The Gut Microbiome and Short-Chain Fatty Acid Metabolites in Sepsis.

The dual sugar absorption test, specifically the lactulose:mannitol test, is used to assess gut health. Lactulose absorption is said to represent gut damage and mannitol absorption is used as a measure of normal small bowel function and serves as normalizing factor for lactulose. A underappreciated limitation of this common understanding of the lactulose:mannitol test is that mannitol is not absorbed to any substantial extent by a transcellular process. Additionally, this interpretation of lactulose:mannitol is not consistent with current understanding of paracellular pathways, where three pathway types exist: pore, leak, and unrestricted. Pore and leak pathways are regulated biological constructions of the small bowel barrier, and unrestricted pathways represent micropathological damage. We analyzed 2334 lactulose:mannitol measurements rigorously collected from 622 young rural Malawian children at high risk for poor gut health in light of the pathway model. An alternative method of normalizing for gut length utilizing autopsy data is described. In our population, absorbed lactulose and mannitol are strongly correlated, r = 0.68 P <0.0001, suggesting lactulose and mannitol are traversing the gut barrier via the same pathways. Considering measurements where pore pathways predominate, mannitol flux is about 14 times that of lactulose. As more leak pathways are present, this differential flux mannitol:lactulose falls to 8:1 and when increased numbers of unrestricted pathways are present, the differential flux of mannitol:lactulose is 6:1. There was no substantial correlation between the lactulose:mannitol and linear growth. Given that mannitol will always pass through a given pathway at a rate at least equal to that of lactulose, and lactulose absorption is a composite measure of flux through both physiologic and pathologic pathways, we question the utility of the lactulose:mannitol test. We suggest using lactulose alone is as informative as lactulose:mannitol in a sugar absorption testing in subclinical gut inflammation. Impact statement Our work integrates the standard interpretation of the lactulose:mannitol test (L:M), with mechanistic insight of intestinal permeability. There are three paracellular pathways in the gut epithelium; pore, leak, and unrestricted. Using thousands of L:M measurements from rural Malawian children at risk for increased intestinal permeability, we predict the differential flux of L and M through the pathways. Our findings challenge the traditional notions that little L is absorbed through a normal epithelial barrier and that M is a normalizing factor for L. Our observations are consistent with pore pathways allowing only M to pass. And that substantial amounts of L and M pass through leak pathways which are normal, regulated, cell-junctional adaptations. So M is a composite measure of all pathways, and L is not a measure solely of pathologic gut damage. Using L alone as a probe will yield more information about gut health than L:M.

The absorption and metabolism of short and long chain fatty acids in puromycin-treated rats

A noninvasive breath test has the potential to improve survival from esophagogastric cancer by facilitating earlier detection. This study aimed to investigate the production of target volatile fatty acids (VFAs) in esophagogastric cancer through analysis of the ex vivo headspace above underivatized tissues and in vivo analysis within defined anatomical compartments, including analysis of mixed breath, isolated bronchial breath, and gastric-endoluminal air. VFAs were measured by PTR-ToF-MS and GC-MS. Levels of VFAs (acetic, butyric, pentanoic, and hexanoic acids) and acetone were elevated in ex vivo experiments in the headspace above esophagogastric cancer compared with the levels in samples from control subjects with morphologically normal and benign conditions of the upper gastrointestinal tract. In 25 patients with esophagogastric cancer and 20 control subjects, receiver-operating-characteristic analysis for the cancer-specific VFAs butyric acid ( P < 0.001) and pentatonic acid ( P = 0.005) within in vivo gastric-endoluminal air gave an area under the curve of 0.80 (95% confidence interval of 0.65 to 0.93, P = 0.01). Compared with mixed- and bronchial-breath samples, all examined VFAs were found in highest concentrations within esophagogastric-endoluminal air. In addition, VFAs were higher in all samples derived from cancer patients compared with in the controls. Equivalence of VFA levels within the mixed and bronchial breath of cancer patients suggests that their origin within breath is principally derived from the lungs and, by inference, from thesystemic circulation as opposed to direct passage from the upper gastrointestinal tract. These findings highlight the potential to utilize VFAs for endoluminal-gas biopsies and noninvasive mixed-exhaled-breath testing for esophagogastric-cancer detection.

