Branched-chain Short-chain Fatty Acids Research Articles

Simple, high-throughput measurement of gut-derived short-chain fatty acids in clinically relevant biofluids using gas chromatography-mass spectrometry

IntroductionThe quantitative measurement of circulating gut bacteria-derived metabolites has increased in recent years due to their associations with health and disease. While much of the previous attention has been placed on metabolites considered as deleterious to health, a shift to the investigation of short-chain fatty acids (SCFAs) as potential health promotors has been observed. ObjectivesTo develop a simple, high-throughput and quantitative assay to measure gut-derived SCFAs in clinically relevant biofluids using gas chromatography-mass spectrometry (GC–MS). MethodsA short (7.5 min) GC–MS assay was optimized for measurement of seven straight- and branched-chain SCFAs and their deuterated isotopes using a wax-based column for analysis without prior derivatization. The assay was validated using routine criteria to assess precision, accuracy, matrix effects, recovery, and extraction reproducibility. Assay applicability was tested in cohorts of healthy individuals and kidney disease patients. ResultsThe assay was demonstrated to be precise, accurate and reproducible with acceptable levels of matrix effect and analyte recovery. Lower limits of detection and quantitation were in the low ng/mL range. An investigation into different blood collection tube chemistries demonstrated that lithium heparin plasma and serum clotting activator tubes are recommended for use in future cross-study comparisons. Kidney disease patient analyses demonstrated variable differences across SCFAs when comparing hemodialysis to earlier stages of chronic kidney disease, demonstrating the suitability of the assay for translation to clinical analyses. ConclusionThe assay has been validated and identified as reliable for use in larger-scale studies for the analysis of SCFAs in human plasma and serum.

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.

A specific synbiotic-containing amino acid-based formula restores gut microbiota in non-IgE mediated cow\u2019s milk allergic infants: a randomized controlled trial

BackgroundAltered gut microbiota is implicated in cow’s milk allergy (CMA) and differs markedly from healthy, breastfed infants. Infants who suffer from severe CMA often rely on cow’s milk protein avoidance and, when breastfeeding is not possible, on specialised infant formulas such as amino-acid based formulas (AAF). Herein, we report the effects of an AAF including specific synbiotics on oral and gastrointestinal microbiota of infants with non-IgE mediated CMA with reference to healthy, breastfed infants.MethodsIn this prospective, randomized, double-blind controlled study, infants with suspected non-IgE mediated CMA received test or control formula. Test formula was AAF with synbiotics (prebiotic fructo-oligosaccharides and probiotic Bifidobacterium breve M-16V). Control formula was AAF without synbiotics. Healthy, breastfed infants were used as a separate reference group (HBR). Bacterial compositions of faecal and salivary samples were analysed by 16S rRNA-gene sequencing. Faecal analysis was complemented with the analysis of pH, short-chain fatty acids (SCFAs) and lactic acids.ResultsThe trial included 35 test subjects, 36 controls, and 51 HBR. The 16S rRNA-gene sequencing revealed moderate effects of test formula on oral microbiota. In contrast, the gut microbiota was substantially affected across time comparing test with control. In both groups bacterial diversity increased over time but was characterised by a more gradual increment in test compared to control. Compositionally this reflected an enhancement of Bifidobacterium spp. and Veillonella sp. in the test group. In contrast, the control-fed infants showed increased abundance of adult-like species, mainly within the Lachnospiraceae family, as well as within the Ruminococcus and Alistipes genus. The effects on Bifidobacterium spp. and Lachnospiraceae spp. were previously confirmed through enumeration by fluorescent in situ hybridization and were shown for test to approximate the proportions observed in the HBR. Additionally, microbial activity was affected as evidenced by an increase of l-lactate, a decrease of valerate, and reduced concentrations of branched-chain SCFAs in test versus control.ConclusionsThe AAF including specific synbiotics effectively modulates the gut microbiota and its metabolic activity in non-IgE mediated CMA infants bringing it close to a healthy breastfed profile.Trial registration Registered on 1 May 2013 with Netherlands Trial Register Number NTR3979.

Simultaneous quantification of straight-chain and branched-chain short chain fatty acids by gas chromatography mass spectrometry

Biomedical research in areas such as metabolic disorders, neuromodulatory, and immunomodulatory conditions involves lipid metabolism and demands a reliable and inexpensive method for quantification of short chain fatty acids (SCFAs). We report a GC–MS method for analysis of all straight-chain and branched-chain SCFAs using pentafluorobenzyl bromide (PFBBr) as derivatization reagent. We optimized the derivatization and GC–MS conditions using a mixture containing all eight SCFA standards, i.e., five straight-chain and three branched-chain SCFAs. The optimal derivatization conditions were derivatization time 90 min, temperature 60 °C, pH 7, and (CH3)2CO:H2O ratio 2:1 (v:v). Comparing the performance of different GC column configurations, a 30 m DB-225ms hyphenated with a 30 m DB-5ms column in tandem showed the best separation of SCFAs. Using the optimized experiment conditions, we simultaneously detected all SCFAs with much improved detection limit, 0.244–0.977 μM. We further applied the developed method to measure the SCFAs in mouse feces and all SCFAs were successfully quantified. The recovery rates of the eight SCFAs ranged from 55.7% to 97.9%.

Effect of lactulose intervention on gut microbiota and short chain fatty acid composition of C57BL/6J mice.

