Determination of six volatile fatty acids in human serum, urine and faeces by low temperature derivatisation combined with HPLC-MS/MS.

A stable isotope dilution-liquid chromatography tandem mass spectrometry method based on a low-temperature derivatization strategy with 3-nitrophenylhydrazine (3-NPH) was developed for the determination of six volatile fatty acids (VFAs) in serum, urine, and feces. Ice acetonitrile was used to precipitate proteins and extract the target analytes. The extract was derivatized with 3-NPH methanol solution at 4 °C. BEH C8 (1.7 µm, 2.1 × 100 mm) column was used for chromatographic separation, and acetonitrile-water (both containing 0.01 % formic acid) were used as the mobile phase with a gradient elution of 10 min. Electrospray ionization source (ESI) in negative ion multiple reaction monitoring (MRM) mode were used for analyte detection. The regression coefficients R2 of the calibration curves for the six VFAs were in the range of 0.9963-0.9994, and the LOQs were in the range of 0.02-0.5 µg mL-1, with the recoveries in the range of 85.3-104.3 %, and the intra- and inter-day precision in the range of 1.8-9.1 %. The method is simple, accurate and reliable, and has been applied in the sensitive determination of VFAs in complex biological samples.

Identification of potential chemosignals in the European water vole Arvicola terrestris.

The water vole Arvicola terrestris is endemic to Europe where its outbreak generates severe economic losses for farmers. Our project aimed at characterising putative chemical signals used by this species, to develop new sustainable methods for population control that could also be used for this species protection in Great Britain. The water vole, as well as other rodents, uses specific urination sites as territorial and sex pheromone markers, still unidentified. Lateral scent glands and urine samples were collected from wild males and females caught in the field, at different periods of the year. Their volatile composition was analysed for each individual and not on pooled samples, revealing a specific profile of flank glands in October and a specific profile of urinary volatiles in July. The urinary protein content appeared more contrasted as males secrete higher levels of a lipocalin than females, whenever the trapping period. We named this protein arvicolin. Male and female liver transcript sequencing did not identify any expression of other odorant-binding protein sequence. This work demonstrates that even in absence of genome, identification of chemical signals from wild animals is possible and could be helpful in strategies of species control and protection.

Effects of Weight-Loss Interventions on Short-Chain Fatty Acid Concentrations in Blood and Feces of Adults: A Systematic Review.

Short-chain fatty acids (SCFAs, mainly acetate, propionate, and butyrate), which are primarily derived from the gut microbiome, may exert anti-inflammatory and immunomodulatory effects, and regulate energy homeostasis. It has been suggested that weight loss may affect SCFA metabolism, but a systematic review of intervention studies is lacking. We aimed to systematically assess the effects of dietary, physical activity-based, and surgical weight-loss interventions among overweight [body mass index (BMI) 25-29.9 kg/m2)] or obese (BMI ≥30 kg/m2) adults (≥18 y) on concentrations of acetate, propionate, butyrate, and total SCFAs in blood, urine, or feces. We conducted a systematic literature search in PubMed, Web of Science, and the Cochrane Central Register of Controlled Trials (CENTRAL) up to April 30, 2018 for randomized and nonrandomized weight-loss trials among overweight or obese adults, in which the concentrations of individual and total SCFAs were assessed. A total of 9 studies consisting of 2 randomized parallel-arm trials, 4 crossover trials, and 3 nonrandomized clinical or surgical trials were included. In the majority of studies, changes in fecal SCFA concentrations were assessed, whereas changes in serum SCFAs were reported from 1 trial. Individual and total SCFA concentrations either remained unchanged or decreased significantly following weight loss. Three of the dietary interventions that resulted in decreased SCFA concentrations were low (≤5% of energy) in total carbohydrates. Most of the studies had a high risk of bias. Decreases in SCFA concentrations may accompany weight loss induced by bariatric surgery or dietary restriction among overweight or obese adults, particularly when carbohydrate intake is reduced. However, findings were inconsistent and based on studies with high to unclear risk of bias, and small sample sizes. Because measurements of fecal SCFAs may not be ideal due to limited sample standardization, well-powered trials with repeated blood measurements of SCFAs are required. This review was registered at PROSPERO as CRD42018088716.

Systemic availability and metabolism of colonic-derived short-chain fatty acids in healthy subjects: a stable isotope study.

