Short-chain Fatty Acids In Fecal Samples Research Articles

Impact of age-related gut microbiota dysbiosis and reduced short-chain fatty acids on the autonomic nervous system and atrial fibrillation in rats.

Aging is the most significant contributor to the increasing prevalence of atrial fibrillation (AF). Dysbiosis of gut microbiota has been implicated in age-related diseases, but its role in AF development remains unclear. This study aimed to investigate the correlations between changes in the autonomic nervous system, short-chain fatty acids (SCFAs), and alterations in gut microbiota in aged rats with AF. Electrophysiological experiments were conducted to assess AF induction rates and heart rate variability in rats. 16S rRNA gene sequences extracted from fecal samples were used to assess the gut microbial composition. Gas and liquid chromatography-mass spectroscopy was used to identify SCFAs in fecal samples. The study found that aged rats exhibited a higher incidence of AF and reduced heart rate variability compared to young rats. Omics research revealed disrupted gut microbiota in aged rats, specifically a decreased Firmicutes to Bacteroidetes ratio. Additionally, fecal SCFA levels were significantly lower in aged rats. Importantly, correlation analysis indicated a significant association between decreased SCFAs and declining heart rate variability in aged rats. These findings suggest that SCFAs, as metabolites of gut microbiota, may play a regulatory role in autonomic nervous function and potentially influence the onset and progression of AF in aged rats. These results provide novel insights into the involvement of SCFAs and autonomic nervous system function in the pathogenesis of AF. These results provide novel insights into the involvement of SCFAs and autonomic nervous system function in the pathogenesis of AF.

Why does increased microbial fermentation in the human colon shift toward butyrate?

The microbial community of the human large intestine mainly ferments dietary fiber to short chain fatty acids (SCFAs), which are efficiently absorbed by the host. The three major SCFAs (acetate, propionate, and butyrate) have different fates within the body and different effects on health. A recent analysis of 10 human volunteer studies established that the proportions of these SCFA in fecal samples significantly shifted towards butyrate as the overall concentration of SCFA increased. Butyrate plays a key role in gut health and is preferentially utilized as an energy source by the colonic epithelium. Here we discuss possible mechanisms that underlie this 'butyrate shift'; these include the selection for butyrate-producing bacteria within the microbiota by certain types of fiber, and the possibility of additional butyrate formation from lactate and acetate by metabolite cross-feeding. However, a crucial factor appears to be the pH in the proximal colon, which decreases as the SCFA concentrations increase. A mildly acidic pH has been shown to have an important impact on microbial competition and on the stoichiometry of butyrate production. Understanding these complex interactions has been greatly aided by the refinement of theoretical models of the colonic microbiota that assume a small number (10) of microbial functional groups (MFGs).

Disordered gut microbiota and changes in short-chain fatty acids and inflammatory processes in stress-vulnerable mice

Long-term exposure to chronic stress increases the incidence of depression. However, chronic stress is an associated risk factor in only a subset of individuals. Inflammation has been identified as a putative mechanism promoting stress vulnerability. Because of the gut microbiota's potential role as a source of inflammatory substances, short-chain fatty acids (SCFAs) may exert their influence on inflammation, emotional states, and cognition via the gut-brain axis. In this study, Classic behavioral tests were used to categorize C57BL/6 J mice into a CUMS-vulnerable and a CUMS-resilient group after they were exposed to chronic unpredictable mild stress (CUMS). We compared the 16S ribosomal RNA (rRNA) gene sequences retrieved from fecal samples between control, CUMS-vulnerable, and CUMS-resilient mice. SCFAs in fecal samples were detected by liquid chromatography and gas chromatography–mass spectrometry. Hippocampal cytokine production and TLR4/MYD88/NF-κB inflammatory pathway activation were evaluated using enzyme-linked immunosorbent assays (ELISAs) and western blotting. Then, we supplemented SCFAs in CUMS mice. we observed depression-like behavior and the expression of TLR4/MYD88/NF-κB inflammatory pathway in hippocampus of SCFAs supplementation mice. Susceptible mice to CUMS showed more severe symptoms of depression and anxiety, α diversity was significantly different, as well as higher expression of interleukin (IL)-1β and TLR4/MYD88/NF-κB inflammatory pathway components in the hippocampus. SCFA levels in the feces were significantly higher in CUMS-resilient mice than in control mice. Depressive behavior was reversed in CUMS-SCFAs group, and the protein level of TLR4/MYD88/NF-κB in hippocampus was decreased. Overall, these results provide new light on the possible involvement of the microbiome in the gut-brain axis development in depressive disorder and provide a theoretical basis for identifying novel therapeutic targets.

