Does metabolomics have a place in microbiome research?

Microbiome research and human health

From the lumen of our gut to the top of our skin and the inside of our lungs, bacteria and other microorganisms live with us. They share our food, experience our environmental exposures, and feed on our waste products. In return, they provide nutrients – some essential, some harmful. Thus, our microbiomes play a crucial role in human health, influencing digestion, metabolism, immune function, and even mental health. Microbiome composition and function have been linked to numerous diseases, including inflammatory bowel disease (IBD), obesity, diabetes, Alzheimer’s disease and autoimmune disorders (Limonciel et al. 2023).

Unlike metagenomics, which reveals the potential functions encoded in microbial DNA, metabolomics sheds light on the actual metabolic activities of the microbiome that provide physical signals for communication with the host. If metabolite activity represents the “message” sent to the host, metabolomics is the most comprehensive tool available to listen to that message. In this article, we’ll explore how metabolomics helps decipher the microbiome’s messages, and how the field is leading the way in 4P medicine – medicine that is predictive, personalized, preventive and participatory

Metabolites are the microbiome’s message to the host

One of the main goals of microbiome research is to understand how microbial communities function and interact with their host environment. Here are a few examples of how the microbiome influences host health using metabolites as its messengers:

• Short-chain fatty acids (SCFAs) like propionic acid or butyric acid provide energy substrates for host cells, freeing resources for higher functions such as maintaining the epithelial barrier in the gut.
• Secondary bile acids such as deoxycholic acid are ligands to numerous host receptors and influence not only their own metabolism, but also energy metabolism, intracellular signaling via cyclic adenosine monophosphate (cAMP) and mitogen-activated protein (MAP) kinases, and immune cell activation.
• Indole derivatives such as 3-indole acetic acid (3-IAA) are synthesized by gut bacteria from the amino acid tryptophan and are ligands to the host aryl hydrocarbon receptor (AhR), governing activity in the immune system.
• Derivatives of kynurenine can be synthesized by both the host and the microbiome. In a mouse model, gut-derived kynurenic acid was shown to activate local macrophages and promote an inflammatory phenotype serving as an animal model of multiple sclerosis (Miyamoto et al. 2023).
• Trimethylamine, the precursor of the infamous TMAO with highly debated links to cardiovascular disease, is also a product of gut bacteria metabolism, often derived from choline.
• Several uremic toxins, which are metabolites that are poorly excreted by the kidneys and accumulate in the blood, are also derived from microbial metabolites. Although their effects are not always understood, they have been shown to associate with neurological diseases such as Parkinson’s disease and with cognitive decline.
• Vitamin K and several B vitamins have also been shown to be synthesized by gut microbiota, providing an additional and modulable source of these essential nutrients besides diet.

Metabolomics meets microbiome research

In microbiota-host communication, metabolites are the easiest message to quantify. Standardized metabolomics gives us access to a deeper functional understanding of the impact of microbial communities on health and allows us to link gene-derived functions to the metabolites that mediate their effects.

•  In immunology, circulating tryptophan derivatives and SCFAs have been shown to play an essential role in host immune training (Belkaid and Harrison 2017).
•  In cardiovascular research, the interplay between indoxyl sulfate, TMAO and SCFAs links the microbiome to kidney damage in a mouse model of hypertension (Avery et al. 2023).
• In gastroenterology, fecal levels of bile acids have been shown to associate with microbial community structures in pediatric patients with Crohn’s disease (Connors et al. 2020).
• In neurology, p-cresol sulfate and other microbiota-derived metabolites have been quantified in the brains of patients with Parkinson’s disease (Kalecký et al. 2023), and SCFAs were found to protect blood-brain barrier function (Knox et al. 2022).
• In pulmonology research, a high-soluble-fiber diet modulated gut microbial communities and plasma levels of corresponding microbial metabolites (SCFAs, p-cresol sulfate) and influenced pulmonary vascular remodelling (Pakhomov et al. 2023).
• In oncology, 3-indole acetic acid could modulate response to chemotherapy in a mouse model of pancreatic cancer (Tintelnot et al. 2023).

Astute metabolomists may be wondering where these changes were measured. Were they measured in feces (as is often the case for metagenomics)? Were they measured in blood (which collects messages from most of our microbiomes and from the host)? Or were they measured in yet another matrix such as a relevant tissue or swab sample? This is a highly relevant question when applying metabolomics to the study of the microbiome and its effects, and the most common choice in the literature may not be the right one for your study. 

