Simple and sensitive LC-MS analysis of short chain fatty acids in cecum content and feces in mice: A derivatization-free approach.

Stable isotope labeling combined with liquid chromatography-tandem mass spectrometry for comprehensive analysis of short-chain fatty acids

Short-chain fatty acids, the end products of fermentation of dietary fibers by the gut microbiota, have been shown to exert multiple effects on mammalian metabolism. For the analysis of short-chain fatty acids, gas chromatography-mass spectrometry is a very powerful and reliable method. Here, we describe a fast, reliable, and reproducible method for the separation and quantification of short-chain fatty acids in mouse feces, cecum content, and blood samples (i.e., plasma or serum) using gas chromatography-mass spectrometry. The short-chain fatty acids analyzed include acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and heptanoic acid.

Quantitative method for intestinal short chain fatty acids based on stable isotope labeling combined with liquid chromatography-mass spectrometry.

A novel LCMSMS method for quantitative measurement of short-chain fatty acids in human stool derivatized with 12C- and 13C-labelled aniline

Short-chain fatty acids (SCFAs) are metabolites derived from gut microbiota and implicated in host homeostasis. Hence, the profiling SCFAs from biological samples plays an important role in revealing the interaction between gut microbiota and pathogens. Previous studies, liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with various derivatization strategies have been performed to obtain the SCFA profiles from biological samples. However, it is poor evidence to compare these derivatization regents and conditions. Thus, we present the evaluation of three major derivatization reagents, namely 3-nitrophenylhydrazine (3-NPH), O-benzylhydroxylamine (O-BHA), and 2-picolylamine (2-PA), for the analysis of eight SCFAs classified as C2-C5 isomers using LC-MS/MS. First, in a reversed-phase LC separation, 3-NPH showed good retention capacity. Although O-BHA derivatization showed higher sensitivity and good retention capacity than 2-PA, only 2-PA derivatization could successfully separate eight SCFAs. The matrix effects in human serum ranged 77.1-99.0% (RSD ≤ 3.4%, n = 6) for 3-NPH derivatives, 91.0-94.6% (RSD ≤ 5.4%, n = 6) for O-BHA derivatives, 81.6-99.5% (RSD ≤ 8.0%, n = 6) for 2-PA derivatives. These compared results showed each characteristic of 3-NPH, O-BHA, and 2-PA for SCFA derivatization based on LC-MS/MS approaches.

Short chain fatty acids (SCFAs) are the main products of dietary fibers that are not digested by the human body, and they have been shown to affect human metabolism and inflammation. The amount of SCFAs in the body is related to many human diseases, and studies have focused on elucidating their roles and target molecules in both metabolic and immune responses. Thus, the quantitation of SCFAs in biological samples becomes crucial in understanding their important roles in the human body. Herein, a facile profiling method of SCFAs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and then applied to biological samples. C2-C6 SCFAs were derivatized while using 4-acetamido-7-mercapto-2,1,3-benzoxadiazole for 5 min. at room temperature prior to LC-MS/MS analysis, and characteristic fragmentation patterns and increased hydrophobicity after chemical derivatization enabled specific discrimination among 12 SCFAs. Derivatization was fast and reliable, and the reaction products were stable for a week at 4 °C. The developed method was applied to measure SCFAs in mouse feces, plasma, and human exhaled breath condensates. This fast and simple method can save labor and effort to profile SCFAs from various biological samples.

