Measurement of short-chain fatty acids in human plasma by means of fast gas chromatography-mass spectrometry.

A rapid and practicable analytical method for the measurement of short-chain fatty acids (SCFAs) in human plasma was developed. The extraction procedure involved the use of acidified water and methyl tert-butyl ether (MTBE), while the separation and detection of SCFAs, including acetic, propionic, and butyric acids was carried out by using gas chromatography-mass spectrometry (GC-MS) technique. The novelty of the research involves reducing the analysis time (less than 7 min) by using the novel fast GC-MS method. A narrow-bore GC capillary column of dimensions 30 m × 0.25 mm ID × 0.25 µm df with acid-modified poly(ethylene glycol) stationary phase was employed for the chromatographic separation. The signals of target compounds were acquired in selected ion monitoring (SIM) mode monitoring a quantifier ion (Q) and two qualifier ions (q1 and q2). Linearity of the method, limits of detection (LoD) and quantification (LoQ) were evaluated. In detail, regression coefficients of the calibration curves were between 0.9960 and 0.9933; LoDs ranged from 0.02 µM to 0.03 µM, while LoQs from 0.06 µM to 0.10 µM.

Engraftment of essential functions through multiple fecal microbiota transplants in chronic antibiotic-resistant pouchitis-a case study using metatranscriptomics.

BACKGROUND: Ileal pouch-anal anastomosis (IPAA) is the standard of care after total proctocolectomy for ulcerative colitis (UC). Around 50% of patients will experience pouchitis, an idiopathic inflammatory condition. Antibiotics are the backbone of treatment of pouchitis; however, antibiotic-resistant pouchitis develops in 5-10% of those patients. It has been shown that fecal microbiota transplantation (FMT) is an effective treatment for UC, but results for FMT antibiotic-resistant pouchitis are inconsistent. METHODS: To uncover which metabolic activities were transferred to the recipients during FMT and helped the remission, we performed a longitudinal case study of the gut metatranscriptomes from three patients and their donors. The patients were treated by two to three FMTs, and stool samples were analyzed for up to 140 days. RESULTS: Reduced expression in pouchitis patients compared to healthy donors was observed for genes involved in biosynthesis of amino acids, cofactors, and B vitamins. An independent metatranscriptome dataset of UC patients showed a similar result. Other functions including biosynthesis of butyrate, metabolism of bile acids, and tryptophan were also much lower expressed in pouchitis. After FMT, these activities transiently increased, and the overall metatranscriptome profiles closely mirrored those of the respective donors with notable fluctuations during the subsequent weeks. The levels of the clinical marker fecal calprotectin were concordant with the metatranscriptome data. Faecalibacterium prausnitzii represented the most active species contributing to butyrate synthesis via the acetyl-CoA pathway. Remission occurred after the last FMT in all patients and was characterized by a microbiota activity profile distinct from donors in two of the patients. CONCLUSIONS: Our study demonstrates the clear but short-lived activity engraftment of donor microbiota, particularly the butyrate biosynthesis after each FMT. The data suggest that FMT triggers shifts in the activity of patient microbiota towards health which need to be repeated to reach critical thresholds. As a case study, these insights warrant cautious interpretation, and validation in larger cohorts is necessary for generalized applications. In the long run, probiotics with high taxonomic diversity consisting of well characterized strains could replace FMT to avoid the costly screening of donors and the risk of transferring unwanted genetic material. Video Abstract.

Can dietary magnesium sources and buffer change the ruminal microbiota composition and fermentation of lactating dairy cows?

