure (Yancey et al. 2014). TMAO metabolizes into trimethylamine (TMA), which is responsible for the pungent smell of degrading seafood.

Recently, interest in TMAO has extended beyond the world of marine biology, with a growing body of research linking it to cardiovascular disease (CVD), insulin resistance, renal dysfunction, cancer, stroke, and inflammation in humans (Velasquez et al. 2016, Ufnal et.al. 2015, Pelletier et al. 2019, Farhangi et al. 2020).

It’s not all bad news though: research points to TMAO having several positive effects too, including circulatory benefits and helping cells maintain volume under stress (Ufnal et al. 2015).

Biosynthesis vs. dietary uptake

TMAO is biosynthesized from TMA, which is generated when gut bacteria metabolize choline, derived from lecithin, L-carnitine and betaine (Janeiro et al. 2018, Rohrmann et al. 2016). TMA is transported to the liver and oxidized into TMAO by the liver enzyme FMO3 (Wang et al. 2011). TMAO is then stored in cells or cleared by the kidneys.

The amount of TMAO found in plasma can be affected by diet, gut microbiota, drugs, and liver enzyme activity (Janeiro et al. 2018). Diet is the main source of TMAO and TMA precursors. TMAO levels increase after consuming foods high in choline, carnitine and lecithin, including red meat, seafood, eggs and dairy (Velasquez et al. 2016). Vegan and vegetarian diets do not seem to select for the gut bacteria needed to metabolize these precursors into TMAO (Koeth et al. 2013).

Small amounts of L-carnitine are synthesized endogenously from lysine and methionine, in the kidney, liver and brain (Cave et al. 2008). Similarly, humans can produce some choline endogenously through hepatic synthesis, though not enough to meet the body’s needs. TMAO is often seen as a waste product of choline, but does appear to play an important role in many biological processes (Ufnal et al. 2015).

TMAO and the microbiome

The composition of the gut microbiome heavily influences TMAO production. TMAO levels are dependent on the presence of specific intestinal microbiota, particularly Deferribacteraceae, Anaeroplasmataceae, Prevotellaceae and Enterobacteriaceae (Velasquez et al. 2016). Plasma levels of TMAO are suppressed when individuals take antibiotics

A short-term crossover feeding trial in healthy young men showed that TMAO production is a function of individual differences in the gut microbiome, and demonstrated differences in how microbiota respond to dietary TMAO and its precursors (Cho et al. 2016). For example, fish consumption led to higher quantities of TMAO metabolites than eggs or beef.

Dietary TMAO was absorbed without needing any processing by gut microbiota. Individuals who produced higher levels of TMAO seemed to have a less diverse gut microbiome.

Is TMAO good or bad?

A major debate in current TMAO research surrounds its role in linking the gut microbiome and cardiovascular disease. The link is there – but literature is unclear on whether TMAO is a bystander or a mediator (Janeiro et al. 2018), and whether it should be sought out or avoided in a healthy diet.

On one hand, TMAO and its precursors are found in foods eaten regularly in heart-healthy Mediterranean diets, particularly seafood. Some research suggests that TMAO could be beneficial for heart function. A study on hypertensive rats found that higher levels of TMAO had no negative effects on circulation, and a low-dose TMAO treatment helped reduce hypertension-related heart disease symptoms (Huc et al. 2018).

However, other studies have shown that increased levels of TMAO are associated with increased risk of CVD. Similarly, higher TMAO levels also appear to correlate with impaired kidney function, and reduced renal clearance of TMAO is associated with an increased risk of CVD.

It is thought that TMAO causes changes in cholesterol and bile acid metabolism leading to cell inflammation, which in turn creates an atherogenic effect, but whether TMAO is a causal factor or not remains unclear (Janeiro et al. 2018, Winther et al. 2021).

There is also a growing body of evidence identifying TMAO as a missing link between gut dysbiosis and other conditions, including colorectal cancer (often linked to red meat consumption). This would suggest TMAO-rich foods should be left off the menu.

TMAO and cardiovascular disease

The connection between TMAO and CVD was first made using an animal model in 2011, which revealed that choline, TMAO and betaine were predictors of CVD risk. When gut bacteria were suppressed, TMAO production was limited, which in turn inhibited atherosclerosis progression (the primary underlying cause of CVD)

Human studies into the relationship between TMAO and atherosclerosis have been generally small or with mixed results (Meyer et al. 2016). In one frequently cited three-year study of over 4000 individuals, high levels of TMAO in plasma correlated with an increased risk of adverse cardiovascular events, independent of traditional risk factors.

However, the validity of these findings has been challenged because of the high prevalence of comorbidities and pre-existing inflammation markers among participants (Meyer et al. 2016).

Interestingly, TMA has not attracted the same attention as a potential risk factor for CVD, despite being a known uremic toxin (Jaworska et al. 2019). Nevertheless, evidence is accumulating to show an association between gut microbes and cardiometabolic disease, and elevated plasma TMAO levels do seem to predict future risk of major adverse cardiac events and CVD (Senthong et al. 2016).

TMAO and diabetes

The emerging association between gut dysbiosis and insulin resistance is another area of interest in TMAO research (Øellgaard et al. 2017). One systematic review and meta-analysis looked at TMAO levels and diabetes risk, with a combined dataset of more than 15,000 participants.