KEY POINTS: The short-chain fatty acids (SCFAs) are bacterial metabolites produced during the colonic fermentation of undigested carbohydrates, such as dietary fibre and prebiotics, and can mediate the interaction between the diet, the microbiota and the host. We quantified the fraction of colonic administered SCFAs that could be recovered in the systemic circulation, the fraction that was excreted via the breath and urine, and the fraction that was used as a precursor for glucose, cholesterol and fatty acids. This information is essential for understanding the molecular mechanisms by which SCFAs beneficially affect physiological functions such as glucose and lipid metabolism and immune function. ABSTRACT: The short-chain fatty acids (SCFAs), acetate, propionate and butyrate, are bacterial metabolites that mediate the interaction between the diet, the microbiota and the host. In the present study, the systemic availability of SCFAs and their incorporation into biologically relevant molecules was quantified. Known amounts of 13 C-labelled acetate, propionate and butyrate were introduced in the colon of 12 healthy subjects using colon delivery capsules and plasma levels of 13 C-SCFAs 13 C-glucose, 13 C-cholesterol and 13 C-fatty acids were measured. The butyrate-producing capacity of the intestinal microbiota was also quantified. Systemic availability of colonic-administered acetate, propionate and butyrate was 36%, 9% and 2%, respectively. Conversion of acetate into butyrate (24%) was the most prevalent interconversion by the colonic microbiota and was not related to the butyrate-producing capacity in the faecal samples. Less than 1% of administered acetate was incorporated into cholesterol and <15% in fatty acids. On average, 6% of colonic propionate was incorporated into glucose. The SCFAs were mainly excreted via the lungs after oxidation to 13 CO2 , whereas less than 0.05% of the SCFAs were excreted into urine. These results will allow future evaluation and quantification of SCFA production from 13 C-labelled fibres in the human colon by measurement of 13 C-labelled SCFA concentrations in blood.

Influence of the type of indigestible carbohydrate on plasma and urine short-chain fatty acid profiles in healthy human volunteers.

BACKGROUND/OBJECTIVES: Health effects of whole grain foods are becoming more evident. In this study, we analysed the short-chain fatty acid profiles in urine and serum derived from the colonic fermentation process of (13)C-barley meals, prepared from barley grown under (13)CO(2) atmosphere. SUBJECTS/METHODS: In a crossover study, five volunteers ingested intact barley kernels (high content of non-starch polysaccharides (NSP) and resistant starch (RS)) and barley porridge (high content of NSP only). Using a newly developed stable isotope technology, we monitored 14 and 24 h postprandially (13)C-acetate, (13)C-propionate and (13)C-butyrate in plasma and urine, respectively. The oro-cecal transit time (OCTT) of the meals was measured with the hydrogen breath test. RESULTS: The OCTT was 6 h and did not differ between the two test meals. An increase of (13)C-acetate was observed already early after ingestion of the meals (<6 h) and was attributed to early fermentation of the test meal. A rise in plasma (13)C-propionate in the fermentation phase could only be detected after the porridge and not after the kernel meal. An increase in (13)C-butyrate was only found in the fermentation phase and was higher after the barley kernels. Urine (13)C-short-chain fatty acids data were consistent with these observations. CONCLUSIONS: The difference in the profiles of (13)C-acetate, (13)C-propionate and (13)C-butyrate indicates that NSP combined with RS results in an altered fermentation profile than dietary fibre alone.

Comparative effects of very low-carbohydrate, high-fat and high-carbohydrate, low-fat weight-loss diets on bowel habit and faecal short-chain fatty acids and bacterial populations.