Magnesium oxide (MgO) is one of the most used Mg supplements in livestock. However, to avoid relying upon only one Mg source, it is important to have alternative Mg sources. Therefore, the objective of this study was to evaluate the effects of the interaction of two Mg sources with buffer use on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. Twenty lactating Holstein cows were blocked by parity and days in milk into five blocks with four cows each, in a 2 × 2 factorial design. Within blocks, cows were assigned to one of four treatments: 1) MgO; 2) MgO + Na sesquicarbonate (MgO+); 3) calcium-magnesium hydroxide (CaMgOH); 4) CaMgOH + Na sesquicarbonate (CaMgOH+). For 60 d, cows were individually fed a corn silage-based diet, and treatments were top-dressed. Ruminal fluid was collected via an orogastric tube, for analyses of the microbiota composition, volatile fatty acids (VFA), lactate, and ammonia nitrogen (NH3-N). The microbiota composition was analyzed using V4/16S rRNA gene sequencing, and taxonomy was assigned using the Silva database. Statistical analysis was carried out following the procedures of block design analysis, where block and cow were considered random variables. Effects of Mg source, buffer, and the interaction between Mg Source × Buffer were analyzed through orthogonal contrasts. There was no interaction effect of the two factors evaluated. There was a greater concentration of NH3-N, lactate, and butyrate in the ruminal fluid of cows fed with CaMg(OH)2, regardless of the buffer use. The increase in these fermentation intermediates/ end-products can be explained by an increase in abundance of micro-organisms of the genus Prevotella, Lactobacillus, and Butyrivibrio, which are micro-organisms mainly responsible for proteolysis, lactate-production, and butyrate-production in the rumen, respectively. Also, dietary buffer use did not affect the ruminal fermentation metabolites and pH; however, an improvement of the apparent total tract digestibility of dry matter (DM), organic matter (OM), neutral fiber detergent (NDF), and acid fiber detergent (ADF) were found for animals fed with dietary buffer. In summary, there was no interaction effect of buffer use and Mg source, whereas buffer improved total tract apparent digestibility of DM and OM through an increase in NDF and ADF digestibility and CaMg(OH)2 increased ruminal concentration of butyrate and abundance of butyrate-producing bacteria.

Magnesium oxide (MgO) is extensively used as a dietary magnesium (Mg) source in dairy cow diets. However, dairy operations can benefit from other Mg sources. Thus, we evaluated the replacement of dietary MgO with calcium­magnesium hydroxide (CaMg(OH)2) in diets with and without ruminal buffer and their effects on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. The study used 20 lactating Holstein cows that were blocked in groups of four and randomly assigned to one of the four treatments. The ruminal content, feed, feces, and urine were collected for analysis of the microbiota composition, ruminal fermentation, nitrogen metabolism, and apparent nutrient digestibility. There was no interaction effect of dietary buffer use and Mg source, while buffer improved total tract apparent digestibility of the dry matter and fiber components; CaMg(OH)2 increased the ruminal concentration of butyrate and the abundance of butyrate-producing bacteria. In summary, we conclude that using CaMg(OH)2 can improve ruminal fermentation regardless of buffer use, which indicates that we can take advantage of the mineral formulation in the diet to modulate the ruminal microbiota composition.

HMOs Exert Marked Bifidogenic Effects on Children's Gut Microbiota Ex Vivo, Due to Age-Related Bifidobacterium Species Composition.

Prebiotics are substrates that are selectively utilized by host microorganisms, thus conferring a health benefit. There is a growing awareness that interpersonal and age-dependent differences in gut microbiota composition impact prebiotic effects. Due to the interest in using human milk oligosaccharides (HMOs) beyond infancy, this study evaluated how HMOs [2'Fucosyllactose (2'FL), Lacto-N-neotetraose (LNnT), 3'Sialyllactose (3'SL), 6'Sialyllactose (6'SL)] and blends thereof affect the microbiota of 6-year-old children (n = 6) and adults (n = 6), compared to prebiotics inulin (IN) and fructooligosaccharides (FOS). The ex vivo SIFR® technology was used, given its demonstrated predictivity in clinical findings. First, HMOs and HMO blends seemed to maintain a higher α-diversity compared to FOS/IN. Further, while 2'FL/LNnT were bifidogenic for both age groups, 3'SL/6'SL and FOS/IN were exclusively bifidogenic for children and adults, respectively. This originated from age-related differences in microbiota composition because while 3'SL/6'SL stimulated B. pseudocatenulatum (abundant in children), FOS/IN enhanced B. adolescentis (abundant in adults). Moreover, all treatments significantly increased acetate, propionate and butyrate (only in adults) with product- and age-dependent differences. Among the HMOs, 6'SL specifically stimulated propionate (linked to Bacteroides fragilis in children and Phocaeicola massiliensis in adults), while LNnT stimulated butyrate (linked to Anaerobutyricum hallii in adults). Indole-3-lactic acid and 3-phenyllactic acid (linked to immune health) and gamma-aminobutyric acid (linked to gut-brain axis) were most profoundly stimulated by 2'FL and HMO blends in both children and adults, correlating with specific Bifidobacteriaceae. Finally, 2'FL/LNnT increased melatonin in children, while 3'SL remarkably increased folic acid in adults. Overall, age-dependent differences in microbiota composition greatly impacted prebiotic outcomes, advocating for the development of age-specific nutritional supplements. HMOs were shown to be promising modulators in the adult, and particularly the children's microbiota. The observed HMO-specific effects, likely originating from their structural heterogeneity, suggest that blends of different HMOs could maximize treatment effects.