This revealed a robust association between high circulating concentrations of TMAO and increased prevalence of diabetes mellitus: people with high circulating concentrations of TMAO were 50% more likely to have diabetes than those with lower levels, and people with diabetes had higher levels of TMAO than nondiabetic people.

In animal models, dietary TMAO impaired glucose tolerance and inhibited insulin signaling. Again, the precise mechanism remains unclear (Zhuang et al. 2019, Winther et al. 2021).

TMAO and chronic kidney disease (CKD)

Gut dysbiosis is linked with an increased risk of kidney disease, particularly when TMAO and choline levels are elevated (Velasquez et al. 2016). In one prospective cohort study of CKD patients, mortality risk was 2.8 times higher in those with higher TMAO levels In another study, patients with end-stage renal disease on dialysis had TMAO levels forty times higher than the control group (Hai et al. 2015).

While CKD is known to alter the gut microbiome, there is mounting evidence to suggest that TMAO is not only a biomarker for CKD, but also contributes to disease progression This points to diet, microbiome, hepatic TMA metabolism, and TMAO behavior as potential therapeutic targets.

A study in germ-free mice versus mice colonized with gut microbiota showed that the gut microbiota influences the host’s development of hypertensive organ damage, with TMAO and other microbial metabolites such as short-chain fatty acids (SCFAs) contributing to the phenotype (Avery et al., 2023).

TMAO and trimethylaminuria (TMAU)

Trimethylaminuria, or ‘fish odor syndrome,’ is caused when a mutation of FMO3 prevents the conversion of TMA into TMAO. Individuals with TMAU excrete TMA, instead of TMAO, which causes a fishy smell in urine, sweat, and breath (Velasquez et al. 2016).

TMAU is inherited when both parents carry FMO3-altering genes, and can be triggered by diet and stress. It also affects more women than men, suggesting a link with sex hormones. Treatment usually focuses on avoiding foods rich in TMA or TMA precursors, suppressing TMA production in the gut with antibiotics, and taking supplements to enhance FMO3 enzyme activity and reduce TMA concentration (National Human Genome Research Institute 2018).

Learn more about the roles of TMAO, TMA, and other microbial metabolites in complex chronic diseases such as cancer, Alzheimer’s disease, depression, inflammatory bowel disease, multiple sclerosis and diabetes in our whitepaper “Complex chronic diseases have a common origin”.

References

• History & Evolution
• Biosynthesis and dietary uptake
• 3-IAA and the gut microbiome
• 3-IAA and cancer
• 3-IAA as a biomarker
• Potential dietary and microbiome-based interventions with 3-IAA
• References

History & Evolution

1925: first synthesized (Majima et al., 1925) | 1933: identified as plant hormone (Weissbach et al., 1959) | 2020: identified as potential mediator of AhR activation (Sadik et al., 2020)

Indole-3-acetic acid, also referred to as 3-indoleacetic acid (3-IAA), is known as the most common plant growth hormone, though it is also found in mammals (Jones et al., 1995). Our understanding of it comes primarily from its role in plant biology, but interestingly, its function as a phytohormone was actually determined using human urine samples, in 1933 (Weissbach et al., 1959).

As a microbial metabolite of tryptophan, 3-IAA is associated with several important biological processes including immune function, inflammation and metabolic homeostasis. There’s a growing body of evidence linking indole derivatives with digestive disorders and toxicity (Zhang et al., 2020). Much current research focuses on 3-IAA’s role in activating the aryl hydrocarbon receptor (AhR) pathway, which presents potential therapeutic options for cancer and autoimmune disease (Shen et al., 2022).

Advances in omics techniques are allowing us to learn more about this metabolite. Researchers can investigate 3-IAA and other indole derivatives as potential biomarkers and mediators of disease, in a way they couldn’t before. This is hugely valuable given the prevalence of diet- and lifestyle-related disease and the role of the microbiome in those diseases.

An interesting recent discovery using omics techniques highlights the role of tryptophan-derived indole alkaloids in cellular responses to environmental change (Chen et al., 2023) . Prenyl indole alkaloids biosynthesized from tryptophan have been found to dramatically accumulate in the fungus Thermomyces dupontii under cold stress. Metabolomics revealed 3-IAA to be among the enriched metabolites, suggesting its involvement in a lipid-mediated fungal response to cold.

Biosynthesis vs. dietary uptake

In mammals, 3-IAA’s precursor indole is metabolized from tryptophan by gut microbiota. Tryptophan is an essential amino acid, which means 3-IAA production largely depends on dietary sources of tryptophan – typically dairy, eggs and meat. As these dietary proteins break down, tryptophan is released and converted into catabolites including indole, tryptamine and kynurenine.

Read our metabolite of the month article on tryptophan.

The vast majority of ingested tryptophan is metabolized to kynurenine in the gut by local immune cells and intestinal epithelial cells expressing the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). An estimated 5% of the tryptophan pool is metabolized by microbial tryptophanase and decarboxylase in the indole pathway, and an even smaller fraction is converted to serotonin and melatonin (Taleb, 2019; Wyatt et al., 2021).

3-IAA can be synthesized from several precusors depending on the enzymatic capacities of the intestinal microbiome. Indole pyruvic acid, indole acetamide and indole acetaldehyde are commonly reported as precursors of 3-IAA (Hendrikx et al., 2019; Taleb, 2019; Wyatt et al., 2021). Small amounts of indole are also metabolized into the uremic toxin indoxyl sulfate.

