Quantification Of Short-chain Fatty Acids Research Articles

A Chiral-LC-MS Method for the Simultaneous Quantification of Short-Chain Fatty Acids and D/L-Lactate in the Ruminal Fluid of Dairy Cows.

Short-chain fatty acids (SCFA) and lactate in ruminal fluid are products resulting from the microbial fermentation of substrates and can be used to reflect the composition and activity of the ruminal microbiome. Determination of SCFA and D-/L-lactate in ruminal fluid currently requires two separate protocols, which is time-consuming and costly. In this study, we have optimised and validated a simple and unified 3-nitrophenylhydrazine (3-NPH) derivatisation protocol and a 20 min chiral-LC-MS method for the simultaneous quantification of all SCFA and D- and L-lactate in ruminal fluid. This method, which requires no sample pretreatment or purification shows adequate sensitivity (limit of detection (LOD): 0.01 µg/mL), satisfactory accuracy (recovery: 88-103%), and excellent reproducibility (relative standard deviation (RSD) for repeated analyses < 3% for most analytes). The application of this method to a cohort of 24 animals allowed us to reveal a large inter-cow variation in ruminal SCFA and lactate level, the concentration range for each species, the widespread correlation between different SCFA, and the strong correlation between D- and L-lactate.

Characterization of the Effects of a Novel Probiotic on Salmonella Colonization of a Piglet-Derived Intestinal Microbiota Using Improved Bioreactor.

The carriage of Salmonella in pigs is a major concern for the agri-food industry and for global healthcare systems. Humans could develop salmonellosis when consuming contaminated pig products. On the other hand, some Salmonella serotypes could cause disease in swine, leading to economic losses on farms. The purpose of the present study was to characterize the anti-Salmonella activity of a novel Bacillus-based probiotic using a bioreactor containing a piglet-derived intestinal microbiota. Two methods of probiotic administration were tested: a single daily and a continuous dose. Salmonella enumeration was performed using selective agar at T24h, T48h, T72h, T96h and T120h. The DNA was extracted from bioreactor samples to perform microbiome profiling by targeted 16S rRNA gene sequencing on Illumina Miseq. The quantification of short-chain fatty acids (SCFAs) was also assessed at T120h. The probiotic decreased Salmonella counts at T96 for the daily dose and at T120 for the continuous one. Both probiotic doses affected the alpha and beta diversity of the piglet-derived microbiota (p < 0.05). A decrease in acetate concentration and an increase in propionate proportion were observed in the continuous condition. In conclusion, the tested Bacillus-based product showed a potential to modulate microbiota and reduce Salmonella colonization in a piglet-derived intestinal microbiota and could therefore be used in vivo.

P179 Decrease in Butyric Acid in fecal matter in patients with Inflammatory Bowel Disease is associated with the levels of secretory Immunoglobulin A and fecal calprotectin