Simvastatin improves learning and memory impairment via gut-brain axis regulation in an ovariectomized/D-galactose Alzheimer's rat model

AimAlzheimer’s disease (AD) is the most prevalent form of dementia with multiple etiology and no effective remedy. Statins are a group of medicines that are basically used to lower cholesterol. However, several studies have recently done to assess the potential relationship between statins use and dementia but presented controversial results. MethodsIn this study, using ovariectomy and D‐galactose injection, a model of AD was induced in female rats, and then the protective effects of oral administration of simvastatin were investigated. shuttle box and Y-maze tests were done to assess the animals’ learning and memory performance. Using GC-MC, ELISA, Immunohistochemistry and tissue staining techniques, changes in the amount of short-chain fatty acids (SCFAs), plasma and hippocampus neuroinflammatory markers and histological changes in the intestine and hippocampus were assessed in sham, disease and treatment groups. Key findingsOral administration of simvastatin improved the gut microbiome activity (increased the amount of SCFAs in fecal samples) and strengthened the tight junctions of intestinal cells. Moreover, simvastatin reduced the amount of TNF-α and IL-1β in plasma and hippocampus. Also, cell death and Amyloid plaques notably decreased in the simvastatin-treated hippocampal tissue. All these physiological changes led to better performance in behavioral tasks in the treatment group in comparison to the disease group. SignificanceThese findings provide evidence that simvastatin may improve gut-brain axis followed by improvement in learning and memory via an anti-inflammatory effect.

Low Concentration of Fecal Valeric Acid at 1 Year of Age Is Linked with Eczema and Food Allergy at 13 Years of Age: Findings from a Swedish Birth Cohort

Background: Short-chain fatty acids (SCFAs) are abundant bacterial metabolites in the gut, with immunomodulatory properties. Hence, they may influence allergy development. Previous studies have linked fecal SCFA pattern during infancy with allergy. However, the association of SCFAs to allergic outcomes in adolescence is not well established. Here, we examined how the fecal SCFA pattern at 1 year of age related to allergy at 13 years of age. Methods: Levels of 8 SCFAs in fecal samples collected at 1 year of age from 110 children were quantified using gas chromatography. The same individuals were evaluated at 13 years of age for allergic symptoms, allergy diagnosis and allergy medication by questionnaire, and for sensitization using skin prick test against egg, milk, fish, wheat and soy, cat, dog, horse, birch, and timothy grass. Results: The concentration of fecal valeric acid at 1 year of age was inversely associated with eczema at 13 years of age (OR 0.6, 95% CI: 0.4–1.0, p = 0.049) and showed a trend for inverse association with food allergy at 13 years of age (OR 0.6, 95% CI: 0.4–1.0, p = 0.057). In a sub-group analysis of children with eczema at 1 year of age, a higher concentration of fecal valeric acid was linked with reduced risk of their eczema remaining at 13 years of age (OR 0.2, 95% CI: 0.0–1.5), although this latter analysis did not reach statistical significance (p = 0.12). Conclusions: Our findings lend further support to the notion of early childhood as a critical period when allergy may be programmed via the gut microbiota. Higher levels of fecal valeric acid may be characteristic of a protective gut microbiota and/or actively contribute to protection from eczema and food allergy.

Accurate and reliable quantitation of short chain fatty acids from human feces by ultra high-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)

Short chain fatty acids (SCFAs), generated from microbial fermentation of dietary fibers, can regulate weight, appetite and energy homeostasis. Therefore, measuring SCFAs in fecal samples is important to understand the relationship between dietary patterns, gut microbial metabolism, and their impact on host metabolism homeostasis. However, due to the chemical complexity of fecal samples and the volatility of these SCFAs, the quantitative measurements of SCFAs remain challenging. In this study, we developed an absolute quantitation method for accurate and reliable analysis of SCFAs using an UPLC-Q Exactive HRMS system. Nine C2-C6 SCFAs were first derivatized and then separated on a reversed-phase CSH C18 column, and quantitated by UPLC-HRMS with targeted-selected ion monitoring (t-SIM) mode. Our calibration plots showed high linearity (R2>0.99) with high quantitation accuracy (from 91.24%–118.42%); additional analyses showed excellent precisions ranging from 1.12 % to 6.13 %, and accurate recoveries between 92.38 % and 109.65 % with relative standard deviations of 0.31 %–6.44 %. Meanwhile, the short-term stability, freeze and thaw stability, and 168 h storage stability were tested and reported from 85.07%–106.44% with RSDs 0.44%–20.00%, 98.99%–128.84% with RSDs 0.77%–19.79%, and 77.53%–104.42% with RSDs 0.92%–18.65%, respectively. Lastly, this quantitative method was applied to determine the SCFA concentrations and compositions in forty fecal samples from a group of study subjects participating in an obesity prevention trial, and a broad range of concentrations was noted for the detected SCFAs.