In addition to exogenous biosynthesis, 3-IAA may also be produced endogenously from tryptophan. For instance, cells transfected with the gene for interleukin-4-induced 1 (IL4I1) metabolized tryptophan to indole-3-pyruvic acid and then to 3-IAA (Zhang et al., 2020).
As a side note, this pathway also involves the decarboxylation of indole acetic acid into skatole, the intestinal metabolite 3-methylindole that sometimes causes an off-flavor in pork from male pigs (Roager et al., 2018).

3-IAA and the gut microbiome

Most of the reactions involved in 3-IAA synthesis occur in the small intestine and colon, with the resulting metabolites entering the blood circulation through the intestinal epithelium (Riazati, N. et al., 2022). In the liver, 3-IAA can combine with glutamine to produce indole acetyl glutamine, or be metabolized by peroxidases to indole-3-aldehyde (Gao, 2018; De Mello et al., 1980).

The synthesis of 3-IAA from dietary tryptophan is particularly associated with enzymatic reactions involving the Clostridium and Bacteriodes species of intestinal bacteria (Russell et al., 2013; Gao, 2018).

In humans, 3-IAA has been detected in blood, feces, urine, saliva and cerebrospinal fluid (Shen et al., 2022) (HMDB). Fecal samples of healthy adult humans contain around 5 µM of 3-IAA (Lamas et al., 2016). Serum concentrations are closer to 1 µM (Rosas, H. et al., 2015). Both blood and urinary levels vary greatly from individual to individual, possibly due to microbiome differences (Rosas et al., 2015; Pavlova et al., 2017).

There is a strong correlation between the gut microbiota and tryptophan metabolism, suggesting that changes in the composition of the gut microbiome could influence tryptophan levels and consequently 3-IAA production (Roager et al., 2018). These findings suggest that manipulating gut microbiota may be a therapeutic possibility for the treatment of many diseases associated with tryptophan metabolism and more specifically, with 3-IAA production.

3-IAA and cancer

Several studies show links between 3-IAA and cancer. Metabolomic analysis has been an invaluable tool in understanding these links and exploring potential treatment options.

For example, a 2023 study by Tintelnot et al. used metagenomic sequencing and targeted metabolomics to investigate factors that could shed light on the poor response to chemotherapy in patients with pancreatic ductal adenocarcinoma (PDAC). Patients who responded were found to have higher levels of 3-IAA. Mouse models showed that treatment with fecal microbiota transplantation, short-term dietary manipulation of tryptophan and oral 3-IAA administration increased the efficacy of chemotherapy (Tintelnot et al., 2023).

Watch the webinar: Joseph Tintelnot, PhD, joins biocrates to present the team’s research and discuss the potential for nutritional interventions in PDAC treatment.

Targeted metabolomic profiling was also used in a 2021 study of prognostic biomarkers in epithelial ovarian cancer (Hishinuma et al., 2021). This showed a correlation between IDO activity and disease risk, and found 3-IAA synthesis to be decreased in tumor-bearing patients. This suggests that IDO inhibitors may be a therapeutic option for patients with increased IDO activity.

In a 2017 study, the biocrates AbsoluteIDQ®p180 kit was used for the quantitative analysis of tryptophan and phenylalanine metabolites in patients with gastric carcinogenesis (Lario et al., 2017). The results showed significant alterations in tryptophan metabolism in patients with the disease, raising the possibility of biomarker candidates from metabolites in this pathway.

3-IAA could also be relevant for cancer therapy due to its cytotoxic effects when oxidized by horseradish peroxidase (HRP) (Folkes et al., 2001). Because 3-IAA is only toxic after oxidized decarboxylation, 3-IAA and HRP could be used as the basis for targeted cancer therapy. By targeting HRP to a tumor, 3-IAA could be activated in the tumor while minimizing systemic toxicity.

3-IAA as a biomarker

As well as being a promising biomarker for cancers, 3-IAA may be a useful diagnostic tool in other diseases, evidenced using metabolomic analysis. For example, males with long-term overweight and individuals with overweight from childhood to adolescence have been found to have lower levels of urinary 3-IAA (Oluwagbemigun et al., 2020). Indole metabolites may therefore act as indicators of cardiometabolic disease risk.

Indoleacetic acid has also been discussed as a marker of renal transporter-mediated drug-drug interactions (Gessner et al., 2023). A cross-sectional study found that higher levels of serum 3-IAA were significantly associated with knee pain scores in patients with osteoarthritis, suggesting its use as a pain marker (Mehta et al., 2023).

Plasma concentrations of 3-IAA have been explored as markers of systemic inflammation and immune activation, given the metabolite’s role in regulating immune activity via the AhR pathway (Riazati et al., 2022). As a ligand of AhR and free radical scavenger, 3-IAA mediates toxicity and inflammation, both limiting production of pro-inflammatory cytokines and modulating the immune cells that trigger anti-inflammatory cytokine production (Krishnan et al., 2018).

Potential dietary and microbiome-based interventions with 3-IAA

The links between diet, the metabolome and disease risk are well-established. Could dietary or microbiome-focused interventions be used to modify 3-IAA concentrations? Could doing so help mitigate disease risk or influence treatment efficacy? Research suggests this may be the case.

A high-fiber-low-protein diet has been found to favor microbial production of 3-IAA (Huang et al., 2023). High-fat diets are associated with lower indole production (Hou et al., 2023). Total protein intake is positively associated with plasma 3-IAA, suggesting that dietary tryptophan may influence health via the microbiome (Riazati et al., 2022).

