Can we estimate prebiotic effects from short-chain fatty acid production?

By Prof. Kristin Verbeke PhD, KU Leuven

Short-chain fatty acids (SCFA), primarily acetate, propionate and butyrate, are the most abundant anions in the large intestine. They are mainly produced from bacterial fermentation of undigested carbohydrates. Since SCFA were found to activate the orphan G-protein coupled receptors GPR-41 and 43 (renamed as free fatty acid receptor ffar-3 and ffar-2), research into their physiological effects on human health has increased exponentially.

SCFA production is proposed to be a mechanism for several health benefits associated with intake of dietary fiber and prebiotics, not only via local effects in the gut but also on distant organs. Molecular mechanisms explaining SCFA effects have mainly been elucidated in cell-based in vitro experiments and animal studies. However, studying the impact of SCFA on human physiology is complicated by the kinetics of these molecules.

Although fecal concentrations of SCFA are relatively easy to measure, consensus has grown that they provide little information. Fecal SCFA do not adequately reflect the production of SCFA in the proximal colon and only represent the fraction of SCFA that has been produced and not used. The capacity of the anion transporters,mainly the monocarboxylate transporter-1 (MCT-1) and sodium-coupled monocarboxylate transporter 1 (SMCT-1), that absorb SCFA into the colonocytes does not seem to be a limiting factor. More bacterial SCFA production results in more uptake of SCFA but not necessarily in a higher fecal excretion. For instance, when we administered colon-delivery capsules containing SCFA in a dose of 250 mmol (equivalent to what is produced from 20 g of fermentable fiber), fecal SCFA concentrations did not increase, indicating nearly complete absorption into the colonocytes (1).

Quantification of SCFA in serum or plasma provides a more relevant alternative, particularly for understanding effects of SCFA on distant organs. Systemic SCFA concentrations are about a 1000-fold lower than fecal concentrations, requiring more sophisticated analytical protocols for measurement. Currently, both GC-MS or LC-MS/MS protocols with or without prior derivatization are available for accurate and reliable SCFA quantification (2). However, it is important to be aware of the ubiquitous nature of acetate and to take sufficient precautions to avoid contamination. For instance, the type of blood tubes used for blood collection should be considered since EDTA-tubes induce contaminations with acetate while separator tubes result in propionate and butyrate concentrations. Also, the type of water used during sample preparation can be a source of acetate contamination, necessitating the measurement of blanks in every run to check for background acetate.

Beyond analytical challenges, uncertainties about when to measure systemic SCFA concentrations also hamper their interpretation in humans. SCFA have a plasma half-life in the order of a few minutes, causing plasma SCFA to vary during the day in response to food intake, particularly fiber. Indeed, postprandial plasma SCFA start to rise about 4 hours after the consumption of a breakfast rich in fermentable fiber and return back to baseline by the end of the day. Measured concentrations therefore depend significantly on the composition and timing of the last meal. Even when using fasting blood samples, it remains important to standardize the evening meal of the previous day to avoid residual fermentation of that meal, known as the second meal effect. Due to their short plasma half-life, SCFA do not accumulate in the circulation, explaining the lack of differences in fasting SCFA concentrations from before to after prebiotic interventions. Additionally, interindividual variation in fasting SCFA concentrations is substantial as shown in a cross-sectional study in 160 individuals (3). The factors contributing to this variability require further investigation but may include dietary habits, microbiota composition, exercise levels or host genetics. In our lab, we prefer measuring postprandial SCFA concentrations during the day and calculating the area-under-the concentration vs time curve as a measure of SCFA production rather than relying on fasting concentrations, despite the increased burden on the participants involved in clinical trials and the associated cost and effort of sample analysis.

Importantly, SCFA production may explain part of the prebiotic activity, but it likely does not provide the complete picture. For example, while the interaction of prebiotics with the immune system may be partly explained by activation of ffar2 and ffar3 receptors on immune cells by SCFA, some prebiotics such as human milk oligosaccharides or specific pectin structures directly activate immune cells via interaction with toll-like receptors 2 and 4 (4). Additionally, by altering the microbiota composition, prebiotics also indirectly alter the microbe-immune interaction. Such effects also need consideration when evaluating prebiotic interactions with host health.