Associations between disordered gut microbiota and changes of neurotransmitters and short-chain fatty acids in depressed mice

Mounting evidence suggests that gut microbiota can play an important role in pathophysiology of depression, but its specific molecular mechanisms are still unclear. This study was conducted to explore the associations between changes in neurotransmitters and short-chain fatty acids (SCFAs) and altered gut microbiota in depressed mice. Here, the chronic restraint stress (CRS) model of depression was built. The classical behavioral tests were conducted to assess the depressive-like behaviors of mice. The 16S rRNA gene sequence extracted from fecal samples was used to assess the gut microbial composition. Liquid and gas chromatography mass spectroscopy were used to identify neurotransmitters in hypothalamus and SCFAs in fecal samples, respectively. Finally, 29 differential bacteria taxa between depressed mice and control mice were identified, and the most differentially abundant bacteria taxa were genus Allobaculum and family Ruminococcaceae between the two groups. The acetic acid, propionic acid, pentanoic acid, norepinephrine, 5-HIAA and 5-HT were significantly decreased in depressed mice compared to control mice. Genus Allobaculum was found to be significantly positively correlated with acetic acid and 5-HT. Taken together, these results provided novel microbial and metabolic frameworks for understanding the role of microbiota-gut-brain axis in depression, and suggested new insights to pave the way for novel therapeutic methods.

Evaluation of metabolites levels in feces of patients with inflammatory bowel diseases

Inflammatory bowel diseases (IBD), which include ulcerative colitis (UC) and Crohn's disease (CD), are chronic intestinal inflammatory disorders with an unknown etiology. They are characterized by chronic recurrent inflammation of the intestinal mucosa and lead to a significant decrease in the quality of life and death of patients. IBD are associated with suppression of normal intestinal microflora, including a decrease in bacteria, producers of short chain fatty acids (SCFAs), exhibiting anti-inflammatory and protective properties. Among the various methods of intestinal microflora correction, fecal microbiota transplantation (FMT), which engrafts the fecal microbiota from a healthy donor into a patient recipient, is of a particular interest. As a result, a positive therapeutic effect is observed, accompanied by the restoration of the normal intestinal microflora of the patient. A significant drawback of the method is the lack of standardization. Metabolites produced by intestinal microflora, namely SCFAs, allow objective assessment of the functional state of the intestinal microbiota and, consequently, the success of the FMT procedure. Using gas chromatography and nuclear magnetic resonance spectroscopy techniques, we have analyzed concentrations and molar ratios of SCFAs in fecal samples of 60 healthy donors. Results were in good accord when comparing two methods as well as with published data. Analysis of SCFAs in feces of patients with UC (19 patients) and CD (17 patients) revealed a general decrease in the concentration of fatty acids in the experimental groups with significant fluctuations in the values in experimental groups compared to control group of healthy donors. On the limited group of IBD patients (6 patients with UC and 5 patients with CD) concentration of SCFAs before and within 30 days of observation after FMT was determined. It was shown that FMT had a significant impact on the SCFAs levels within 1 month term; tendency to reach characteristics of healthy donors is unambiguously traced for both diseases.

Profiling of Polar Metabolites in Mouse Feces Using Four Analytical Platforms to Study the Effects Of Cathelicidin-Related Antimicrobial Peptide in Alcoholic Liver Disease.

Alterations in gut bacterial homeostasis result in changes in intestinal metabolites. To investigate the effects of alcohol on fecal metabolites and the role of cathelicidin-related antimicrobial peptide (CRAMP) in alcoholic liver disease (ALD), CRAMP knockout (KO) and their control wild type (WT) mice were fed a Lieber-DeCarli liquid diet with or without alcohol. Polar metabolites in mouse feces were analyzed by GC × GC-MS and 2DLC-MS, and the concentrations of short chain fatty acids (SCFAs) were measured by GC-MS. A total of 95 and 190 metabolites were detected by GC × GC-MS and 2DLC-MS, respectively. Among the significantly changed metabolites, taurine and nicotinic acid were decreased in WT mice fed alcohol, which were also down-regulated in KO mice fed without alcohol. Interestingly, these two metabolites were increased in KO mice fed alcohol compared to them in WT controls. Additionally, SCFAs were significantly decreased in WT mice fed alcohol and in KO mice fed without alcohol, whereas two branched-chain SCFAs were increased by alcohol treatment in KO mice. In summary, the analytical platforms employed in this study successfully dissected the alterations of polar metabolites and SCFAs in fecal samples, which helped understand the effects of alcohol consumption and CRAMP in intestinal metabolism and alcohol-induced liver injury.