Patients with inflammatory bowel disease (IBD) have been found to have reduced fecal concentrations of 3-IAA, and in mice, oral administration of indole and indole derivatives appeared to mediate colonic inflammation (Roager et al., 2018). This suggests that 3-IAA supplementation may be a therapeutic option for IBD patients.

Read the whitepaper: Learn more IBD through the lens of metabolomics in our whitepaper on the use of metabolomics to study complex chronic diseases.

Also in mice, supplementation with 3-IAA has been found to attenuate behaviors associated with depression and stress, enhance serotonin synthesis in the brain and gut, and alter gut microbiota (Chen et al., 2022).

Administration of 3-IAA via fecal transplant may be another route to influence gut microbiota in certain diseases. As discussed above, Tintelnot et al. demonstrated this in relation to cancer (Tintelnot et al., 2023). In another study, Ji et al. assessed the effects of 3-IAA in mice with high-fat diet-induced nonalcoholic fatty liver disease (NAFLD) (Ji et al., 2019). 3-IAA administered by intraperitoneal injection alleviated markers of insulin resistance and suggested a protective effect on liver damage.

While more research is needed, particularly in human subjects, these findings suggest promising avenues for exploring the use of 3-IAA as a biomarker and in therapeutic interventions.

References

Chen, Y. et al.: Indole Acetic Acid Exerts Anti-Depressive Effects on an Animal Model of Chronic Mild Stress. (2022) Nutrients | https://doi.org/10.3390/nu14235019

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Folkes, L. et al.: Oxidative activation of indole-3-acetic acids to cytotoxic species— a potential new role for plant auxins in cancer therapy. (2001) Biochemical Pharmacology | https://doi.org/10.1016/S0006-2952(00)00498-6

Gao, J.: Impact of the Gut Microbiota on Intestinal Immunity Mediated by Tryptophan Metabolism. (2018) Front Cell Infect Microbiol. | https://doi.org/10.3389/fcimb.2018.00013

Gessner, A. et al.: A Metabolomic Analysis of Sensitivity and Specificity of 23 Previously Proposed Biomarkers for Renal Transporter-Mediated Drug-Drug Interactions. (2023) Clin Pharmacol Ther., | https://doi.org/10.1002/cpt.3017

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Huang, Z. et al.: Impact of High-Fiber or High-Protein Diet on the Capacity of Human Gut Microbiota To Produce Tryptophan Catabolites. (2023) J Agric Food Chem., | https://doi.org/10.1021/acs.jafc.2c08953

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• History & Evolution
• Biosynthesis and dietary uptake
• p-cresol sulfate and nephrology
• p-cresol sulfate and cardiometabolic diseases
• p-cresol sulfate, microbiota and the gut
• p-cresol sulfate and neurology
• References

History & Evolution

Also referred to as p-cresyl sulfate, this metabolite is a sulfate conjugate of the bacterial metabolite p-cresol, which is a uremic toxin. p-Cresol sulfate originates when gut bacteria ferment proteins in the large intestine. Researchers should bear in mind that sample preparation can promote deconjugation back to the p-cresol precursor, so high levels of p-cresol but not p-cresol sulfate in blood samples can be considered an indicator of sample degradation (Loor et al. 2005). Research on the effects of temperature and humidity in gestating cattle has also shown that the levels of p-cresol sulfate, amongst other metabolites, are influenced in both cow and calf (Halli et al. 2023).

Biosynthesis and dietary uptake

When dietary proteins reach our large intestine, their amino acids can be fermentation substrates for commensal bacteria. Some of the resulting metabolites are essential nutrients, while others can be detrimental to our health. p-Cresol sulfate is a product of tyrosine fermentation by bacteria of the Coriobacteriaceae or Clostridium genera (Saito et al. 2018), followed by sulfation by the host’s cells (Wikoff et al. 2009). This sulfation step is part of phase II detoxification mechanisms that promote the elimination of harmful toxins by the kidneys. Once in the blood, p-cresol sulfate binds with high affinity to albumin, but is also found to a small extent in its free form (Viaene et al. 2013).

p-cresol sulfate and nephrology

When the kidneys are well-functioning, p-cresol sulfate can be excreted in the urine. However, as kidney function declines, the blood level of p-cresol sulfate increases, hence its classification as a uremic toxin (Glassock 2008). Unsurprisingly, we see higher levels of p-cresol sulfate in the blood as chronic kidney disease (CKD) progresses and renal function declines (Wu et al. 2011; Liabeuf et al. 2010). Because p-cresol sulfate is mainly protein-bound in the blood, most of it escapes artificial blood filtration by dialysis and remains in the patient’s body, which complicates CKD management (Ma et al. 2020).

In renal cells, p-cresol sulfate has been linked to damage to tubular cells mediated by oxidative stress (Watanabe et al. 2013), and activation of epithelial-to-mesenchymal transition which can lead to fibrosis (Sun et al. 2012). It’s also associated with inflammation and overall kidney damage (Vanholder et al. 2014), and is also linked to one of the main co-morbidities of CKD: cardiovascular disease (Meijers et al. 2010).

p-cresol sulfate, microbiota and the gut

A 2009 metabolomic comparison of the plasma of conventional vs. germ-free mice identified hundreds of features altered by the intestinal microbiome, including p-cresol sulfate and its precursor, tyrosine (Wikoff et al. 2009). The study also identified sulfation as a mechanism to modify metabolites resulting from the fermentation of tyrosine (p-cresol sulfate), phenylalanine (phenyl sulfate), tryptophan (indoxyl sulfate) and flavones (equol sulfate and methyl equol sulfate). A 2011 comparison of dialysis patients with and without a colon (following colectomy), confirmed the colonic origin of p-cresol sulfate and indoxyl sulfate in humans (Aronov et al. 2011).