Epithelial tight junctions define the paracellular permeability of the intestinal barrier. Molecules can cross the tight junctions via two distinct size-selective and charge-selective paracellular pathways: the pore pathway and the leak pathway. These can be distinguished by their selectivities and differential regulation by immune cells. However, permeability increases measured in most studies are secondary to epithelial damage, which allows non-selective flux via the unrestricted pathway. Restoration of increased unrestricted pathway permeability requires mucosal healing. By contrast, tight junction barrier loss can be reversed by targeted interventions. Specific approaches are needed to restore pore pathway or leak pathway permeability increases. Recent studies have used preclinical disease models to demonstrate the potential of pore pathway or leak pathway barrier restoration in disease. In this Review, we focus on the two paracellular flux pathways that are dependent on the tight junction. We discuss the latest evidence that highlights tight junction components, structures and regulatory mechanisms, their impact on gut health and disease, and opportunities for therapeutic intervention.

Alcohol use disorder is associated with increased mortality in septic patients. Murine studies demonstrate that ethanol/sepsis is associated with changes in gut integrity. This study examined intestinal permeability after ethanol/sepsis and investigated mechanisms responsible for alterations in barrier function. Mice were randomized to drink either 20% ethanol or water for 12 weeks and then were subjected to either sham laparotomy or cecal ligation and puncture (CLP). Intestinal permeability was disproportionately increased in ethanol/septic mice via the pore, leak, and unrestricted pathways. Consistent with increased permeability in the leak pathway, jejunal myosin light chain (MLC) kinase (MLCK) expression and the ratio of phospho-MLC to total MLC were both increased in ethanol/CLP. Gut permeability was altered in MLCK -/- mice in water/CLP; however, permeability was not different between WT and MLCK -/- mice in ethanol/CLP. Similarly, jejunal IL-1β levels were decreased while systemic IL-6 levels were increased in MLCK -/- mice in water/CLP but no differences were identified in ethanol/CLP. While we have previously shown that mortality is improved in MLCK -/- mice after water/CLP, mortality was significantly worse in MLCK -/- mice after ethanol/CLP. Consistent with an increase in the pore pathway, claudin 4 levels were also selectively decreased in ethanol/CLP WT mice. Furthermore, mRNA expression of jejunal TNF and IFN-γ were both significantly increased in ethanol/CLP. The frequency of CD4 + cells expressing TNF and IL-17A and the frequency of CD8 + cells expressing IFN-γ in Peyer's Patches were also increased in ethanol/CLP. Thus, there is an ethanol-specific worsening of gut barrier function after CLP that impacts all pathways of intestinal permeability, mediated, in part, via changes to the tight junction. Differences in the host response in the setting of chronic alcohol use may play a role in future precision medicine approaches toward the treatment of sepsis.

Short Chain Fatty Acids (SCFAs) are produced by the gut microbiota and are present in varying concentrations in the intestinal lumen, in feces but also in the circulatory system. By interacting with different cell types in the body, they have a great impact on host metabolism and their exact quantification is indispensable. Here, we present a derivatization-free method for the gas chromatography mass spectrometry (GC-MS) based quantification of SCFAs in plasma, feces, cecum, liver and adipose tissue. SCFAs were extracted using ethanol and concentrated by alkaline vacuum centrifugation. To allow volatility for separation by GC, samples were acidified with succinic acid. Analytes were detected in selected ion monitoring (SIM) mode and quantified using deuterated internal standards and external calibration curves. Method validation rendered excellent linearity (R2 > 0.99 for most analytes), good recovery rates (95–117%), and good reproducibility (RSD: 1–4.5%). Matrix effects were ruled out in plasma, feces, cecum, liver and fat tissues where most abundant SCFAs were detected and accurately quantified. Finally, applicability of the method was assessed using samples derived from conventionally raised versus germ-free mice or mice treated with antibiotics. Altogether, a reliable, fast, derivatization-free GC-MS method for the quantification of SCFAs in different biological matrices was developed allowing for the study of the (patho)physiological role of SCFAs in metabolic health.

Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro: effect of SCFA chain length and pH

Short chain fatty acids inhibit endotoxin-induced uveitis and inflammatory responses of retinal astrocytes

A short-chain fatty acid (SCFA) burst feeding strategy to dry cows (periodically high SCFA concentration in the ruminal fluid) has been hypothesized to increase the ruminal surface area and absorptive capacity. The present paper deals with the effects of a SCFA burst feeding strategy on epithelial transport function in dry cows. The epithelial transport capacity for [14C]butyrate and Na+ across the rumen epithelium in vitro was significantly increased by the SCFA burst feeding strategy, compared with the controls. The transport of Cl- was significantly increased by the SCFA burst feeding strategy at the high feeding level, but non-significantly decreased at the low feeding level. The increase in transport capacity could not be attributed to changes in epithelial resistance, to the concentration of Na+/K+-ATP-ase or to the epithelial surface area and structure (data on epithelial surface area and structure were published by Andersen et al., Report No. 23 from the National Institute of Animal Science, Denmark, 1994). The observed changes in transport must reflect a change in either composition of the cell membranes or processes that facilitate transport through the cell, including metabolic processes. The presented results support the existence of a saturable SCFA transport system in the epithelial cells and the results show that the capacity of this transport system can be the subject for feed-induced regulation.

Short-chain fatty acids (SCFAs) from gastrointestinal disorders, metabolism, epigenetics, central nervous system to cancer - A mini-review

Tight junctions (TJs) control the paracellular transport of ions, solutes, and macromolecules across epithelial barriers. There is evidence that claudin-based ion transport (the pore pathway) and the paracellular transport of macromolecules (the leak pathway) are controlled independently. However, how leak pathway flux is regulated is unclear. Here, we have identified the Cdc42/Rac GTPase-activating protein ARHGAP12 as a specific activator of the leak pathway. ARHGAP12 is recruited to TJs via an interaction between its Src homology (SH3) domain and the TJ protein ZO-2 to suppress N-WASP-mediated F-actin assembly. This dampens junctional tension and promotes the paracellular transport of macromolecules without affecting ion flux. Mechanistically, we demonstrate that the ARHGAP12 tandem WW domain interacts directly with PPxR motifs in the proline-rich domain of N-WASP and thereby attenuates SH3-domain-mediated N-WASP oligomerization and Arp2/3-driven F-actin assembly. Collectively, our data indicate that branched F-actin networks regulate junctional tension to fine-tune the TJ leak pathway.

<h3>Objective:</h3> To evaluate whether short-chain fatty acids (SCFAs) will benefit the long-term recovery of traumatic brain injury (TBI) in mice. <h3>Background:</h3> SCFAs, including acetate and butyrate, are the main metabolites generated by gut microbiota consuming dietary fiber in the colon. SCFAs have pleotropic effects throughout the body including influences on immunity and neurogenesis. We hypothesized that SCFAs could influence recovery from TBI. <h3>Design/Methods:</h3> Adult C57BL6/J mice (n=4/group) were randomized to standard high-amylose maize starch (HAMS) chow or acetylated- and butyrylated-HAMS (HAMS-AB). Fermentation of HAMS-AB by gut bacteria releases acetate and butyrate in the intestinal lumen. The controlled cortical impact (CCI) model of TBI (6m/s, 2.2mm depth, 50msec dwell) was performed. Sham mice received anesthesia and skin incision without craniotomy. At 6 months post-injury, mice were sacrificed and single-cell mRNA from pericontusional tissue sequenced following the 10X Genomics protocol. After pre-processing by <i>CellRanger</i> and SCT normalization by <i>Seurat</i>, cells were annotated based on canonical markers. Cell subtype gene modules were clustered by weighted gene co-expression network analysis (WGCNA) and enriched at gene-ontology (GO) pathways. The GO pathway and regulon activity were evaluated by <i>AUCell</i> and <i>SCENIC</i> respectively. <h3>Results:</h3> In a total of 116,134 cells, consisting of 12 cell types, from 3 groups (41,509 from sham_control, 36,409 from injury_control, 38,216 from injury_SCFA,) passed the quality control and doublet filtering. Both the cell composition and transcriptome were different in each group. SCFAs decreased gene expression associated with neuronal activity and increased the immature neuron frequency. During immature neuron differentiation, SCFAs downregulated neurodegenerative disease-related genes expression in injured mice. In addition, SCFAs decreased chronic microglia/macrophage activation and promoted an M2-like phenotype. Lastly, SCFAs attenuated T cell activation and cytotoxic-related pathways. <h3>Conclusions:</h3> SCFAs impact gene expression in multiple cell types at 6 months after TBI. The pathways affected suggest a beneficial role for gut-derived SCFA administration for recovery after TBI. <b>Disclosure:</b> Dr. Xiong has nothing to disclose. Mrs. Nelson has nothing to disclose. Mr. Sneiderman has nothing to disclose. Ms. Feldman has nothing to disclose. Patrick M. Kochanek, MD, MCCM has received personal compensation in the range of $10,000-$49,999 for serving as an Expert Witness for Johns Hopkins Health System. The institution of Patrick M. Kochanek, MD, MCCM has received research support from Chuck Noll Foundation. The institution of Patrick M. Kochanek, MD, MCCM has received research support from NIH. Patrick M. Kochanek, MD, MCCM has received publishing royalties from a publication relating to health care. Dr. Rajasundaram has nothing to disclose. Dr. Jha has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Biogen. An immediate family member of Dr. Jha has received personal compensation in the range of $5,000-$9,999 for serving as an Expert Witness for Legal fees. The institution of Dr. Jha has received research support from NIH/NINDS, Chuck Noll Foundation, University of Pittsburgh, Barrow Neurological Foundation. The institution of Dr. Kohanbash has received research support from NIH. Dr. Simon has nothing to disclose.