Very low-carbohydrate diets are often used to promote weight loss, but their effects on bowel health and function are largely unknown. We compared the effects of a very low-carbohydrate, high-fat (LC) diet with a high-carbohydrate, high-fibre, low-fat (HC) diet on indices of bowel health and function. In a parallel study design, ninety-one overweight and obese participants (age 50.6 (sd 7.5) years; BMI 33.7 (sd 4.2) kg/m(2)) were randomly assigned to either an energy-restricted (about 6-7 MJ, 30 % deficit) planned isoenergetic LC or HC diet for 8 weeks. At baseline and week 8, 24 h urine and faecal collections were obtained and a bowel function questionnaire was completed. Compared with the HC group, there were significant reductions in the LC group for faecal output (21 (sd 145) v. - 61 (sd 147) g), defecation frequency, faecal excretion and concentrations of butyrate ( - 0.5 (sd 10.4) v. - 3.9 (sd 9.7) mmol/l) and total SCFA (1.4 (sd 40.5) v. - 15.8 (sd 43.6) mmol/l) and counts of bifidobacteria (P < 0.05 time x diet interaction, for all). Urinary phenols and p-cresol excretion decreased (P < or = 0.003 for time) with no difference between diets (P > or = 0.25). Faecal form, pH, ammonia concentration and numbers of coliforms and Escherichia coli did not change with either diet. No differences between the diets were evident for incidences of adverse gastrointestinal symptoms, which suggests that both diets were well tolerated. Under energy-restricted conditions, a short-term LC diet lowered stool weight and had detrimental effects on the concentration and excretion of faecal SCFA compared with an HC diet. This suggests that the long-term consumption of an LC diet may increase the risk of development of gastrointestinal disorders.

Probiotic modulation of symbiotic gut microbial-host metabolic interactions in a humanized microbiome mouse model.

The transgenomic metabolic effects of exposure to either Lactobacillus paracasei or Lactobacillus rhamnosus probiotics have been measured and mapped in humanized extended genome mice (germ-free mice colonized with human baby flora). Statistical analysis of the compartmental fluctuations in diverse metabolic compartments, including biofluids, tissue and cecal short-chain fatty acids (SCFAs) in relation to microbial population modulation generated a novel top-down systems biology view of the host response to probiotic intervention. Probiotic exposure exerted microbiome modification and resulted in altered hepatic lipid metabolism coupled with lowered plasma lipoprotein levels and apparent stimulated glycolysis. Probiotic treatments also altered a diverse range of pathways outcomes, including amino-acid metabolism, methylamines and SCFAs. The novel application of hierarchical-principal component analysis allowed visualization of multicompartmental transgenomic metabolic interactions that could also be resolved at the compartment and pathway level. These integrated system investigations demonstrate the potential of metabolic profiling as a top-down systems biology driver for investigating the mechanistic basis of probiotic action and the therapeutic surveillance of the gut microbial activity related to dietary supplementation of probiotics.

System level metabolic effects of a Schistosoma japonicum infection in the Syrian hamster.

A metabolic profiling strategy was used to investigate the metabolic responses of Syrian hamsters (SLAC) to a Schistosoma japonicum infection using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy and pattern recognition. In two independent experiments, male hamsters were each infected with 100 S. japonicum cercariae. At days 34-36 post-infection, urine was obtained from hamsters housed individually in metabolism cages. At the same time, urine was collected from age- and sex-matched infection-free control hamsters. The main biochemical effects of a S. japonicum infection in the hamster consisted of reduced levels of urinary tricarboxylic acid cycle intermediates, including citrate and succinate and increased levels of pyruvate. In addition, a range of microbial-related metabolites, such as hippurate, p-cresol glucuronide, phenylacetylglycine and trimethylamine were also associated with a S. japonicum infection. Most of the observed biochemical effects were in common with those previously characterized for a S. mansoni infection in a mouse host. The major distinguishing consequence of a S. japonicum infection in the hamster was the inhibition of manufacture or utilization of short-chain fatty acids, when compared to a S. mansoni infection in the mouse.

In situ derivatisation and extraction of volatile fatty acids entrapped on anion-exchange resin from aqueous solutions and urine as a test matrix using pentafluorobenzyl bromide in supercritical carbon dioxide.

The simultaneous extraction and derivatisation of anion-exchange resin-trapped volatile fatty acids (C2-C5) as their pentafluorobenzyl esters has successfully been performed under CO2 supercritical fluid extraction conditions. Volatile fatty acid standards of acetic, propionic and n-butyric acids (at 20 and 100 ppm) as their ester derivatives were recovered at 78.0-101.5% (C.V. 3.5-7.5%, n=6 and 7). Likewise, acrylic acid recoveries were 57.0-61.0% (C.V. 5.5-5.6%, n=6 and 7). This methodology was applied to the quantitation of acetic, propionic, n-butyric and n-valeric acids in spiked urine as a test matrix. Initial clean-up of phosphate and sulfate in the urine was required prior to anion-exchange application and this was achieved by barium salt precipitation. Recoveries ranged from 36 to 66.5% (C.V. 5.9-14.4%, n=9 and 6).