Clostridium butyricum Prevents Dysbiosis and the Rise in Blood Pressure in Spontaneously Hypertensive Rats.

Hypertension is accompanied by dysbiosis and a decrease in the relative abundance of short-chain fatty acid (SCFA)-producing bacteria. However, there is no report to examine the role of C. butyricum in blood pressure regulation. We hypothesized that a decrease in the relative abundance of SCFA-producing bacteria in the gut was the cause of spontaneously hypertensive rats (SHR)-induced hypertension. C. butyricum and captopril were used to treat adult SHR for six weeks. C. butyricum modulated SHR-induced dysbiosis and significantly reduced systolic blood pressure (SBP) in SHR (p < 0.01). A 16S rRNA analysis determined changes in the relative abundance of the mainly SCFA-producing bacteria Akkermansia muciniphila, Lactobacillus amylovorus, and Agthobacter rectalis, which increased significantly. Total SCFAs, and particularly butyrate concentrations, in the SHR cecum and plasma were reduced (p < 0.05), while C. butyricum prevented this effect. Likewise, we supplemented SHR with butyrate for six weeks. We analyzed the flora composition, cecum SCFA concentration, and inflammatory response. The results showed that butyrate prevented SHR-induced hypertension and inflammation, and the decline of cecum SCFA concentrations (p < 0.05). This research revealed that increasing cecum butyrate concentrations by probiotics, or direct butyrate supplementation, prevented the adverse effects of SHR on intestinal flora, vascular, and blood pressure.

Alteration of the Gut Microbiome in Inflammatory Bowel Disease.

BACKGROUND: Alteration of the gut microbial structure and function (dysbiosis) is associated with the pathogenesis of various disorders including inflammatory bowel disease (IBD). SUMMARY: Under normal conditions, ß-oxidation of butyrate consumes oxygen in colonocytes and maintains the anaerobic environment in the lumen. Depletion of butyrate-producing bacteria results in anaerobic glycolysis in colonocytes and increases oxygen diffusion into the lumen, leading to a luminal facultative anaerobe expansion. Dysbiosis in IBD is characterized by the reduced abundance of the phylum Firmicutes (e.g., Faecalibacterium, Roseburia, and Ruminococcus) and an increase of the phylum Proteobacteria (e.g., Enterobacteriaceae). The overall structure of the gut mycobiome differs markedly in IBD patients, particularly Crohn's disease (CD), compared with healthy individuals. An increase in the genus Candida is a major contributory factor in the alteration of the mycobiome in Japanese CD patients, but an increase in the genus Saccharomyces is characteristic in Western patients. The gut virome, which is mainly composed of bacteriophages (phages), influences gut homeostasis and pathogenic conditions via an interaction with the gut bacterial community. Alterations in the gut virome have been suggested in patients with IBD. This may alter either the immunogenicity of bacteria, thus affecting the bacteria-host interactions, or the bacterial functions such as antibiotic resistance and toxin synthesis. KEY MESSAGE: Advances in DNA sequencing technology and bioinformatics have revolutionized our understanding of the microbiome in the gut.

Genetic Predictors for Fecal Propionate and Butyrate-Producing Microbiome Pathway Are Not Associated with Colorectal Cancer Risk: A Mendelian Randomization Analysis.

BACKGROUND: Mechanistic data indicate the benefit of short-chain fatty acids (SCFA) produced by gut microbial fermentation of fiber on colorectal cancer, but direct epidemiologic evidence is limited. A recent study identified SNPs for two SCFA traits (fecal propionate and butyrate-producing microbiome pathway PWY-5022) in Europeans and showed metabolic benefits. METHODS: We conducted a two-sample Mendelian randomization analysis of the genetic instruments for the two SCFA traits (three SNPs for fecal propionate and nine for PWY-5022) in relation to colorectal cancer risk in three large European genetic consortia of 58,131 colorectal cancer cases and 67,347 controls. We estimated the risk of overall colorectal cancer and conducted subgroup analyses by sex, age, and anatomic subsites of colorectal cancer. RESULTS: We did not observe strong evidence for an association of the genetic predictors for fecal propionate levels and the abundance of PWY-5022 with the risk of overall colorectal cancer, colorectal cancer by sex, or early-onset colorectal cancer (diagnosed at <50 years), with no evidence of heterogeneity or pleiotropy. When assessed by tumor subsites, we found weak evidence for an association between PWY-5022 and risk of rectal cancer (OR per 1-SD, 0.95; 95% confidence intervals, 0.91-0.99; P = 0.03) but it did not surpass multiple testing of subgroup analysis. CONCLUSIONS: Genetic instruments for fecal propionate levels and the abundance of PWY-5022 were not associated with colorectal cancer risk. IMPACT: Fecal propionate and PWY-5022 may not have a substantial influence on colorectal cancer risk. Future research is warranted to comprehensively investigate the effects of SCFA-producing bacteria and SCFAs on colorectal cancer risk.