High-performance liquid chromatographic analysis of serum short-chain fatty acids by direct derivatization

Butyric acid and sphingomyelin (SM) affect colonic tumorigenesis. We examined the potential link between butyrate stimulation and SM metabolism in colonic and hepatic cancer cell lines. After incubating HT29 and HepG2 cells with butyrate and other short-chain fatty acids, we found that butyrate increased acid but not neutral or alkaline sphingomyelinase (SMase) activity by 10- to 20-fold. The effects occurred after 16 h of incubation and were associated with reduced SM and phosphatidylcholine contents and increased ceramide levels. Northern blotting showed increased acid SMase mRNA levels in these cells after butyrate stimulation. Propionate was less potent, and acetate had no effect. No similar changes of acid phosphatase could be identified. At concentrations that increased acid SMase expression, butyrate inhibited cell proliferation, activated caspase 3, and induced apoptosis. However, the antiproliferative and apoptotic effects of butyrate preceded the changes of acid SMase and were not affected by knocking down acid SMase expression by small, interfering RNA. In addition, butyrate-induced acid SMase expression was not affected by blocking the caspase pathway. In conclusion, butyrate regulates SM metabolism by stimulating acid SMase expression in colon and liver cancer cells, but the increased acid SMase is not a critical mechanism for initiating the anticancer effects of butyrate.

Short chain fatty acids (SCFAs) are important metabolites of the gut microbiota. It has been shown that the microbiota and its metabolic activity in children are highly influenced by the type of diet and age. Our aim was to analyse the concentration of fecal SCFAs over two years of life and to evaluate the influence of feeding method on the content of these compounds in feces. We searched PubMed/MEDLINE/Embase/Ebsco/Cinahl/Web of Science from the database inception to 02/23/2021 without language restriction for observational studies that included an analysis of the concentration of fecal SCFAs in healthy children up to 3 years of age. The primary outcome measures-mean concentrations-were calculated. We performed a random-effects meta-analysis of outcomes for which ≥2 studies provided data. A subgroup analysis was related to the type of feeding (breast milk vs. formula vs. mixed feeding) and the time of analysis (time after birth). The initial search yielded 536 hits. We reviewed 79 full-text articles and finally included 41 studies (n = 2,457 SCFA analyses) in the meta-analysis. We found that concentrations of propionate and butyrate differed significantly in breastfed infants with respect to time after birth. In infants artificially fed up to 1 month of age, the concentration of propionic acid, butyric acid, and all other SCFAs is higher, and acetic acid is lower. At 1–3 months of age, a higher concentration of only propionic acid was observed. At the age of 3–6 months, artificial feeding leads to a higher concentration of butyric acid and the sum of SCFAs. We concluded that the type of feeding influences the content of SCFAs in feces in the first months of life. However, there is a need for long-term evaluation of the impact of the observed differences on health later in life and for standardization of analytical methods and procedures for the study of SCFAs in young children. These data will be of great help to other researchers in analyzing the relationships between fecal SCFAs and various physiologic and pathologic conditions in early life and possibly their impact on health in adulthood.

An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in human feces by liquid chromatography–tandem mass spectrometry

Fermentation metabolites of Diamond V Original XPC™ (XPC), a biological product derived from yeast fermentation, were evaluated for their ability to reduce the Salmonella Typhimurium population using an in vitro mixed anaerobic culture system containing cecal microbiota to simulate chicken hindgut conditions. Four different samples were prepared: anaerobic mixed culture containing (1) feed only, (2) cecal only (ceca were harvested from 42 days old broiler chickens), (3) feed and cecal contents, and (4) feed, cecal contents, and 1% XPC. Two experimental conditions were investigated: Group 1, in which the cecal content was added at the same time as a S. Typhimurium marker strain and Group 2, in which the cecal content was preincubated for 24 h prior to the inoculation with the S. Typhimurium marker strain. The mixed cultures were incubated anaerobically at 37°C, and the S. Typhimurium marker strain was enumerated at 0, 24, and 48 h. Analysis of short chain fatty acids was also conducted for 24 h. In the Group 1 experiment, adding XPC did not exhibit significant reduction of S. Typhimurium. However, the presence of XPC resulted in rapid reduction of S. Typhimurium in Group 2. S. Typhimurium was reduced from 6.81 log10 CFU/ml (0 h) to 3.73 log10 CFU/ml and 1.19 log10 CFU/ml after 24 and 48 h, respectively. These levels were also 2.47 log10 and 2.72 log10 lower than the S. Typhimurium level recovered from the control culture with feed and cecal contents, but without XPC. Based on these results, it appears that the ability of XPC to reduce S. Typhimurium requires the presence of the cecal microbiota. Short chain fatty acid analysis indicated that acetate and butyrate concentrations of cultures containing XPC were twofold greater than the control cultures by 24 h of anaerobic growth. Results from the present study suggest that dietary inclusion of XPC may influence cecal microbiota fermentation and has the potential to reduce Salmonella in the cecum. Implications of these findings suggest that XPC may decrease preharvest levels of Salmonella in broilers and layers.