Abstract Background Accumulating evidence shows that the development of IBD is always accompanied by dysbiosis of the intestinal microbiota. Short-chain fatty acids (SCFAs) are produced by prebiotic intestinal bacteria, with mportant anti-inflammatory functions, delaying the clinical progression of IBD. The aim of this study is to compare SCFA levels in fecal matter in patients with IBD versus patients with Spondyloarthritis (SpA) and healthy controls. Methods 24 patients were evaluated for the presence of gastrointestinal symptoms, demographic data and inflammatory markers: fecal calprotectin, C-reactive protein (CRP), secretory IgA (SIgA) and erythrocyte sedimentation rate (ESR). The quantification of SCFAs (acetic-AC, propionic-AP, butyric-AB and total SCFAs-ACCC-T) from fecal matter was analyzed by ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-Q/ TOFMS). Results 33.3% of patients with IBD were men, with body mass index &amp;gt;25 in 16.7%; 83.3% presented more than 2 gastrointestinal symptoms at the time of sample collection, 33.3% had high levels of fecal calprotectin, 50% had CRP levels higher than 3mg/L and high ESR in 33.3% and a mean SIgA. of 27.73±5.03 g/mL. Very decreased levels of butyric acid (BA)ug/mL and T-SCFA were observed in IBD (9.45±4.89 and 121.73±65.94, respectively), compared to CS (BA 20.84±8.83 and total SCFA.12±122.09)., SpA SG (AB 13.35±10.44 and total AGCC 150.73±128.52) and SpA without OS (AB 21.13±5.68 and AGCC-T 219.51±229.65). Significant differences were found in fecal BA levels between the study groups (p=0.027). Interestingly, this metabolite in the CS group and the SpA group without OS did not show differences between them, but compared to the IBD group p=0.015 and 0.009 respectively, with a decrease in IBD. And interestingly, no differences were evident between fecal SCFA levels between the IBD group and the SpA group with OS even without a diagnosis of IBD. A principal component analysis was performed for the IBD group where two components were identified that group where COMP.1 was defined by a high correlation between the levels of each of the ACCC (AB CCF= 0.994, AA CCF= 0.935, AP= 0.610 and AGCC-TCCF= 0.910) that had an inverse relationship with COMP.2 which was made up of SIgA (CCF=0.988) and fecal calprotectin (CCF= 0.988). Conclusion SCFAs play a key role in maintaining immune homeostasis both locally and systemically. BA levels were significantly lower in patients with IBD compared to CS and those with SpA. This evidence clearly supports the hypothesis that relates the lack of BA with an exacerbated inflammatory response that could give rise to a dysfunctional intestinal epithelial barrier by regulating the production of mucosal antibodies such as SIgA.

Association between gut health and gut microbiota in a polluted environment

Animals host complex bacterial communities in their gastrointestinal tracts, with which they share a mutualistic interaction. The numerous effects these interactions grant to the host include regulation of the immune system, defense against pathogen invasion, digestion of otherwise undigestible foodstuffs, and impacts on host behaviour. Exposure to stressors, such as environmental pollution, parasites, and/or predators, can alter the composition of the gut microbiome, potentially affecting host-microbiome interactions that can be manifest in the host as, for example, metabolic dysfunction or inflammation. However, whether a change in gut microbiota in wild animals associates with a change in host condition is seldom examined. Thus, we quantified whether wild bank voles inhabiting a polluted environment, areas where there are environmental radionuclides, exhibited a change in gut microbiota (using 16S amplicon sequencing) and concomitant change in host health using a combined approach of transcriptomics, histological staining analyses of colon tissue, and quantification of short-chain fatty acids in faeces and blood. Concomitant with a change in gut microbiota in animals inhabiting contaminated areas, we found evidence of poor gut health in the host, such as hypotrophy of goblet cells and likely weakened mucus layer and related changes in Clca1 and Agr2 gene expression, but no visible inflammation in colon tissue. Through this case study we show that inhabiting a polluted environment can have wide reaching effects on the gut health of affected animals, and that gut health and other host health parameters should be examined together with gut microbiota in ecotoxicological studies.

An UHPLC-QE-Orbitrap-MS Method for Accurate Quantification of Short-Chain Fatty Acids in Serum From an Older Chinese Population

An UHPLC-QE-Orbitrap-MS Method for Accurate Quantification of Short-Chain Fatty Acids in Serum From an Older Chinese Population

Gut Microbiota and Derived Short-Chain Fatty Acids Are Linked to Evolution of Heart Failure Patients.