The impact of almonds and almond processing on gastrointestinal physiology, luminal microbiology, and gastrointestinal symptoms: a randomized controlled trial and mastication study.

BACKGROUND: Almonds contain lipid, fiber, and polyphenols and possess physicochemical properties that affect nutrient bioaccessibility, which are hypothesized to affect gut physiology and microbiota. OBJECTIVES: To investigate the impact of whole almonds and ground almonds (almond flour) on fecal bifidobacteria (primary outcome), gut microbiota composition, and gut transit time. METHODS: Healthy adults (n = 87) participated in a parallel, 3-arm randomized controlled trial. Participants received whole almonds (56 g/d), ground almonds (56 g/d), or an isocaloric control in place of habitual snacks for 4 wk. Gut microbiota composition and diversity (16S rRNA gene sequencing), SCFAs (GC), volatile organic compounds (GC-MS), gut transit time (wireless motility capsule), stool output and gut symptoms (7-d diary) were measured at baseline and endpoint. The impact of almond form on particle size distribution (PSD) and predicted lipid release was measured (n = 31). RESULTS: Modified intention-to-treat analysis was performed on 79 participants. There were no significant differences in mean ± SD abundance of fecal bifidobacteria after consumption of whole almonds (8.7% ± 7.7%), ground almonds (7.8% ± 6.9%), or control (13.0% ± 10.2%; q = 0.613). Consumption of almonds (whole and ground pooled) resulted in higher mean ± SD butyrate (24.1 ± 15.0 µmol/g) than control (18.2 ± 9.1 µmol/g; P = 0.046). There was no effect of almonds on gut microbiota at the phylum level or diversity, gut transit time, stool consistency, or gut symptoms. Almond form (whole compared with ground) had no effect on study outcomes. Ground almonds resulted in significantly smaller PSD and higher mean ± SD predicted lipid release (10.4% ± 1.8%) than whole almonds (9.3% ± 2.0%; P = 0.017). CONCLUSIONS: Almond consumption has limited impact on microbiota composition but increases butyrate in adults, suggesting positive alterations to microbiota functionality. Almonds can be incorporated into the diet to increase fiber consumption without gut symptoms.This trial was registered at clinicaltrials.gov as NCT03581812.

Metabolic Modeling and Bidirectional Culturing of Two Gut Microbes Reveal Cross-Feeding Interactions and Protective Effects on Intestinal Cells.

The gut microbiota is constituted by thousands of microbial interactions, some of which correspond to the exchange of metabolic by-products or cross-feeding. Inulin and xylan are two major dietary polysaccharides that are fermented by members of the human gut microbiota, resulting in different metabolic profiles. Here, we integrated community modeling and bidirectional culturing assays to study the metabolic interactions between two gut microbes, Phocaeicola dorei and Lachnoclostridium symbiosum, growing in inulin or xylan, and how they provide a protective effect in cultured cells. P. dorei (previously belonging to the Bacteroides genus) was able to consume inulin and xylan, while L. symposium only used certain inulin fractions to produce butyrate as a major end product. Constrained-based flux simulations of refined genome-scale metabolic models of both microbes predicted high lactate and succinate cross-feeding fluxes between P. dorei and L. symbiosum when growing in each fiber. Bidirectional culture assays in both substrates revealed that L. symbiosum growth increased in the presence of P. dorei. Carbohydrate consumption analyses showed a faster carbohydrate consumption in cocultures compared to monocultures. Lactate and succinate concentrations in bidirectional cocultures were lower than in monocultures, pointing to cross-feeding as initially suggested by the model. Butyrate concentrations were similar across all conditions. Finally, supernatants from both bacteria cultured in xylan in bioreactors significantly reduced tumor necrosis factor-α-induced inflammation in HT-29 cells and exerted a protective effect against the TcdB toxin in Caco-2 epithelial cells. Surprisingly, this effect was not observed in inulin cocultures. Overall, these results highlight the predictive value of metabolic models integrated with microbial culture assays for probing microbial interactions in the gut microbiota. They also provide an example of how metabolic exchange could lead to potential beneficial effects in the host. IMPORTANCE Microbial interactions represent the inner connections in the gut microbiome. By integrating mathematical modeling tools and microbial bidirectional culturing, we determined how two gut commensals engage in the exchange of cross-feeding metabolites, lactate and succinate, for increased growth in two fibers. These interactions underpinned butyrate production in cocultures, resulting in a significant reduction in cellular inflammation and protection against microbial toxins when applied to cellular models.