Butyrate, propionate and acetate are short chain fatty acids (SCFA), important for maintaining a healthy colon and are considered as protective in colorectal carcinogenesis. However, they may also regulate immune responses and the composition of the intestinal microbiota. Consequently, their importance in a variety of chronic inflammatory diseases is emerging. To review the physiology and metabolism of SCFA in humans, cellular and molecular mechanisms by which SCFA may act in health and disease, and approaches for therapeutic delivery of SCFA. A PubMed literature search was conducted for clinical and pre-clinical studies using search terms: 'dietary fibre', short-chain fatty acids', 'acetate', 'propionate', 'butyrate', 'inflammation', 'immune', 'gastrointestinal', 'metabolism'. A wide range of pre-clinical evidence supports roles for SCFA as modulators of not only colonic function, but also multiple inflammatory and metabolic processes. SCFA are implicated in many autoimmune, allergic and metabolic diseases. However, translating effects of SCFA from animal studies to human disease is limited by physiological and dietary differences and by the challenge of delivering sufficient amounts of SCFA to the target sites that include the colon and the systemic circulation. Development of novel targeted approaches for colonic delivery, combined with postbiotic supplementation, may represent desirable strategies to achieve adequate targeted SCFA delivery. There is a large array of potential disease-modulating effects of SCFA. Adequate targeted delivery to the sites of action is the main limitation of such application. The ongoing development and evaluation of novel delivery techniques offer potential for translating promise to therapeutic benefit.

Short-chain fatty acids (SCFAs), the main bacterial fermentation products in the hindgut of hindgut fermenters, are also present in the foregut lumen. We discuss the impact of SCFAs in the duodenal defense mechanisms and in the gastrointestinal (GI) pathogenesis. Luminal SCFAs augment the duodenal mucosal defenses via release of serotonin (5-HT) and glucagon-like peptide-2 (GLP-2) from enteroendocrine cells. Released GLP-2 protects the small intestinal mucosa from nonsteroidal anti-inflammatory drug-induced enteropathy. SCFAs are also rapidly absorbed via SCFA transporters and interact with afferent and myenteric nerves. Excessive SCFA signals with 5-HT3 receptor overactivation may be implicated in the pathogenesis of irritable bowel syndrome symptoms. SCFA production exhibits diurnal rhythms with host physiological responses, suggesting that oral SCFA treatment may adjust the GI clocks. SCFAs are not only a source of energy but also signaling molecules for the local regulation of the GI tract and systemic regulation via release of gut hormones. Targeting SCFA signals may be a novel therapeutic for GI diseases and metabolic syndrome.

The COVID-19 Pandemic and Post-Infection Irritable Bowel Syndrome: What Lies Ahead for Gastroenterologists