There is a lack of direct evidence regarding gut microbiota dysbiosis and changes in short-chain fatty acids (SCFAs) in heart failure (HF) patients. We sought to assess any association between gut microbiota composition, SCFA production, clinical parameters, and the inflammatory profile in a cohort of newly diagnosed HF patients. In this longitudinal prospective study, we enrolled eighteen newly diagnosed HF patients. At admission and after 12 months, blood samples were collected for the assessment of proinflammatory cytokines, monocyte populations, and endothelial dysfunction, and stool samples were collected for analysis of gut microbiota composition and quantification of SCFAs. Twelve months after the initial HF episode, patients demonstrated improved clinical parameters and reduced inflammatory state and endothelial dysfunction. This favorable evolution was associated with a reversal of microbiota dysbiosis, consisting of the increment of health-related bacteria, such as genus Bifidobacterium, and levels of SCFAs, mainly butyrate. Furthermore, there was a decrease in the abundance of pathogenic bacteria. In vitro, fecal samples collected after 12 months of follow-up exhibited lower inflammation than samples collected at admission. In conclusion, the favorable progression of HF patients after the initial episode was linked to the reversal of gut microbiota dysbiosis and increased SCFA production, particularly butyrate. Whether restoring butyrate levels or promoting the growth of butyrate-producing bacteria could serve as a complementary treatment for these patients deserves further studies.

Development and validation of derivatization-based LC-MS/MS method for quantification of short-chain fatty acids in human, rat, and mouse plasma

Short-chain fatty acids (SCFAs), the end products of gut microbial fermentation of dietary fibers and non-digestible polysaccharides, act as a link between the microbiome, immune system, and inflammatory processes. The importance of accurately quantifying SCFAs in plasma has recently emerged to understand their biological role. In this work, a sensitive and reproducible LC-MS/MS method is reported for SCFAs quantification in three different matrices such as human, rat and mouse plasma via derivatization, using as derivatizing agent O-benzylhydroxylamine (O-BHA), coupled with liquid-liquid extraction. First, the instrumental parameters of the mass spectrometer and then the chromatographic conditions were optimized using previously SCFAs derivatives synthetized and used as standards. After that, the best conditions for derivatization and extraction from plasma were studied and a series of determinations were performed on human, rat, and mouse plasma aliquots to validate the overall method (derivatization, extraction, and LC-MS/MS determination). The method showed good performance in terms of recovery (> 80%), precision (RSD <14%), accuracy (RE < ± 10%) and sensitivity (LOQ of 0.01 µM for acetic, butyric, propionic and isobutyric acid) in all plasma samples. The method thus developed and validated was applied to the quantification of major SCFAs in adult and aged mice, germ-free mice and in germ-free recipient mice subjected to fecal transplant from adult and aged donors. Results highlighted how plasma concentrations of SCFAs are correlated with age further highlighting the importance of developing a method that is reliable for the quantification of SCFAs to study their biological role.

From In Vitro to In Vivo: A Rational Flowchart for the Selection and Characterization of Candidate Probiotic Strains in Intestinal Disorders.

Experimental and clinical evidence has demonstrated the potential of probiotic strains in the prevention or treatment of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, there is little data on what the methodology leading to the identification of such strains should be. In this work, we propose a new flowchart to identify strains with probiotic potential for the management of IBS and IBD, which we tested on a collection of 39 lactic acid bacteria and Bifidobacteria strains. This flowchart included in vitro tests of immunomodulatory properties on intestinal and peripheral blood mononuclear cells (PBMCs), assessment of the barrier-strengthening effect by measuring transepithelial electric resistance (TEER) and quantification of short-chain fatty acids (SCFAs) and aryl hydrocarbon receptor (AhR) agonists produced by the strains. The in vitro results were then combined in a principal component analysis (PCA) to identify strains associated with an anti-inflammatory profile. To validate our flowchart, we tested the two most promising strains identified in the PCA in mouse models of post-infectious IBS or chemically induced colitis to mimic IBD. Our results show that this screening strategy allows the identification of strains with potential beneficial effects on colonic inflammation and colonic hypersensitivity.

HPLC-MS-MS quantification of short-chain fatty acids actively secreted by probiotic strains.