Gut microbiota have strong connections with health. Lactulose has been shown to regulate gut microbiota and benefit host health. In this study, the effect of short‐term (3 week) intervention of lactulose on gut microbiota was investigated. Gut microbiota were detected from mouse feces by 16S rRNA high‐throughput sequencing, and short chain fatty acids (SCFAs) were detected by gas chromatography‐mass spectrometry (GC‐MS). Lactulose intervention enhanced the α‐diversity of the gut microbiota; increased the abundance of hydrogen‐producing bacteria Prevotellaceae and Rikenellaceae, probiotics Bifidobacteriaceae and Lactobacillaceae, and mucin‐degrading bacteria Akkermansia and Helicobacter; decreased the abundance of harmful bacteria Desulfovibrionaceae and branched‐chain SCFAs (BCFAs). These results suggest that lactulose intervention effectively increased the diversity and improved the structure of the intestinal microbiota, which may be beneficial for host health.

Extract of Moringa oleifera leaves improves feed utilization of lactating Nubian goats

The present experiment aimed to assess the effect of providing oral doses of Moringa oleifera leaf extract on feed intake, nutrient digestion, and ruminal and blood serum measurements in goats. Sixteen lactating Nubian does (36.5±0.6kg) were used in a quadruplicated 4×4 Latin square design over an 88-day period. An aqueous M. oleifera extract was supplemented orally to each doe at doses of 0 (Control treatment), 10 (ME10 treatment), 20 (ME20 treatment) or 40mL (ME40 treatment). Compared with control, M. oleifera extract linearly increased (P<0.01) nutrient intake and digestibility of dry matter, organic matter, and neutral detergent fiber, without affecting digestibility of crude protein and ether extract. Without affecting ruminal pH and ammonia-N, M. oleifera extract increased (P<0.05) total short-chain fatty acids (SCFA), branched-chain SCFA, and propionic acid concentrations; however, the extract linearly decreased (P<0.01) acetic/propionic ratio and calculated methane production. Increased (P<0.01) serum albumin and glucose concentrations, and decreased (P<0.05) cholesterol, triglycerides, glutamic oxaloacetic transaminase and glutamic pyruvic transaminase concentrations were noted with the inclusion of M. oleifera extract. It is concluded that an oral dose of M. oleifera extract enhanced feed intake and digestibility and ruminal fermentation in lactating Nubian does. Although further research is needed, performance responses associated with increasing the dose of M. oleifera extract to 40mL/doe were not large; thus, the 20mL dose is recommended for practical use.

A polyphenol-enriched diet and Ascaris suum infection modulate mucosal immune responses and gut microbiota composition in pigs.

Polyphenols are a class of bioactive plant secondary metabolites that are thought to have beneficial effects on gut health, such as modulation of mucosal immune and inflammatory responses and regulation of parasite burdens. Here, we examined the interactions between a polyphenol-rich diet supplement and infection with the enteric nematode Ascaris suum in pigs. Pigs were fed either a basal diet or the same diet supplemented with grape pomace (GP), an industrial by-product rich in polyphenols such as oligomeric proanthocyanidins. Half of the animals in each group were then inoculated with A. suum for 14 days to assess parasite establishment, acquisition of local and systemic immune responses and effects on the gut microbiome. Despite in vitro anthelmintic activity of GP-extracts, numbers of parasite larvae in the intestine were not altered by GP-supplementation. However, the bioactive diet significantly increased numbers of eosinophils induced by A. suum infection in the duodenum, jejunum and ileum, and modulated gene expression in the jejunal mucosa of infected pigs. Both GP-supplementation and A. suum infection induced significant and apparently similar changes in the composition of the prokaryotic gut microbiota, and both also decreased concentrations of isobutyric and isovaleric acid (branched-chain short chain fatty acids) in the colon. Our results demonstrate that while a polyphenol-enriched diet in pigs may not directly influence A. suum establishment, it significantly modulates the subsequent host response to helminth infection. Our results suggest an influence of diet on immune function which may potentially be exploited to enhance immunity to helminths.

Degradation of branched-chain amino acids and their derived 2-oxo acids and fatty acids in human and rat heart and skeletal muscle

Studies were made on the oxidation of the branched-chain amino acids, leucine and valine, and their derived 2-oxo acids and branched-chain short-chain fatty acids in intact muscular preparations and homogenates of man and rat. In rat hemidiaphragm transamination rates of the amino acids are higher than oxidative decarboxylation rates and oxidative decarboxylation rates of the 2-oxo acids are higher than those of the corresponding amino acids at the same concentration. The last phenomenon also was observed in rat- and human-heart slices. Transaminase and 2-oxo acid dehydrogenase activities are much higher in homogenates than in intact preparations from the same muscle. Homogenates from heart muscle have generally a higher transaminase and 2-oxo acid dehydrogenase activity than those from skeletal muscle and those from rat muscle have higher activities than those from comparable human muscle. Homogenates from rat heart and liver have similar 2-oxo acid dehydrogenase activities. In homogenates and mitochondria, CO 2 was only produced by oxidative decarboxylation of the branched-chain 2-oxo acids and no CO 2 was produced from the corresponding branched-chain short-chain fatty acids. In all intact muscular preparations the branched-chain 2-oxo acids were oxidized more to completeness and CO 2 production from the coresponding short-chain fatty acids was observed. The further degradation of the branched-chain 2-oxo acids beyond α-decarboxylation appeared to be limited, for 3-methyl-2-oxobutanoate still more than for 4-methyl-2-oxopentanoate.