The most relevant metabolomics for microbiome research

Most microbiome studies rely on fecal samples. However, the usefulness of collecting samples directly within the intestine has become obvious in later years. Composition of both microbiota and metabolites is drastically different before and after digestion and excretion, so it’s important to be clear about what you want to investigate.

If your main interest is what is excreted or not absorbed by the host, feces is most likely the best option. However, note that feces is a notoriously difficult matrix to homogenize. Normalization across a study is a particular challenge due to varying water contents. In addition, elaborate sample preparation and storage can alter metabolite levels. Careful consideration must be given to ensuring the sampling method and sample preparation are as standardized as possible.

If you want to track the messages that have entered the host’s system, circulating blood is a more appropriate matrix. Circulating blood is rich in microbial metabolites. These are often hypothesized to come from the gut microbiome due to its high absorptive function, but other microbiomes are likely to contribute as well. Note that blood contains the microbial metabolites that passed through the cells that line the intestine (although it will not reflect the ones that were consumed or stored by these cells). Many of the remaining metabolites are quantifiable in whole blood, dried blood, plasma and serum.

Assuming that you’ve used the best possible matrix for your study, what sort of discoveries or innovations can you expect from working on the microbiome? While a few of the examples above reveal how and where microbial metabolites affect our biology, others shed light on promising new therapeutic strategies that use metabolomics insights.

Metabolomics supports microbiome-related innovation

The quantification of microbial metabolites and their effects on the host has provided a wealth of new therapeutic targets and approaches to provide better preventive, predictive, personalized and even participatory medicine (4P medicine). Let’s look at a few examples.

Drug development

The effects of a drug or treatment are influenced by many factors, from a person’s genetics and how they metabolize the drug, to their exposures, their diet, and of course, their microbiome. In a 2023 publication, Tintelnot et al. show how microbial metabolism influences the efficacy of chemotherapy treatment in a cohort of patients with pancreatic cancer.

Fecal microbiota transplant (FMT)

In the early days of microbiome research, two important facts were discovered: firstly, the gut microbiome strongly influences our health; and secondly, no straightforward connection has been found (yet) between microbial composition and functional effects on the host. As a result, the simplest way to test the transferability of a “good” gut microbiome from one individual to another was to transfer the microbial community directly via fecal microbiota transplants. Although FMT has shown promise for patients with IBD and non-gastrointestinal diseases such as cancer, the response varies from patient to patient, and not all respond positively. In a recent study, metabolomics identified predictive markers of patient response to FMT which may improve the effectiveness of this approach (Routy et al. 2023).

Prebiotics, probiotics & postbiotics

Microbiome research has had a distinct trajectory, running parallel to and sometimes even ahead of traditional drug development. What we eat has a direct impact on our microbiome without the need for therapeutic intervention. What’s more, it can even modulate the efficacy or toxicity of therapeutic intervention, as we saw in the previous examples. Consuming prebiotics, probiotics and postbiotics can mediate the effect of our food on our microbiome. As these products are not currently regulated as pharmaceuticals and freely available over the counter, this approach aligns well with the goals of participatory and personalized medicine.

Prebiotics are the compounds in our food that feed our gut microbiome, with potential impact on our health. For example, dietary fiber acts as a substrate for the gut microbiome’s synthesis of SCFAs, affecting both our gut and our immune system (Pakhomov et al. 2023).

Probiotics are selected microbiota strains that are commonly taken as over-the-counter supplements. They can help restore a healthier gut microbiome, though their effects are difficult to predict and should be sustained with appropriate prebiotics. General practitioners are becoming more comfortable discussing probiotics with patients, although medical education remains quite limited.

Postbiotics are metabolites generated by the microbiome that may be ingested as supplements to compensate for lacks in microbial metabolic activities. These supplements have the potential to influence disease progression, for example for complex diseases with a strong metabolic background. One example is the SCFA propionic acid, which has been shown to have clinical potential in patients with multiple sclerosis (Duscha et al. 2020).

While our understanding of the complex relationship between pre-, pro- and post-biotics remains in the early stages, it’s clear that supplementation could play a major role in improving health and supporting 4P medicine, as discussed below.