Mild intermittent hypoxia exposure alters gut microbiota composition in men with overweight and obesity.

Results from high altitude studies in humans and controlled animal experiments suggest that hypoxia exposure induces alterations in gut microbiota composition, which may in turn affect host metabolism. However, well-controlled studies investigating the effects of normobaric hypoxia exposure on gut microbiota composition in humans are lacking. The aim of this study was to explore the impact of mild intermittent hypoxia (MIH) exposure on gut microbiota composition in men with overweight and/or obesity. We performed a randomised, single-blind crossover study, in which participants were exposed to MIH (FiO2: 15%, 3×2 h per day) and normoxia (FiO2: 21%) for seven consecutive days. Following the MIH and normoxia exposure regimens, faecal samples were collected for determination of faecal microbiota composition using 16S rRNA gene-amplicon sequencing in the morning of day 8. Paired faecal samples were available for five individuals. Furthermore, tissue-specific insulin sensitivity was determined using the gold-standard two-step hyperinsulinemic-euglycemic clamp. MIH did not affect microbial alpha and beta-diversity but reduced the relative abundance of Christensenellaceae and Clostridiaceae bacterial families. MIH significantly increased the abundances of obligate anaerobic bacterial genera including Fusicatenibacter, Butyricicoccus and Holdemania, whilst reducing Christensenellaceae R-7 group and Clostridium sensu stricto 1, although these findings were not statistically significant after correction for multiple testing. Furthermore, MIH-induced alterations in abundances of several genera were associated with changes in metabolic parameters such as adipose and peripheral insulin sensitivity, plasma levels of insulin, fatty acids, triacylglycerol and lactate, and substrate oxidation. In conclusion, we demonstrate for the first time that MIH exposure induces modest effects on faecal microbiota composition in humans, shifting several bacterial families and genera towards higher abundances of anaerobic butyrate-producing bacteria. Moreover, MIH-induced effects on faecal microbial composition were associated with parameters related to glucose and lipid homeostasis, supporting a link between MIH-induced alterations in faecal microbiota composition and host metabolism. The study was registered at the Netherlands Trial Register: NL7120/NTR7325.

Omics profiles of fecal and oral microbiota change in irritable bowel syndrome patients with diarrhea and symptom exacerbation.

BACKGROUND: Irritable bowel syndrome (IBS) is a disorder of gut-brain interaction, including dysregulation of the hypothalamic-pituitary-adrenal axis with salivary cortisol changes. However, the role of gastrointestinal microbiota during IBS symptom exacerbation remains unclear. We tested the hypothesis that the microbial species, gene transcripts, and chemical composition of fecal and oral samples are altered during the exacerbation of IBS symptoms. METHODS: Fecal, salivary, and dental plaque samples were collected at baseline from 43 men with IBS with diarrhea (IBS-D) and 40 healthy control (HC) men. Samples in the IBS-D patients were also collected during symptom exacerbation. The composition of the fecal microbiota was determined by analyzing the 16S rRNA gene, RNA-based metatranscriptome, and metabolites in samples from HC and IBS patients with and without symptom exacerbation. Oral samples were also analyzed using omics approaches. RESULTS: The fecal microbiota during IBS symptom exacerbation exhibited significant differences in the phylogenic pattern and short-chain fatty acid compared with fecal samples during defecation when symptoms were not exacerbated. Although there were no significant differences in the phylogenic pattern of fecal microbiota abundance between HCs and IBS-D patients, significant differences were detected in the expression patterns of bacterial transcriptomes related to butyrate production and neuroendocrine hormones, including tryptophan-serotonin-melatonin synthesis and glutamine/GABA. The composition of plaque microbiota was different between HC and IBS-D patients during normal defecation. CONCLUSIONS: Our findings suggest that colonic host-microbial interactions are altered in IBS-D patients during exacerbation of symptoms. There were no overlaps between feces and oral microbiomes.

Characteristics of Gut Microbiome and Its Metabolites, Short-Chain Fatty Acids, in Children With Idiopathic Short Stature.