Short-chain fatty acids (SCFAs) are the main by-products of microbial fermentations occurring in the human intestine and are directly involved in the host's physiological balance. As impaired gut concentrations of acetic, propionic, and butyric acids are often associated with systemic disorders, the administration of SCFA-producing microorganisms has been suggested as attractive approach to solve symptoms related to SCFA deficiency. In this research, nine probiotic strains (Bacillus clausii NR, OC, SIN, and T, Bacillus coagulans ATCC 7050, Bifidobacterium breve DSM 16604, Limosilactobacillus reuteri DSM 17938, Lacticaseibacillus rhamnosus ATCC 53103, and Saccharomyces boulardii CNCM I-745) commonly included in commercial formulations were tested for their ability to secrete SCFAs by using an improved protocol in high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS). The developed method was highly sensitive and specific, showing excellent limits of detection and quantification of secreted SCFAs. All tested microorganisms were shown to secrete acetic acid, with only B. clausii and S. boulardii additionally able to produce propionic and butyric acids. Quantitative differences in the secretion of SCFAs were also evidenced. The experimental approach described in this study may contribute to the characterization of probiotics as SCFA-producing organisms, a crucial stage toward their application to improve SCFA deficiency.

Quantification of short-chain fatty acids in human stool samples by LC-MS/MS following derivatization with aniline analogues

The gut microbiome produces a range of short chain fatty acids (SCFA) crucially linked with diet and nutrition, metabolism, gastrointestinal health and homeostasis. SCFA are primarily measured using gas or liquid chromatography-mass spectrometry (LC/MS) after undergoing chemical derivatization. Here we assess the merits of a derivatization protocol using aniline and two aniline analogues (3-phenoxyaniline and 4-(benzyloxy)aniline) for the targeted LC-MS/MS quantification of nine SCFA (acetic, propionic, butyric, valeric, caproic acid, isobutyric, isovaleric, 2-methylbutyric, and 2-ethylbutyric acid). Evaluation of product ion spectra and optimization of MS detection conditions, provided superior detection sensitivity for 3-phenoxyaniline and 4-(benzyloxy)aniline compared to aniline. We developed a facile SCFA derivatization method using 3-phenoxyaniline under mild reaction conditions which allows for the simultaneous quantification of these SCFA in human stool samples in under eleven minutes using multiple reaction monitoring LC-MS/MS. The method was successfully validated and demonstrates intra- and inter-day accuracy (88.5–103% and 86.0–109%) and precision (CV of 0.55–7.00% and 0.33–9.55%) with recoveries (80.1–87.2% for LLOQ, 88.5–93.0% for ULOQ) and carry-over of (2.68–17.9%). Selectivity, stability and matrix effects were also assessed and satisfied validation criteria. Method applicability was demonstrated by analysing SCFA profiles in DNA-stabilized human stool samples from newly diagnosed colorectal cancer patients prior to surgery. The development of this improved method and its compatibility to measure SCFAs from DNA-stabilized stool will facilitate studies investigating the gut microbiome in health and disease.

Sensitive quantification of short-chain fatty acids combined with global metabolomics in microbiome cultures.

The microbiome has been identified to have a key role for the physiology of their human host. One of the major impacts is the clearance of bacterial pathogens. We have now developed a chemoselective probe methodology for the absolute quantification of short-chain fatty acids at low nM concentrations, with high reproducibility and spiked isotope labelled internal standards. Immobilization to magnetic beads allows for separation from the matrix and the tagged metabolites upon bioorthogonal cleavage can be analyzed via UHPLC-MS. The major advantage of our sensitive method is the simple combination with global metabolomics analysis as only a small sample volume is required. We have applied this chemical metabolomics strategy for targeted SCFA analysis combined with global metabolomics on gut microbiome co-cultures with Salmonella and investigated the effect of antibiotic treatment.

The effect of phenobarbital treatment on behavioral comorbidities and on the composition and function of the fecal microbiome in dogs with idiopathic epilepsy.