Studies, preferably conducted in the target host (e.g. humans), that aim to elucidate the qualitative and quantitative contribution of SCFA to the host health benefits of prebiotics (i.e. dose-effect relationships, fraction of health benefit explained by SCFA) are highly warranted. Only then can we establish the value of SCFA as markers of prebiotic activity.

Episode 25: The effects of metabolites in the colon


The Science, Microbes & Health Podcast 

This podcast covers emerging topics and challenges in the science of probiotics, prebiotics, synbiotics, postbiotics and fermented foods. This is the podcast of The International Scientific Association for Probiotics and Prebiotics (ISAPP), a nonprofit scientific organization dedicated to advancing the science of these fields.

The effects of metabolites in the colon, with Prof. Kristin Verbeke PhD

Episode summary:

In this episode, the ISAPP podcast hosts talk about colonic metabolites with Prof. Kristin Verbeke PhD, from KU Leuven, Belgium. She talks about characterizing microbial metabolism in the colon and the consequences of producing various metabolites, both beneficial ones (such as short-chain fatty acids) and potentially detrimental ones.

Key topics from this episode:

  • Prof. Verbeke is a pharmacist by training, and now leads hospital breath testing and carries out research on microbial metabolites in the gastrointestinal tract, including how prebiotics and probiotics can change bacterial metabolism.
  • The majority of protein in the diet is digested in the small intestine, but about 5% of animal protein and 10-15% of plant protein reaches the large intestine to be fermented by the microbiota. This produces metabolites, which are shown in vitro to be toxic. However, in vivo there is less evidence of toxicity; the negative effects of these metabolites may be reduced by the interactions of different compounds in the colon.
  • Short-chain fatty acids (SCFAs) are produced when the body digests dietary fiber, and Prof. Verbeke’s group and others are investigating whether they are responsible for the benefits of eating fiber.
  • Most SCFAs are quickly absorbed in the large intestine, and they serve as an energy source for the cells. They then travel to the liver via portal circulation, where they have additional functions. What’s left over reaches systemic circulation.
  • The difficulty is knowing how many SCFAs are produced in the colon, and how many reach systemic circulation. In one experiment, they labeled the SCFAs that were administered to the colon via capsule; 36% ended up in systemic circulation. Further, when SCFAs were administered at physiological doses the subjects receiving them (compared to placebo) showed a lower cortisol response to stress.
  • SCFAs also affect fat oxidation and fat synthesis in the liver. Their relevance to non-alcoholic fatty liver disease are being investigated.
  • It’s important to eat fiber, and lots of different types. After fiber consumption, SCFAs increase in a sustained manner and take about 8h to get back to baseline. But with SCFA delivery via capsule they spike quickly and then disappear.
  • As for coatings to deliver to the colon, some coatings are time-dependent, pH dependent, etc. and this is an area for further exploration.

Episode links:

About Prof. Kristin Verbeke PhD:

Kristin Verbeke graduated from the KU Leuven, Belgium as a pharmacist in 1991. She obtained a PhD in Pharmaceutical Sciences at the Laboratory of Radiopharmaceutical Chemistry in 1995 and subsequently spend a postdoctoral period in developing radioactively labelled compounds. In 2002, she was appointed at the department of gastroenterology of the Medical Faculty of the Leuven University where she got involved in the use of stable isotope labelled compounds to evaluate gastrointestinal functions. Within the University Hospitals Leuven, she is responsible for the clinical application of diagnostic 13C- and H2-breath tests. Her current research interest specifically addresses the microbial bacterial metabolism in the human colon. Her team has developed several analytical techniques based on mass spectrometry and stable isotope or radioisotope technologies to evaluate several aspects of intestinal metabolism and function in humans (transit time, intestinal permeability, carbohydrate fermentation, protein fermentation, metabolome analysis). Collaborative research has allowed showing an aberrant bacterial metabolism in patient groups with end stage renal failure, inflammatory bowel diseases, irritable bowel disorders and alcohol abuse. These collaborations all have resulted in high quality peer-reviewed papers. In addition, she showed the impact of dietary interventions (modulation of macronutrient composition, pre- or probiotic interventions) on the microbial metabolism and its impact on health. As a PI, she acquired grant support from the university and different funding bodies and successfully completed these projects. Similarly, she supervised several PhD projects that all resulted in the achievement of a PhD degree. Her research resulted in over 200 full research papers. Together with colleague Prof. J. Delcour, she was the beneficiary of the W.K. Kellogg Chair in Cereal Sciences and Nutrition (2010-2020). She is the president of the Belgian Nutrition Society, the vice-chair of the Leuven Food Science and Nutrition Center, and the co-chair of the Prebiotic task force at ILSI Europe. Furthermore, Kristin Verbeke is the editor of the journal Gut Microbiome and member of the editorial board of Gastrointestinal Disorders. Kristin joined the ISAPP Board of Directors in 2023.

The small intestinal ‘mysteriome’: A potentially important but uncharted microbiome

By Eamonn MM Quigley MD FRCP FACP MACG FRCPI, Lynda K and David M Underwood, Center for Digestive Disorders, Division of Gastroenterology and Hepatology, Houston Methodist Hospital, Houston, Texas, USA


Over recent years, countless publications have documented the status of the microbiota of the gastrointestinal tract by examining fecal samples. While this approach does provide a “snapshot” or representation of what is going on in the gut, and especially in the colon, it is a crude measure of the complex interactions between micro-organisms in the gut, as well as between these same microorganisms and us (their hosts). Fecal samples comprise a terminal microbial ecosystem, characterized by depletion of readily fermentable substrates, with a concomitant change in microbial composition, even compared to those farther upstream in the colon. It is unlikely, for example, that studies using fecal samples provide a full picture of what happens when bacteria (or other microorganisms) “talk” to the lining of the gut (the mucosa) or interact with the immune system of the intestine. Even less likely is that they provide any insights into bacterial populations in the small intestine, where most of the digestion of food and absorption of nutrients takes place. The small intestine also possesses the most abundant immune tissue of the entire gastrointestinal tract.

Yet, details of which bacteria actually inhabit this long and important organ, the small intestine, are sketchy. This lack of knowledge has apparently not restricted much theorizing and speculation about the role of an overgrowth of colonic-type bacteria (referred to as small intestinal bacterial overgrowth – SIBO) in the small intestine in many symptoms, disorders, and diseases. According to one especially popular theory – the “leaky gut” hypothesis – the list of conditions is nearly endless. The “leaky gut” hypothesizes that dysbiosis in the small intestine (in other words SIBO) and a disruption of the gut barrier leads to “leakage” of bacteria and bacterial products into the circulation causing inflammation, allergy, and autoimmunity.

There are several leaps of faith involved in “leaky gut” including, of course, the definition and diagnosis of SIBO. Traditional methods of diagnosing SIBO (obtaining fluid samples directly from the upper small intestine or a variety of breath tests) are fraught with problems and, in essence, have precluded a universally accepted definition of SIBO.

Fundamental to this dilemma is the definition of the normal small intestinal microbiome – how can we diagnose abnormal when we do not know the limits of normality? I would contend that, while there are situations where it is undoubted (based on the clinical context and various laboratory and other findings) that SIBO is an issue, there are countless more instances where SIBO is over-diagnosed and incorrectly implicated as the cause of an individual’s symptoms. This is an important issue as it can lead to the inappropriate use of antibiotics – something we all wish to avoid.

There is some good news – clever techniques exist for obtaining uncontaminated fluid samples from the small intestine, a capsule technology that permits live sampling of intestinal gases (generated by bacteria) as it traverses the intestine and the application, at last, of high-throughput sequencing, metagenomics, metabolomics, and metatranscriptomics to small intestinal microbiota suggest that the accurate definition of the normal small intestinal microbiome is not far off. At that time, we can all agree on an accurate and clinically meaningful definition of SIBO.