Background: The gut microbiome is important for host nutrition and metabolism. Whether the gut microbiome under normal diet regulate human height remains to be addressed. Our study explored the possible relationship between gut microbiota, its metabolic products and the pathogenesis of idiopathic short stature disease (ISS) by comparing the gut microbiota between children with ISS and of normal height, and also the short-chain fatty acids (SCFAs) produced by the gut microbiota. Methods: The subjects of this study were 32 prepubescent children aged 4-8 years. The fecal microbial structure of the subjects was analyzed by 16S rRNA high-throughput sequencing technology. The concentrations of SCFAs in feces were determined by gas chromatography-mass spectrometry. Results: The richness of gut microbiota in ISS group was decreased, and the composition of gut microbiota was significantly different between ISS group and control group. The relative abundance of nine species including family Ruminococcaceae and genera Faecalibacterium and Eubacterium, in ISS group was significantly lower than that in control group (P<0.05). The relative abundance of 10 species, such as those belonging to genus Parabacteroides and genus Clostridium, in ISS group was significantly higher than that in control group (P<0.05). The concentration of total SCFAs and butyrate in ISS group was significantly lower than that in control group. The correlation analysis among different species, clinical indicators, and SCFAs showed that the relative abundance of family Ruminococcaceae and genera Faecalibacterium and Eubacterium was positively correlated with the standard deviation score of height. Furthermore, the concentrations of total SCFAs and butyrate were positively correlated with serum insulin-like growth factor 1 (IGF-1)-SDS. Disease prediction model constructed based on the bacteria who abundance differed between healthy children and ISS children exhibited high diagnostic value (AUC: 0.88). Conclusions: The composition of gut microbiota and the change in its metabolite levels may be related to ISS pathogenesis. Strains with increased or decreased specificity could be used as biomarkers to diagnose ISS.

Association of gut microbiota characteristics and metabolites reveals the regulation mechanisms under cadmium consumption circumstance.

BACKGROUND: Cadmium is a non-biodegradable heavy metal with a long biological half-life. Although its negative impact on human health has been previously reported, the association of cadmium consumption overdose with changes in the gut microbiota and its corresponding metabolites has not been fully elucidated so far. RESULTS: Cadmium consumption overdose led to a reduced body weight gain accompanied by an enhanced level of the proinflammatory cytokine tumor necrosis factor-α, interleukin-6, and histamine in the serum of the rats in comparison with normal rats. Furthermore, hepatotoxicity was also observed to be induced by cadmium, which was consistent with abnormal hepatic activities of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase and oxidative stress. In contrast, Lactobacillus rhamnosus-fermented Ganoderma lucidum (FGL) slice supplementation improved the aforementioned physiological properties. More importantly, microbiome and metabolites analysis indicated cadmium exposure significantly reduced the generation of short-chain fatty acids in the gut, particularly butyrate. However, rats in the FGL group had the highest level of butyrate in the feces, characterized with significantly enriched probiotics (Lactobacillus, Bifidobacterium) and butyrate-producing bacteria (Roseburia). CONCLUSION: The targeted regulation of the gut microbial community and its metabolites might be the essential association for attenuating body dysfunction induced by cadmium. The supplementation of FGL, as evidenced in this study, might highlight a novel approach to this field. © 2022 Society of Chemical Industry.

Quantitative analysis of short-chain fatty acids in human plasma and serum by GC-MS.

Short-chain fatty acids (SCFAs) are volatile fatty acids produced by gut microbial fermentation of dietary nondigestible carbohydrates. Acetate, propionate, and butyrate SCFA measures are important to clinical and nutritional studies for their established roles in promoting healthy immune and gut function. Additionally, circulating SCFAs may influence the metabolism and allied function of additional tissues and organs. The accurate quantification of SCFAs in plasma/serum is critical to understanding the biological role of SCFAs. The low concentrations of circulating SCFAs and their volatile nature present challenges for quantitative analysis. Herein, we report a sensitive method for SCFA quantification via extraction with methyl tert-butyl ether after plasma/serum acidification. The organic extract of SCFAs is injected directly with separation and detection using a polar GC column coupled to mass spectrometry. The solvent-to-sample ratio, plasma volume, and amount of HCl needed for SCFA protonation were optimized. Method validation shows good within-day and inter-day repeatability. The limit of detection was 0.3-0.6 µg/mL for acetate and 0.03-0.12 µg/mL for propionate and butyrate. Successful application of this method on clinical plasma and serum samples was demonstrated in six datasets. By simplifying the sample preparation procedure, the present method reduces the risk of contamination, lowers the cost of analysis, increases throughput, and offers the potential for automated sample preparation.