Phenobarbital (PB) is one of the most important antiseizure drugs (ASDs) to treat canine idiopathic epilepsy (IE). The effect of PB on the taxonomic changes in gastrointestinal microbiota (GIM) and their functions is less known, which may explain parts of its pharmacokinetic and pharmacodynamic properties, especially its antiseizure effect and drug responsiveness or drug resistance as well as its effect on behavioral comorbidities. Fecal samples of 12 dogs with IE were collected prior to the initiation of PB treatment and 90 days after oral PB treatment. The fecal samples were analyzed using shallow DNA shotgun sequencing, real-time polymerase chain reaction (qPCR)-based dysbiosis index (DI), and quantification of short-chain fatty acids (SCFAs). Behavioral comorbidities were evaluated using standardized online questionnaires, namely, a canine behavioral assessment and research questionnaire (cBARQ), canine cognitive dysfunction rating scale (CCDR), and an attention deficit hyperactivity disorder (ADHD) questionnaire. The results revealed no significant changes in alpha and beta diversity or in the DI, whereas only the abundance of Clostridiales was significantly decreased after PB treatment. Fecal SCFA measurement showed a significant increase in total fecal SCFA concentration and the concentrations of propionate and butyrate, while acetate concentrations revealed an upward trend after 90 days of treatment. In addition, the PB-Responder (PB-R) group had significantly higher butyrate levels compared to the PB-Non-Responder (PB-NR) group. Metagenomics of functional pathway genes demonstrated a significant increase in genes in trehalose biosynthesis, ribosomal synthesis, and gluconeogenesis, but a decrease in V-ATPase-related oxidative phosphorylation. For behavioral assessment, cBARQ analysis showed improvement in stranger-directed fear, non-social fear, and trainability, while there were no differences in ADHD-like behavior and canine cognitive dysfunction (CCD) scores after 90 days of PB treatment. While only very minor shifts in bacterial taxonomy were detected, the higher SCFA concentrations after PB treatment could be one of the key differences between PB-R and PB-NR. These results suggest functional changes in GIM in canine IE treatment.

Escherichia/Shigella, SCFAs, and Metabolic Pathways-The Triad That Orchestrates Intestinal Dysbiosis in Patients with Decompensated Alcoholic Cirrhosis from Western Mexico.

Gut microbiota undergoes profound alterations in alcohol cirrhosis. Microbiota-derived products, e.g., short chain fatty acids (SCFA), regulate the homeostasis of the gut-liver axis. The objective was to evaluate the composition and functions of the intestinal microbiota in patients with alcohol-decompensated cirrhosis. Fecal samples of 18 patients and 18 healthy controls (HC) were obtained. Microbial composition was characterized by 16S rRNA amplicon sequencing, SCFA quantification was performed by gas chromatography (GC), and metagenomic predictive profiles were analyzed by PICRUSt2. Gut microbiota in the cirrhosis group revealed a significant increase in the pathogenic/pathobionts genera Escherichia/Shigella and Prevotella, a decrease in beneficial bacteria, such as Blautia, Faecalibacterium, and a decreased α-diversity (p < 0.001) compared to HC. Fecal SCFA concentrations were significantly reduced in the cirrhosis group (p < 0.001). PICRUSt2 analysis indicated a decrease in acetyl-CoA fermentation to butyrate, as well as an increase in pathways related to antibiotics resistance, and aromatic amino acid biosynthesis. These metabolic pathways have been poorly described in the progression of alcohol-related decompensated cirrhosis. The gut microbiota of these patients possesses a pathogenic/inflammatory environment; therefore, future strategies to balance intestinal dysbiosis should be implemented. These findings are described for the first time in the population of western Mexico.

Rapid Quantification of Gut Microbial Short-Chain Fatty Acids by pDART-MS.

Short-chain fatty acids (SCFAs) are small molecules ubiquitous in nature. In mammalian guts, SCFAs are mostly produced by anaerobic intestinal microbiota through the fermentation of dietary fiber. Levels of microbe-derived SCFAs are closely relevant to human health status and indicative to gut microbiota dysbiosis. However, the quantification of SCFA using conventional chromatographic approaches is often time consuming, thus limiting high-throughput screening tests. Herein, we established a novel method to quantify SCFAs by coupling amidation derivatization of SCFAs with paper-loaded direct analysis in real time mass spectrometry (pDART-MS). Remarkably, SCFAs of a biological sample were quantitatively determined within a minute using the pDART-MS platform, which showed a limit of detection at the μM level. This platform was applied to quantify SCFAs in various biological samples, including feces from stressed rats, sera of patients with kidney disease, and fermentation products of metabolically engineered cyanobacteria. Significant differences in SCFA levels between different groups of biological practices were promptly revealed and evaluated. As there is a burgeoning demand for the analysis of SCFAs due to an increasing academic interest of gut microbiota and its metabolism, this newly developed platform will be of great potential in biological and clinical sciences as well as in industrial quality control.