Development of a sequential injection-liquid microextraction procedure with GC-FID for analysis of short-chain fatty acids in palm oil mill effluent

A sensitive GC/MS detection method for analyzing microbial metabolites short chain fatty acids in fecal and serum samples

Gastroesophageal reflux disease (GERD) is a condition characterized by persistent symptoms and complications resulting from reflux of gastric contents into the esophagus. Short-chain fatty acids (SCFAs) are fermentation products of dietary fibres by the gut microbiota and are often studied for their anti-inflammatory and anticancer effects. The presence of SCFAs in the upper gastrointestinal tract, including in patients with GERD, has not been previously studied. The aim of this study was to investigate the relationship between the concentrations of SCFAs in the saliva of different age groups of patients with GERD. The study included 86 patients diagnosed with GERD, divided into two groups according to age: under and over 60 years of age, treated in the Gastroenterology and Hepatology Outpatient Clinic of the University Hospital in Cracow and 39 patients without gastrointestinal tract diseases. After clinical examination, blood was drawn to determine complete blood count, haemoglobin, and CRP. The oral cavity was examined, and unstimulated mixed saliva was collected. The SCFAs analysis was made by liquid chromatography-tandem mass spectrometry after facile derivatization coupled with liquid-liquid extraction. Of the six SCAFs studied, the highest median concentrations of acetic acid and propionic acid were observed in the saliva of patients with GERD and in the control group, in both the younger and older groups of patients. The concentrations of acetic acid and propionic acid were also higher compared with the four other fatty acids in the saliva of patients with GERD and in the control subjects. There were no correlations between salivary SCFAs levels and selected clinical and endoscopic parameters, including chronic inflammatory changes of the esophagus and stomach. In conclusions: SCFAs are present in the saliva of patients with GERD and in the control healthy persons. With the exception of valeric and isovaleric acids, salivary levels of SCFAs were significantly higher in patients with GERD compared to the control group. The highest concentrations of acetic acid and propionic acid were observed in patients with GERD and in both the younger and older patient groups. There were no differences in the concentrations of SCFAs in the saliva of female and male groups. We found no correlations between salivary SCFAs levels and selected clinical, laboratory and endoscopic changes of the oesophagus and stomach.

This study developed a method for quantifying eight short-chain fatty acids (SCFAs) in mouse fecal samples using solid-phase microextraction (SPME) coupled with triple quadrupole gas chromatography tandem mass spectrometry. Furthermore, significant factors affecting SCFA analysis, including SPME fiber selection, pH, salting-out agent, and sample collection time, were investigated. Contrary to previous studies, we found that the CAR/PDMS fiber had the highest extraction efficiency for all SCFAs. The optimal extraction efficiency was observed at pH 2.0, particularly for low-molecular-weight SCFAs. NaH2PO4 showed a more effective extraction efficiency than NaCl, owing to its pH stability and less interference with the solvent matrix. Additionally, our results showed that the SCFA concentration increased over collection time. The composition ratio of the eight SCFAs was maintained for up to 24 h; thus, we concluded that samples should be collected within four hours to obtain reliable results. Our findings may improve laboratory methods for SCFA extraction and mouse fecal sample analysis.Graphical Supplementary informationThe online version contains supplementary material available at 10.1007/s00216-023-04895-1.