Increased circulating butyrate and ursodeoxycholate during probiotic intervention in humans with type 2 diabetes.

BACKGROUND: An increasing body of evidence implicates the resident gut microbiota as playing a critical role in type 2 diabetes (T2D) pathogenesis. We previously reported significant improvement in postprandial glucose control in human participants with T2D following 12-week administration of a 5-strain novel probiotic formulation ('WBF-011') in a double-blind, randomized, placebo controlled setting (NCT03893422). While the clinical endpoints were encouraging, additional exploratory measurements were needed in order to link the motivating mechanistic hypothesis - increased short-chain fatty acids - with markers of disease. RESULTS: Here we report targeted and untargeted metabolomic measurements on fasting plasma (n = 104) collected at baseline and end of intervention. Butyrate and ursodeoxycholate increased among participants randomized to WBF-011, along with compelling trends between butyrate and glycated haemoglobin (HbA1c). In vitro monoculture experiments demonstrated that the formulation's C. butyricum strain efficiently synthesizes ursodeoxycholate from the primary bile acid chenodeoxycholate during butyrogenic growth. Untargeted metabolomics also revealed coordinated decreases in intermediates of fatty acid oxidation and bilirubin, potential secondary signatures for metabolic improvement. Finally, improvement in HbA1c was limited almost entirely to participants not using sulfonylurea drugs. We show that these drugs can inhibit growth of formulation strains in vitro. CONCLUSION: To our knowledge, this is the first description of an increase in circulating butyrate or ursodeoxycholate following a probiotic intervention in humans with T2D, adding support for the possibility of a targeted microbiome-based approach to assist in the management of T2D. The efficient synthesis of UDCA by C. butyricum is also likely of interest to investigators of its use as a probiotic in other disease settings. The potential for inhibitory interaction between sulfonylurea drugs and gut microbiota should be considered carefully in the design of future studies.

Inulin-grown Faecalibacterium prausnitzii cross-feeds fructose to the human intestinal epithelium.

Many chronic diseases are associated with decreased abundance of the gut commensal Faecalibacterium prausnitzii. This strict anaerobe can grow on dietary fibers, e.g., prebiotics, and produce high levels of butyrate, often associated to epithelial metabolism and health. However, little is known about other F. prausnitzii metabolites that may affect the colonic epithelium. Here, we analyzed prebiotic cross-feeding between F. prausnitzii and intestinal epithelial (Caco-2) cells in a "Human-oxygen Bacteria-anaerobic" coculture system. Inulin-grown F. prausnitzii enhanced Caco-2 viability and suppressed inflammation- and oxidative stress-marker expression. Inulin-grown F. prausnitzii produced excess butyrate and fructose, but only fructose efficiently promoted Caco-2 growth. Finally, fecal microbial taxonomy analysis (16S sequencing) from healthy volunteers (n = 255) showed the strongest positive correlation for F. prausnitzii abundance and stool fructose levels. We show that fructose, produced and accumulated in a fiber-rich colonic environment, supports colonic epithelium growth, while butyrate does not.

Production of butyrate and branched-chain amino acid catabolic byproducts by CHO cells in fed-batch culture enhances their specific productivity.

Chinese hamster ovary (CHO) cells in fed-batch cultures produce several metabolic byproducts derived from amino acid catabolism, some of which accumulate to growth inhibitory levels. Controlling the accumulation of these byproducts has been shown to significantly enhance cell proliferation. Interestingly, some of these byproducts have physiological roles that go beyond inhibition of cell proliferation. In this study, we show that, in CHO cell fed-batch cultures, branched-chain amino acid (BCAA) catabolism contributes to the formation of butyrate, a novel byproduct that is also a well-established specific productivity enhancer. We further show that other byproducts of BCAA catabolism, namely isovalerate and isobutyrate, which accumulate in CHO cell fed-batch cultures, also enhance specific productivity. Lastly, we show that the rate of production of these BCAA catabolic byproducts is negatively correlated with glucose uptake and lactate production rates. Thus, limiting glucose supply to suppress glucose uptake and lactate production, as in the case of fed-batch cultures employing high-end pH-controlled delivery of glucose (HiPDOG) technology, significantly enhances BCAA catabolic byproduct accumulation, resulting in higher specific productivities.

Metagenomic analysis revealed the potential role of gut microbiome in gout.