Contribution of Short Chain Fatty Acids to the Growth of Pseudomonas aeruginosa in Rhinosinusitis.

Background: Chronic rhinosinusitis (CRS) is characterized by complex bacterial infections with persistent inflammation. Based on our rabbit model of sinusitis, blockage of sinus ostia generated a shift in microbiota to a predominance of mucin degrading microbes (MDM) with acute inflammation at 2 weeks. This was followed by conversion to chronic sinus inflammation at 3 months with a robust increase in pathogenic bacteria (e.g., Pseudomonas). MDMs are known to produce acid metabolites [short chain fatty acids (SCFA)] that have the potential to stimulate pathogen growth by offering a carbon source to non-fermenting sinus pathogens (e.g., Pseudomonas). The objective of this study is to evaluate the concentrations of SCFA within the mucus and its contribution to the growth of P. aeruginosa.Methods: Healthy and sinusitis mucus from the rabbit model were collected and co-cultured with the PAO1 strain of P. aeruginosa for 72 h and colony forming units (CFUs) were determined with the targeted quantification of three SCFAs (acetate, propionate, butyrate). Quantification of SCFAs in healthy and sinusitis mucus from patients with P. aeruginosa was also performed via high performance liquid chromatography.Results: To provide evidence of fermentative activity, SCFAs were quantified within the mucus samples from rabbits with and without sinusitis. Acetate concentrations were significantly greater in sinusitis mucus compared to controls (4.13 ± 0.53 vs. 1.94 ± 0.44 mM, p < 0.01). After 72 h of co-culturing mucus samples with PAO1 in the presence of mucin medium, the blue-green pigment characteristic of Pseudomonas was observed throughout tubes containing sinusitis mucus. CFUs were higher in cultures containing mucus samples from sinusitis (8.4 × 109 ± 4.8 × 107) compared to control (1.4 × 109 ± 2.0 × 107) or no mucus (1.5 × 109 ± 2.1 × 107) (p < 0.0001). To provide evidence of fermentative activity in human CRS with P. aeruginosa, the presence of SCFAs in human mucus was analyzed and all SCFAs were significantly higher in CRS with P. aeruginosa compared to controls (p < 0.05).Conclusion: Given that SCFAs are solely derived from bacterial fermentation, our evidence suggests a critical role for mucin-degrading bacteria in generating carbon-source nutrients for pathogens. MDM may contribute to the development of recalcitrant CRS by degrading mucins, thus providing nutrients for potential pathogens like P. aeruginosa.

Probiotic low-fat fermented goat milk with passion fruit by-product: In vitro effect on obese individuals’ microbiota and on metabolites production

This study aimed to evaluate the impact of a two-week treatment period with probiotic low-fat fermented goat milk by Lactobacillus casei Lc-1, supplemented with passion fruit by-product (1%), on the modulation of gut microbiota from obese individuals using the Simulator of Human Intestinal Microbial Ecosystem (SHIME) system. The effects were carried out through the study of gut microbiota composition, using 16S rRNA next generation sequencing, quantification of short-chain fatty acids (SCFA) and ammonium ions. The microbiota composition changed across three vessels representing the colon regions, because of fermented milk treatment. Fermented goat milk administration caused a reduction of bacteria belonging to genera Prevotella, Megamonas and Succinivibrio, which can produce SCFA, and an increase of Lactobacillus and Bifidobacterium genera in all simulated colon regions. There was no effect on SCFA and on ammonium ions concentration during treatment period. Fermented milk shifted the obese donors’ microbiota without changing metabolites production. It happens, possibly, due to a balance in abundances among bacterial genera that can produce or not SCFA, and among bacterial genera with high or low proteolytic activity. Our outcomes help to clarify the effects of the ingestion of a probiotic low-fat fermented goat milk product on colon microbiota composition.