The gut microbiota is a highly competitive environment. Metabolites produced by commensal bacteria can act as bacteriostatic molecules, i.e. compounds that impede the growth of other bacteria. Clostridium difficile, one of the most studied bacteria living in our intestine, uses p-cresol to reduce bacterial diversity in its environment (Passmore et al. 2018; Selmer and Andrei 2001). This bacteriostatic effect is also visible when adding millimolar concentrations of p-cresol to sewage water, impacting bacteria, protozoa and metazoan (Erb et al. 1997).

Comparing the microbiota of patients with end stage renal disease (ESRD) to that of healthy subjects showed a large difference in microbial diversity. ESRD patients had more bacteria expressing hydroxyphenylacetate decarboxylase, an enzyme responsible for the conversion of tyrosine to p-cresol, and less bacteria capable of converting dietary fiber to short-chain fatty acids (Wong et al. 2014). In both human and mouse, p-cresol sulfate was identified as a promising biomarker of antibiotic treatment and fecal microbiota transplantation in urine (Zhou et al. 2023).

p-cresol sulfate and neurology

Although uremic toxins are thought to contribute to cognition complications in CKD patients (Watanabe et al. 2014), few studies have looked at the role of p-cresol sulfate in neurological disorders. A 2020 study conducted on Parkinson’s disease (PD) patients revealed that levels of p-cresol sulfate in cerebrospinal fluid (CSF) were higher than circulating levels (Sankowski et al. 2020), thus demonstrating the capacity of the metabolite to cross the blood-brain-barrier (BBB) in a significant amount. The authors suggest that this is due to a damaged BBB, but cannot exclude the possibility that p-cresol sulfate crosses the BBB via organic anion transporters (OATs), which are known to process the secretion of p-cresol sulfate in renal tubular cells (Miyamoto et al. 2011).

In an in vitro screening of uremic toxins, p-cresol sulfate was one of the least toxic molecules in cultured mouse hippocampal neuronal cells (Kimio Watanabe et al. 2021). Although it did not induce cell death at the concentrations tested, this does not rule out the possibility that p-cresol sulfate could affect the physiology and functions of the cells, even at the concentrations tested.

Another study looked at the association of plasmatic levels of uremic toxins with cognitive impairment in patients undergoing hemodialysis. This found a link with indoxyl sulfate but not p-cresol sulfate, although it only investigated the association with free circulating levels of these mostly albumin-bound molecules (Yi-Ting Lin et al.). Further exploration of the role of p-cresol sulfate in these mechanisms could have implications for the understanding and treatment of multiple neurological conditions.

p-cresol sulfate and cardiometabolic diseases

There is a well-established link between high blood levels of p-cresol sulfate and mortality from cardiovascular disease (Barreto et al. 2009; Cheng-Jui Lin et al. 2015). However, there are question marks around whether p-cresol sulfate and other uremic toxins are reliable predictors of cardiovascular disease. A 2017 clinical trial suggests that their predictive power is relevant in patients who also have a low level of circulating albumin, but does not hold up when applied to a broader population (Shafi et al. 2017).

On the other hand, levels of p-cresol sulfate appear to be negatively correlated with both peak cardiac power and aerobic exercise capacity in male CKD patients (Chinnappa et al. 2018). At the cellular level, p-cresol sulfate and other uremic toxins have been shown to induce the calcification of vascular smooth muscle cells (Hénaut et al. 2018). The mechanism involves the production of oxidative stress and inflammation, leading to a phenotypic switch from a contractile to a synthetic phenotype, including proliferation, senescence and calcification (Chang et al. 2020). In epithelial cells, p-cresol sulfate and indoxyl sulfate (a metabolite of tryptophan produced through bacterial fermentation in the intestine) are also thought to induce epithelial-to-mesenchymal transition, leading to fibrosis and calcification of the vessels (Opdebeeck et al. 2020).

In a CKD rat model, a 7-week exposure to p-cresol sulfate or indoxyl sulfate was shown to induce arterial calcification. Exposure also triggered a prodiabetic state characterized by impaired glucose homeostasis and a decrease in GLUT1 expression (Opdebeeck et al. 2019). Interestingly, one difference between rats that did and did not experience calcification was the activation of lipid-regulating nuclear receptors LXR, FXR and PPAR. This suggests a possible protection mechanism against p-cresol sulfate-induced effects. The administration of p-cresol sulfate to healthy mice also triggers insulin resistance and redistribution of lipids in the muscle and liver tissues (Koppe et al. 2013).

Learn more about the roles of p-cresol sulfate and other microbial metabolites in complex chronic diseases such as cancer, Alzheimer’s disease, depression, inflammatory bowel disease, multiple sclerosis and diabetes in our whitepaper “Complex chronic diseases have a common origin”.