Emerging evidence indicates an association between gut microbiome and arthritis diseases including gout. However, how and which gut bacteria affect host urate degradation and inflammation in gout remains unclear. Here we performed a metagenome analysis on 307 fecal samples from 102 gout patients and 86 healthy controls. Gout metagenomes significantly differed from those of healthy controls. The relative abundances of Prevotella, Fusobacterium, and Bacteroides were increased in gout, whereas those of Enterobacteriaceae and butyrate-producing species were decreased. Functionally, gout patients had greater abundances for genes in fructose, mannose metabolism and lipid A biosynthesis, and lower for genes in urate degradation and short chain fatty acid production. A three-pronged association between metagenomic species, functions and clinical parameters revealed that decreased abundances of species in Enterobacteriaceae were associated with reduced amino acid metabolism and environmental sensing, which together contribute to increased serum uric acid and C-reactive protein levels in gout. A random forest classifier based on three gut microbial genes showed high predictivity for gout in both discovery and validation cohorts (0.91 and 0.80 accuracy), with high specificity in the context of other chronic disorders. Longitudinal analysis showed that uric-acid-lowering and anti-inflammatory drugs partially restored gut microbiota after 24-week treatment. Comparative analysis with obesity, type 2 diabetes, ankylosing spondylitis and rheumatoid arthritis indicated that gout metagenomes were more similar to those of autoimmune than metabolic diseases. Our results suggest that gut dysbiosis was associated with dysregulated host urate degradation and systemic inflammation and may be used as non-invasive diagnostic markers for gout.

Dynamic Colonization of Microbes and Their Functions after Fecal Microbiota Transplantation for Inflammatory Bowel Disease.

For fecal microbiota transplantation (FMT) to be successful in immune diseases like inflammatory bowel disease, it is assumed that therapeutic microbes and their beneficial functions and immune interactions must colonize a recipient patient and persist in sufficient quantity and for a sufficient period of time to produce a clinical benefit. Few studies, however, have comprehensively profiled the colonization and persistence of transferred microbes along with the transfer of their microbial functions and interactions with the host immune system. Using 16S, metagenomic, and immunoglobulin A (IgA) sequencing, we analyzed hundreds of longitudinal microbiome samples from a randomized controlled trial of 12 patients with ulcerative colitis who received fecal transplant or placebo for 12 weeks. We uncovered diverse competitive dynamics among donor and patient strains, showing that persistence of transferred microbes is far from static. Indeed, one patient experienced a dramatic loss of donor bacteria 10 weeks into the trial, coinciding with a bloom of pathogenic bacteria and worsening symptoms. We evaluated the transfer of microbial functions, including desired ones, such as butyrate production, and unintended ones, such as antibiotic resistance. By profiling bacteria coated with IgA, we identified bacteria associated with inflammation and found that microbial interactions with the host immune system can be transferred across people, which could play a role in gut microbiome therapeutics for immune-related diseases. Our findings shed light on the colonization dynamics of gut microbes and their functions in the context of FMT to treat a complex disease-information that may provide a foundation for developing more-targeted therapeutics. IMPORTANCE Fecal microbiota transplantation (FMT)-transferring fecal microbes from a healthy donor to a sick patient-has shown promise for gut diseases such as inflammatory bowel disease. Unlike pharmaceuticals, however, fecal transplants are complex mixtures of living organisms, which must then interact with the microbes and immune system of the recipient. We sought to understand these interactions by tracking the microbes of 12 inflammatory bowel disease patients who received fecal transplants for 12 weeks. We uncovered a range of dynamics. For example, one patient experienced successful transfer of donor bacteria, only to lose them after 10 weeks. We similarly evaluated transfer of microbial functions, including how they interacted with the recipient's immune system. Our findings shed light on the colonization dynamics of gut microbes, as well as their functions in the context of FMT-information that may provide a critical foundation for the development of more-targeted therapeutics.

Towards a standard operating procedure for revealing hidden volatile organic compounds in breath: the Fourier-transform IR spectroscopy case.

Human breath contains a large amount of small volatile organic compounds (VOCs) and could therefore be used as a carrier of metabolic information for medical diagnostics. Still, in spite of several promising techniques that have been applied during the last decades to study breath content, there is a lack of breath-based diagnostic tools available for physicians. Among several promising techniques, infrared (IR) spectroscopy has already proved its potential for reliable detection of VOCs in the breath. However, due to the large dynamic range of molecular concentrations and overlapping absorption spectra of different VOCs, many low-absorption molecules stay hidden in spectroscopic measurements. To overcome this obstacle, we propose the Matryoshka method for removing masking effects and revealing the buried spectral structures in any bio-fluid in the gas phase. By exploiting both physical and digital removal steps, we demonstrate how the method reveals methane, acetone, aldehyde, and methyl butyrate in a real breath.