Quantification of Fecal Short Chain Fatty Acids by Liquid Chromatography Tandem Mass Spectrometry-Investigation of Pre-Analytic Stability.

Short chain fatty acids (SCFAs) are generated by the degradation and fermentation of complex carbohydrates, (i.e., dietary fiber) by the gut microbiota relevant for microbe–host communication. Here, we present a method for the quantification of SCFAs in fecal samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) upon derivatization to 3-nitrophenylhydrazones (3NPH). The method includes acetate, propionate, butyrate, and isobutyrate with a run time of 4 min. The reproducible (coefficients of variation (CV) below 10%) quantification of SCFAs in human fecal samples was achieved by the application of stable isotope labelled internal standards. The specificity was demonstrated by the introduction of a quantifier and qualifier ions. The method was applied to investigate the pre-analytic stability of SCFAs in human feces. Concentrations of SCFA may change substantially within hours; the degree and kinetics of these changes revealed huge differences between the donors. The fecal SCFA level could be preserved by the addition of organic solvents like isopropanol. An analysis of the colon content of mice either treated with antibiotics or fed with a diet containing a non-degradable and -fermentable fiber source showed decreased SCFA concentrations. In summary, this fast and reproducible method for the quantification of SCFA in fecal samples provides a valuable tool for both basic research and large-scale studies.

Analysis of Short-Chain Fatty Acids in Fecal Samples by Headspace-Gas Chromatography

The level of short-chain fatty acids (SCFAs) is related to health benefits. In this study, a static headspace (HS) gas chromatography method was developed and validated for analyzing SCFAs in fecal samples. SCFAs were injected onto a column through an HS injector through an HS procedure and salting-out technology. The limits of detection and quantification (LOQ) of this method are 0.02–0.08 µg L− 1 (0.4–1.6 µg g− 1 sample) and 0.005–0.020 µg L− 1 (0.1–0.4 µg g− 1 sample), respectively. The calibration curves show good linearity over the range (LOQ–200 µg L− 1 or LOQ–4000 µg g− 1 sample) of these compounds, and the correlation coefficient is > 0.99. The recovery of the method is between 80 and 105%, and matrix effects were not observed. The proposed method was successfully applied to analyze SCFAs in fecal samples from rats.

Sensitive and Simplified Detection of Antibiotic Influence on the Dynamic and Versatile Changes of Fecal Short-Chain Fatty Acids.

Short-chain fatty acids (SCFAs), produced by anaerobic fermentation of mainly indigestible dietary carbohydrates by gut microbiota, have a profound influence on intestinal function and host energy metabolism. Antibiotics may seriously disturb the balance of fecal SCFAs. To evaluate the impacts of antibiotics on fecal SCFAs produced by gut microbiota, a simple, reproducible and accurate gas chromatography (GC) method, which can simultaneously analyze seven SCFAs in fecal samples, was developed and validated. The ranges of detection and quantitation of the SCFAs reached 0.0868 ~ 0.393 and 0.261 ~ 1.18 μg·mL-1 respectively, in an optimized protocol for SCFAs extraction and analysis that used 10 mL 75% ethanol aqueous solution containing 1% HCl, without ultrasonication. The technique exhibited excellent intra-day (relative standard deviation (RSD) ≤ 2.54%) and inter-day (RSD ≤ 4.33%) precisions for all the SCFAs. Later, we administered broad-spectrum antibiotics, cefdinir or azithromycin to rats and analyzed the alterations in fecal SCFAs. The total amount, types and distribution of nearly all fecal SCFAs were significantly altered during the administration and even after withdrawal of the antibiotics in rats. The effects of cefdinir on the SCFAs were more pronounced than those of azithromycin. Our findings suggest SCFAs may serve as sensitive indicators to monitor the influences of antibiotics on SCFAs originated by intestinal bacteria. Our improved SCFAs analysis method is a potential platform for a standard clinical test of the effects of new antibiotics on SCFAs.