References

Aronov, Pavel A. et al.: Colonic Contribution to Uremic Solutes. (2011) Journal of the American Society of Nephrology | https://doi.org/10.1681/ASN.2010121220

Barreto, Fellype C. et al.: Serum Indoxyl Sulfate Is Associated with Vascular Disease and Mortality in Chronic Kidney Disease Patients (2009) Clinical journal of the American Society of Nephrology | https://doi.org/10.2215/CJN.03980609

Chang, Jia-Feng et al.: Scavenging Intracellular ROS Attenuates p-Cresyl Sulfate-Triggered Osteogenesis through MAPK Signaling Pathway and NF-κB Activation in Human Arterial Smooth Muscle Cells (2020) Toxins | https://doi.org/10.3390/toxins12080472

Cheng-Jui Lin et al.: Meta-Analysis of the Associations of p-Cresyl Sulfate (PCS) and Indoxyl Sulfate (IS) with Cardiovascular Events and All-Cause Mortality in Patients with Chronic Renal Failure.(2020) PLOS ONE | https://doi.org/10.1371/journal.pone.0132589

Chinnappa, S.et al.: Association between Protein-Bound Uremic Toxins and Asymptomatic Cardiac Dysfunction in Patients with Chronic Kidney Disease.(2018) Toxins | https://doi.org/10.3390/toxins10120520

Erb, R. W. et al.: Bioprotection of microbial communities from toxic phenol mixtures by a genetically designed pseudomonad.(1997) Nature biotechnology | https://doi.org/10.1038/nbt0497-378

Halli et al. Effects of temperature-humidity index on blood metabolites of German dairy cows and their female calves. (2023) Journal of Dairy Science | https://doi.org/10.3168/jds.2022-22890

Hénaut, L. et al.: The Impact of Uremic Toxins on Vascular Smooth Muscle Cell Function.(2018) Toxins | https://doi.org/10.3390/toxins10060218

Glassock R.: Uremic toxins: what are they? An integrated overview of pathobiology and classification (2008) Journal of renal nutrition | https://doi.org/10.1053/j.jrn.2007.10.003 

Kimio Watanabe et al.: Effect of uremic toxins on hippocampal cell damage: analysis in vitro and in rat model of chronic kidney disease. (2021) Heliyon | https://doi.org/10.1016/j.heliyon.2021.e06221

Koppe, L. et al.: p-Cresyl sulfate promotes insulin resistance associated with CKD. (2013) Journal of the American Society of Nephrology | https://doi.org/10.1681/ASN.2012050503

Liabeuf, S. et al.: Free p-cresylsulphate is a predictor of mortality in patients at different stages of chronic kidney disease. (2010) Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association | https://doi.org/10.1093/ndt/gfp592

Loor, Henriette de et al.: Gas chromatographic-mass spectrometric analysis for measurement of p-cresol and its conjugated metabolites in uremic and normal serum. (2005) Clinical chemistry | https://doi.org/10.1373/clinchem.2005.050781

Ma, Y. R. et al.: An LC-MS/MS analytical method for the determination of uremic toxins in patients with end-stage renal disease. (2020) Journal of pharmaceutical and biomedical analysis | https://doi.org/10.1016/j.jpba.2020.113551

Meijers, B. K. et al.: p-Cresol and cardiovascular risk in mild-to-moderate kidney disease. (2010) Clinical journal of the American Society of Nephrology | https://doi.org/10.2215/CJN.07971109

Miyamoto, Y. et al.: Organic anion transporters play an important role in the uptake of p-cresyl sulfate, a uremic toxin, in the kidney. (2011) Nephrology, dialysis, transplantation | https://doi.org/10.1093/ndt/gfq785

Opdebeeck, B. et al.: Indoxyl Sulfate and p-Cresyl Sulfate Promote Vascular Calcification and Associate with Glucose Intolerance. (2019) Journal of the American Society of Nephrology | https://doi.org/10.1681/ASN.2018060609

Opdebeeck, Britt et al.: Molecular and Cellular Mechanisms that Induce Arterial Calcification by Indoxyl Sulfate and P-Cresyl Sulfate. (2020) Toxins | https://doi.org/10.3390/toxins12010058

Passmore, I. J. et al.: Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria. (2018) PLoS pathogens | https://doi.org/10.1371/journal.ppat.1007191

Saito, Yuki et al.: Identification of phenol- and p-cresol-producing intestinal bacteria by using media supplemented with tyrosine and its metabolites. (2018) FEMS Microbiol Ecol | https://doi.org/10.1093/femsec/fiy125

Sankowski, Bartłomiej et al.: Higher cerebrospinal fluid to plasma ratio of p-cresol sulfate and indoxyl sulfate in patients with Parkinson’s disease. (2020) Clinica Chimica Acta | https://doi.org/10.1016/j.cca.2019.10.038

Selmer, T. et al.: p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of p-cresol. (2001) European journal of biochemistry | https://doi.org/10.1046/j.1432-1327.2001.02001.x

Shafi, T. et al. (2017): Results of the HEMO Study suggest that p-cresol sulfate and indoxyl sulfate are not associated with cardiovascular outcomes. (2017) Kidney international | https://doi.org/10.1016/j.kint.2017.05.012.