A sensitive method for the quantification of short-chain fatty acids by benzyl chloroformate derivatization combined with GC-MS.

Short-chain fatty acids (SCFAs) were identified as critical markers in the diagnosis of chronic and metabolic diseases, but a sensitive and stable method to determine SCFAs in feces is a challenge for analysts due to the high volatility. Herein, a sensitive and accurate method to determine SCFAs adopting precolumn derivatization coupled with gas chromatography-mass spectrometry (GC-MS) has been developed. Benzyl chloroformate (BCF) was chosen as the reaction reagent and emulsified derivatization was applied to homogenize the reaction system. Higher sensitivity, wider application and satisfactory derivatization efficiency were obtained using the developed method. An excellent method validation showed a good linearity ranging from 0.9947 to 0.9998. At the same time, the intra-day and inter-day precision were achieved in the range of 0.56% to 13.07%. The lower limits of detection of all target analytes varied from 0.1 to 5 pg. The recovery ranged from 80.87% to 119.03%, and storage stability under three different conditions was also determined. This method was also successfully applied to the analysis of SCFAs in mice fecal samples to illustrate the significant differences between normal and type 2 diabetes mellitus mice.

Kansuiphorin C and Kansuinin A ameliorate malignant ascites by modulating gut microbiota and related metabolic functions.

Euphorbia kansui is a toxic Chinese herbal medicine and exhibits promising treatment to the malignant ascites (MA) in its traditional use. Ingenane-type and jastrophane-type diterpenes are demonstrated to be responsible for the toxicity and efficacy of kansui. Two representative compounds, kansuiphorin C (KPC) and kansuinin A (KA) in each type were proved to effectively reduce the ascites. The biological and toxicological effects are closely associated with the gastrointestinal tract, but the possible mechanism and related metabolic functions of KPC and KA treating MA through modulating the gut microbiota remain unclear. To investigate the possible mechanism and related metabolism of KPC and KA ameliorating malignant ascites through modulating gut microbiota. MA rats and normal rats were divided into different groups and administrated with KPC, KA, and positive drug, respectively. 16S rDNA gene sequencing and metagenomes analysis combined with the quantification of short-chain fatty acids of feces were performed to reflect the modulation of gut microbiota. Then, the metabolites of KPC and KA in rat feces under the normal and pathological circumstances were detected by ultra-fast liquid chromatography coupled with MS/MS detector (UFLC-MS/MS) to explore the in-vivo bacterial biotransformation. KPC and KA were modulatory compounds for gut microbiota. The richness of Lactobacillus and the decreased abundance of Helicobacter involved in the carbohydrate metabolism and amino acid metabolism could be responsible for their prohibitory effects on malignant ascites. KPC exhibited stronger modulation of gut microbiota through making the abundance of Helicobacter about 3.5 times lower than KA. Besides, in-vivo microbial biotransformation of KPC and KA contained oxidation, hydrolysis, dehydration, and methylation to form metabolites of lower polarity. Besides, at the dosage of 10mgkg-1, the toxicity of both compounds had weaker influences on the gut microbiota of normal rats. KPC and KA could ameliorate malignant ascites by modulating gut microbiota mainly containing the increase of Lactobacillus and the decrease of Helicobacter and related carbohydrate and amino acid metabolism, providing a basis for their promising clinical usage.