Effect of different volatile fatty acids mixtures on energy metabolism in cattle.

Four Friesian steers (mean BW = 282 kg) were given mixtures of VFA and casein by intragastric infusion to give a total energy input of 675 kJ/kg BW.75. Casein supplied 16.3% of the energy and 777 mg N/kg BW.75. The molar proportion of butyric acid was held constant at 8 mol/100 mol, and the acetic and propionic acids varied inversely. Acetic acid was varied in 12 increments from 11 to 91 mol/100 mol and propionic acid proportion varied inversely. Heat production, blood (urea, insulin, beta-hydroxybutyrate, free fatty acids) and urine metabolites (urea, N, VFA) were measured. There were no differences (P greater than .05) in heat production until the acetic acid proportions exceeded approximately 90 mol/100 mol, at which point there was a decrease in heat production (P less than .05) accompanied by a considerable excretion of acetic acid in the urine. Above 80 mol/100 mol acetic acid, beta-hydroxybutyrate was greatly elevated, accompanied by a small decrease in blood glucose and blood insulin together with an increase in blood free fatty acid concentration. There was also an elevation of N excretion in the urine. When the proportion of propionic acid exceeded 76 mol/100 mol there were some metabolic disturbances resulting in blood hemolysis, an increase in N excretion in the urine, and nervous disposition of the animals. It is concluded that differences in heat production between roughage and concentrate diets are not likely to be a result of differences in the energetic response to different proportions of VFA. Differences in activity during standing, feeding, and ruminating may, therefore, be more important.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of volatile fatty acids in urine by gas chromatography on a new stationary phase.

A rapid gas chromatographic method for determination of volatile fatty acids in urine is described. Acids up to five carbon atoms, including their isomers can be separated in less than six minutes on the new stationary phase Fluorad FC430. Good separation and sensitivity is obtained with isothermal conditions.

A rapid method for the determination of volatile fatty acids in biological fluids.

A rapid and simple method for the determination of volatile fatty acids in plasma and urine without any pretreatment is described. The gas chromatographic method reported here allows us to detect and quantitate in approx. 20 min biological VFA characteristics of various metabolic diseases. Conditions used are very mild so to avoid as much as possible any thermal decomposition of biological compounds.

Jamaican vomiting sickness. Biochemical investigation of two cases.

We identified methylenecyclopropylacetic acid, a known metabolite of hypoglycin A, in the urine of two patients with Jamaican vomiting sickness. Excretion of unusual dicarboxylic acids such as 2-ethylmalonic, 2-methylsuccinic, glutaric, adipic and dicarboxylic acids with eight and 10 carbon chains were also detected in both patients. The amounts of these dicarboxylic acids were 70 to 1000 times higher than normal. These metabolites have also been identified in urine of hypoglycin-treated rats. This evidence links hypoglycin A to Jamaican vomiting sickness as its causative agent. Urinary excretion of short-chain fatty acids was also increased up to 300 times higher than normal. These results indicate that, despite their clinical and histologic similarities, the cause and biochemical mechanisms of Jamaican vomiting sickness differ distinctly from those of Reye's syndrome in which these abnormal urinary metabolites are not appreciably increased.

Jamaican vomiting sickness: biochemical investigation of two cases

We identified methylenecyclopropylacetic acid, a known metabolite of hypoglycin A, in the urine of two patients with Jamaican vomiting sickness. Excretion of unusual dicarboxylic acids such as 2-ethylmalonic, 2-methylsuccinic, glutaric, adipic and dicarboxylic acids with eight and 10 carbon chains were also detected in both patients. The amounts of these dicarboxylic acids were 70 to 1000 times higher than normal. These metabolities have also been identified in urine of hypoglycin-treated rats. This evidence links hypoglycin A to Jamaican vomiting sickness as its causative agent. Urinary excretion of short-chain fatty acids was also increased up to 300 times higher than normal. These results indicate that, despite their clinical and histological similarities, the cause and biochemical mechanisms of Jamaican vomiting sickness differ distinctly from those of Reye's syndrome in which these abnormal urinary metabolities are not appreciably increased.(AU)