Sun, C. Y. et al.: Uremic toxins induce kidney fibrosis by activating intrarenal renin-angiotensin-aldosterone system associated epithelial-to-mesenchymal transition. (2012) PloS one | https://doi.org/10.1371/journal.pone.0034026

Vanholder, Raymond et al.: The Uremic Toxicity of Indoxyl Sulfate and p-Cresyl Sulfate: A Systematic Review. (2014) Journal of the American Society of Nephrology | https://doi.org/10.1681/ASN.2013101062

Viaene, L. et al.: Albumin is the main plasma binding protein for indoxyl sulfate and p-cresyl sulfate. (2013) Biopharmaceutics & drug disposition | https://doi.org/10.1002/bdd.1834

Watanabe, H. et al. (2013): p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase. (2013) Kidney international | https://doi.org/10.1038/ki.2012.448

Watanabe, K. et al.: Cerebro-renal interactions: Impact of uremic toxins on cognitive function. (2014) NeuroToxicology | https://doi.org/10.1016/j.neuro.2014.06.014

Wikoff, William R. et al.: Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. (2009) Proceedings of the National Academy of Sciences of the United States of America | https://doi.org/10.1073/pnas.0812874106

Wong, J. et al.: Expansion of urease- and uricase-containing, indole- and p-cresol-forming and contraction of short-chain fatty acid-producing intestinal microbiota in ESRD. (2014) American journal of nephrology | https://doi.org/10.1159/000360010

Wu, I-Wen et al.: p-Cresyl sulphate and indoxyl sulphate predict progression of chronic kidney disease. (2011) Nephrol Dial Transplant | https://doi.org/10.1093/ndt/gfq580

Yi-Ting Lin et al.: Protein-bound uremic toxins are associated with cognitive function among patients undergoing maintenance hemodialysis. (2019) Sci Rep | https://doi.org/10.1038/s41598-019-57004-7

Zhou et al.: p-Cresol Sulfate Is a Sensitive Urinary Marker of Fecal Microbiota Transplantation and Antibiotics Treatments in Human Patients and Mouse Models. (2023) International Journal of Molecular Sciences | https://doi.org/10.3390/ijms241914621

History & Evolution

2000 BC: cinnamon is used to embalm mummies (Abdel-Maksoud et al. 2011) | 1834: isolation of cinnamaldehyde from cinnamon oil (Dumas et al. 1834) | 1854: first synthesis from unrelated compounds (Richmond 1947)

As the name suggests, cinnamaldehyde is a compound found in cinnamon, contributing to cinnamon’s flavor, aroma and potential health benefits. Cinnamaldehyde has antimicrobial, antioxidant and anti-inflammatory properties, and is also studied for its potential effects on cardiovascular and metabolic diseases.

Cinnamon is prepared from the inner bark of Asian evergreen trees, with Sri Lanka its primary producer. Tree bark is typically removed from the branches of mature trees and left to dry in the sun without additional treatment (Shang et al. 2021). Dried bark curls into cinnamon sticks and may be ground into powdered form. Different species of cinnamon tree contain different amounts of cinnamaldehyde and other metabolites. Cinnamomum verum (native to Sri Lanka and later introduced in other countries of the Indian subcontinent) is considered the original cinnamon tree for international trade.

Metabolomics has been used to find signatures of the different cinnamon tree species in cinnamon samples (Avula et al. 2015; Wang et al. 2020; Zhang et al. 2022). Cinnamon from C. verum is typically high in cinnamaldehyde and low in coumarin (Wang et al. 2020). Metabolic profiling can differentiate ‘true’ cinnamon from C. verum from other plants used to produce cinnamon such as C. cassia, simply by measuring the proportion of cinnamaldehyde, coumarin and other metabolites in the samples (Zhang et al. 2022). C. cassia and other species growing in China are prevalent in traditional Chinese medicine.

Cinnamaldehyde is also synthesized by a broad range of microorganisms that exploit its antibacterial and antifungal properties (Gan et al. 2020). In addition, bacteria (e.g., E. coli) can be engineered to synthesize cinnamaldehyde from phenylalanine (Bang et al. 2016).

Biosynthesis vs. dietary uptake

In plants, cinnamaldehyde is synthesized via the Shikimate pathway, a pathway that also yields aromatic amino acids and folates (Morrow 2013). Starting with phosphoenolpyruvate (PEP), this pathway generates aromatic amino acids that are precursors to cinnamaldehyde. Interestingly, bacteria and other microorganisms can also synthesize cinnamaldehyde through this pathway, for example, from phenylalanine.

In C. verum, phenylalanine ammonia-lyase catalyzes the conversion of phenylalanine into trans-cinnamic acid, a compound with antioxidant and anti-inflammatory properties also responsible for some of cinnamon’s biological activities.

It has also been suggested that cinnamic acid plays a role in improving insulin sensitivity (Stevens et al. 2022) and in protection of the cardiovascular system, making it potentially beneficial for people with diabetes and early-stage metabolic disease. Kinetic analysis in rat blood showed that cinnamaldehyde was quickly converted to cinnamic acid via a protein-driven mechanism (Yuan J. et al. 1992).

Cinnamaldehyde and infectious diseases

Cinnamaldehyde and its derivatives have attracted attention for their antimicrobial potential, for example in the development of tuberculosis treatment (Nordqvist et al. 2011). Metabolomics has shown that exposing cultures of Mycobacterium tuberculosis (the strain responsible for the disease) to cinnamon essential oil alters small molecules, including biotin levels and tetrahydrofolate biosynthesis, which is essential for optimal one-carbon metabolism (Sieniawska et al. 2020).