837 Short Chain Fatty Acid Profiles Are Altered by Fecal Microbiota Transplantation for the Treatment of Inflammatory Bowel Disease and Recurrent Clostridioides difficile Infection

INTRODUCTION: Recurrent C. difficile infection (rCDI) is a major challenge among patients with inflammatory bowel disease (IBD). Perturbation of microbiota-mediated metabolism of short chain fatty acids (SCFA) has been reported in IBD patients. Fecal microbiota transplantation (FMT), an established therapy for rCDI, alters gut microbiota composition, but effects on SCFA are unclear. Accordingly, this study assessed SCFA profiles in IBD patients with rCDI pre- and post-FMT. METHODS: This open-label, prospective, single-arm multi-center cohort study enrolled patients from 4 tertiary care centers. Patients with IBD and ≥2 episodes of CDI received a single colonoscopic FMT from a universal stool bank. The primary outcome was CDI recurrence up to week 8 defined as diarrhea and EIA-positive toxin testing for C. difficile. Stool for metabolomic profiling was collected pre-FMT and week 1, 8 and 12 weeks post-FMT. A targeted gas chromatography-mass spectrometry protocol was used for the identification and quantification of SCFA. SCFA concentrations were analyzed via univariate analysis, comparing groups (e.g. pre- vs post-FMT). RESULTS: 37 participants were enrolled, with mean age of 37.6 years (range 20-76) and primarily female (n = 21, 57%). 14 had Crohn’s disease (CD) (mean HBI = 6.4) and 23 had ulcerative colitis (UC) (mean Partial Mayo Score = 4.5). Mean baseline fecal calprotectin was 1804.8 +/- 2307.7 Overall, 3 participants (8%) experienced FMT failure with confirmed EIA-positive stool, and the there were no related serious adverse events. Among the nine SCFAs assessed, including two branched chain fatty acids (isovalerate and isobutyrate) most were significant altered by FMT, with rapid and sustained changes (with the exception of acetate, in which no change was appreciated) (Figure 1). Additionally, 2-hydroxybutyrate and lactate, two markers of metabolic stress, were significantly reduced after FMT. Furthermore, the baseline stool samples of those who went on to recur were compared to those who did not (Figure 2); though not significant, there was a trend towards reduced baseline levels for butyrate, propionate and caproate among those who recurred. CONCLUSION: To our knowledge, this is the largest prospective trial to assess the effect of FMT in patients with IBD-CDI. The data suggests the efficacy and safety profile is better than previous reports. SCFAs were increased significantly post-FMT, and may offer insight into mechanism of clinically efficacy seen in this study.

Gut Microbiota and Fecal Levels of Short-Chain Fatty Acids Differ Upon 24-Hour Blood Pressure Levels in Men.

Gut microbiota may influence blood pressure (BP), namely via end products of carbohydrate fermentation. After informed consent, male volunteers were prospectively categorized into 3 groups upon European Society of Hypertension criteria based on 24-hour ambulatory BP measurements: (1) hypertension, (2) borderline hypertension, and (3) normotension. Stool, urine and serum samples were collected in fasting conditions. Gut microbiota was characterized by 16S amplicon sequencing. Metabolomics, including quantification of short-chain fatty acids, was conducted using nuclear magnetic resonance. Two-way ANOVA combined with Tukey post hoc test, as well as multiple permutation test and Benjamini-Hochberg-Yekutieli false discovery rate procedure, was used. The cohort included 54 males: 38 hypertensive (including 21 under treatment), 7 borderline, and 9 normotensive. No significant difference was observed between groups concerning age, body mass index, smoking habits, and weekly alcohol consumption. The genus Clostridium sensu stricto 1 positively correlated with BP levels in nontreated patients (n=33). This correlation was significant after multiple permutation tests but was not substantiated following false discovery rate adjustment. Short-chain fatty acid levels were significantly different among groups, with higher stool levels of acetate, butyrate, and propionate in hypertensive versus normotensive individuals. No difference was observed in serum and urine metabolomes. Correlation between stool metabolome and 24-hour BP levels was evidenced, with R2 reaching 0.9. Our pilot study based on 24-hour ambulatory BP measurements, 16S amplicon sequencing, and metabolomics supports an association between gut microbiota and BP homeostasis, with changes in stool abundance of short-chain fatty acids.