The same study revealed a significant effect on many lipid classes, with most changes seen in phospholipids (primarily phosphatidylethanolamines and phosphatidylglycerols) and glycerophospholipids (primarily triglycerides and monoglycerides).

Cinnamaldehyde is not the only antibacterial compound in cinnamon; other metabolites such as eugenol may contribute to the antimicrobial effects of cinnamon essential oil and extracts (Vasconcelos et al. 2018).

Cinnamaldehyde and metabolic disease

Cinnamon has been long considered a beneficial food for patients with type 2 diabetes. There is mounting evidence that cinnamon and its metabolites may improve glycemic and lipidemic indicators (Silva et al. 2022). For instance, a 2007 study in male rats with streptozotocin-induced diabetes showed that a 45-day treatment with 20 mg/kg bw of cinnamaldehyde reduced plasma glucose and glycosylated hemoglobin levels, serum total cholesterol and triglyceride levels while increasing insulin, high-density lipoprotein (HDL) cholesterol and liver glycogen levels (Subash Babu et al. 2007).

Randomized controlled clinical trials have investigated the effects of cinnamon and shown that 1 to 3 g of cinnamon per day could reduce glycosylated hemoglobin levels (Silva et al. 2022). Clinical trials also confirmed its anti-inflammatory effect in humans (Davari et al. 2020).

Of note, while C. verum is the plant of choice for culinary cinnamon, many studies focus on C. cassia, C. zeylanicum and others. Whether this is due to easier access, a higher prevalence of those species in traditional Chinese medicine, or a higher therapeutic potential in those species is unclear.

Nevertheless, there appear to be large differences in the effects and required doses depending on the tree of origin for the cinnamon used in these trials. This may explain why a recent meta-analysis of epidemiological studies found no associations between cinnamon intake and levels of low-density lipoprotein (LDL) cholesterol, HDL cholesterol or glycosylated hemoglobin (Krittanawong et al. 2022). Thus, more work is needed to fully understand this spice.

Cinnamaldehyde and chronic diseases

Finally, research into the health benefits of cinnamon points to potential to address multiple complex chronic diseases, even beyond its anti-inflammatory effect. For example, cinnamaldehyde may have applications in cancer, owing to its capacity to induce apoptosis in cancer cells (Sadeghi et al. 2019). Cinnamon’s anti-inflammatory properties and unique flavor have been hypothesized to help breast cancer survivors better adhere to a Mediterranean diet (Zuniga et al. 2019).

Cinnamon’s effects on the immune system have also made it a spice of interest in the field of autoimmune diseases (Pahan et al. 2020). There are also ongoing trials focusing on the effects of cinnamon in various chronic diseases, from Alzheimer’s disease to autoimmune diseases. These encouraging findings suggest that cinnamon and its key metabolites could play an important role in redressing the metabolic imbalance at the origin of many complex chronic diseases (Hariri et al. 2016).

References

Abdel-Maksoud et al. 2011: A review on the materials used during the mummification processes in ancient Egypt | A review on the materials used during mummification processes in Ancient Egypt

Avula et al. 2015: Authentication of true cinnamon (Cinnamon verum) utilising direct analysis in real time (DART)-QToF-MS. Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment | https://doi.org/10.1080/19440049.2014.981763

Bang et al. 2016: Metabolic engineering of Escherichia coli for the production of cinnamaldehyde. Microbial Cell Factories | https://doi.org/10.1186/s12934-016-0415-9

Davari et al. 2020: Effects of cinnamon supplementation on expression of systemic inflammation factors, NF-kB and Sirtuin-1 (SIRT1) in type 2 diabetes: a randomized, double blind, and controlled clinical trial. Nutrition Journal | https://doi.org/10.1186/s12937-019-0518-3

Dumas & Péligot 1834: Recherches de chimie organique – Sur l’huile de cannelle, l’acide hippurique et l’acide sébacique. Annales de chimie et de physique | https://gallica.bnf.fr/ark:/12148/bpt6k6568974z/f311.image.r

Gan et al. 2020: Synthesis and Antifungal Activities of Cinnamaldehyde Derivatives against Penicillium digitatum Causing Citrus Green Mold. Journal of Food Quality | https://doi.org/10.1155/2020/8898692

Hariri & Ghiasvand 2016: Cinnamon and Chronic Diseases. Advances in experimental medicine and biology | https://doi.org/10.1007/978-3-319-41342-6_1

Krittanawong et al. 2022: Association Between Cinnamon Consumption and Risk of Cardiovascular Health: A Systematic Review and Meta-Analysis. The American journal of medicine | https://doi.org/10.1016/j.amjmed.2021.07.019

Nordqvist et al. 2011: Synthesis of functionalized cinnamaldehyde derivatives by an oxidative Heck reaction and their use as starting materials for preparation of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase inhibitors. The Journal of Organic Chemistry | https://doi.org/10.1021/jo201715x

Pahan & Prahan 2020 : Can cinnamon spice down autoimmune diseases? Journal of clinical & experimental immunology | https://doi.org/10.33140/jcei.05.06.01

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Sadeghi et al. 2019: Anti-cancer effects of cinnamon: Insights into its apoptosis effects. European journal of medicinal chemistry | https://doi.org/10.1016/j.ejmech.2019.05.067

Shang et al. 2021: Beneficial effects of cinnamon and its extracts in the management of cardiovascular diseases and diabetes. Food & Function | https://doi.org/10.1039/D1FO01935J

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