Clinical, experimental and pathophysiological effects of Yaq-001: a non-absorbable, gut-restricted adsorbent in models and patients with cirrhosis.

OBJECTIVE: Targeting bacterial translocation in cirrhosis is limited to antibiotics with risk of antimicrobial resistance. This study explored the therapeutic potential of a non-absorbable, gut-restricted, engineered carbon bead adsorbent, Yaq-001 in models of cirrhosis and acute-on-chronic liver failure (ACLF) and, its safety and tolerability in a clinical trial in cirrhosis. DESIGN: Performance of Yaq-001 was evaluated in vitro. Two-rat models of cirrhosis and ACLF, (4 weeks, bile duct ligation with or without lipopolysaccharide), receiving Yaq-001 for 2 weeks; and two-mouse models of cirrhosis (6-week and 12-week carbon tetrachloride (CCl4)) receiving Yaq-001 for 6 weeks were studied. Organ and immune function, gut permeability, transcriptomics, microbiome composition and metabolomics were analysed. The effect of faecal water on gut permeability from animal models was evaluated on intestinal organoids. A multicentre, double-blind, randomised, placebo-controlled clinical trial in 28 patients with cirrhosis, administered 4 gr/day Yaq-001 for 3 months was performed. RESULTS: Yaq-001 exhibited rapid adsorption kinetics for endotoxin. In vivo, Yaq-001 reduced liver injury, progression of fibrosis, portal hypertension, renal dysfunction and mortality of ACLF animals significantly. Significant impact on severity of endotoxaemia, hyperammonaemia, liver cell death, systemic inflammation and organ transcriptomics with variable modulation of inflammation, cell death and senescence in the liver, kidneys, brain and colon was observed. Yaq-001 reduced gut permeability in the organoids and impacted positively on the microbiome composition and metabolism. Yaq-001 regulated as a device met its primary endpoint of safety and tolerability in the clinical trial. CONCLUSIONS: This study provides strong preclinical rationale and safety in patients with cirrhosis to allow clinical translation. TRIAL REGISTRATION NUMBER: NCT03202498.

Optimization of Diffusion-Ordered NMR Spectroscopy Experiments for High-Throughput Automation in Human Metabolic Phenotyping.

The diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY) experiment allows the calculation of diffusion coefficient values of metabolites in complex mixtures. However, this experiment has not yet been broadly used for metabolic profiling due to lack of a standardized protocol. Here we propose a pipeline for the DOSY experimental setup and data processing in metabolic phenotyping studies. Due to the complexity of biological samples, three experiments (a standard DOSY, a relaxation-edited DOSY, and a diffusion-edited DOSY) have been optimized to provide DOSY metabolic profiles with peak-picked diffusion coefficients for over 90% of signals visible in the one-dimensional 1H general biofluid profile in as little as 3 min 36 s. The developed parameter sets and tools are straightforward to implement and can facilitate the use of DOSY for metabolic profiling of human blood plasma and urine samples.

Sevoflurane but not propofol enhances ovarian cancer cell biology through regulating cellular metabolic and signaling mechanisms.

Perioperative risk factors, including the choice of anesthetics, may influence ovarian cancer recurrence after surgery. Inhalational anesthetic sevoflurane and intravenous agent propofol might affect cancer cell metabolism and signaling, which, in turn, may influence the malignancy of ovarian cancer cells. The different effects between sevoflurane and propofol on ovarian cancer cell biology and underlying mechanisms were studied. Cultured ovarian cancer cells were exposed to 2.5% sevoflurane, 4 µg/mL propofol, or sham condition as the control for 2 h followed by 24-h recovery. Glucose transporter 1 (GLUT1), mitochondrial pyruvate carrier 1 (MPC1), glutamate dehydrogenase 1 (GLUD1), pigment epithelium-derived factor (PEDF), p-Erk1/2, and hypoxia-inducible factor 1-alpha (HIF-1α) expressions were determined with immunostaining and/or Western blot. Cultured media were collected for 1H-NMR spectroscopy-based metabolomics analysis. Principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used to analyze metabolomics data. Sevoflurane increased the GLUT1, MPC1, GLUD1, p-Erk1/2, and HIF-1α expressions but decreased the PEDF expression relative to the controls. In contrast to sevoflurane, propofol decreased GLUT1, MPC1, GLUD1, p-Erk1/2, and HIF-1α but increased PEDF expression. Sevoflurane increased metabolite isopropanol and decreased glucose and glutamine energy substrates in the media, but the opposite changes were found after propofol treatment. Our data indicated that, unlike the pro-tumor property of sevoflurane, propofol negatively modulated PEDF/Erk/HIF-1α cellular signaling pathway and inhibited ovarian cancer metabolic efficiency and survival, and hence decreased malignancy. The translational value of this work warrants further study. • Sevoflurane promoted but propofol inhibited ovarian cancer cell biology. • Sevoflurane upregulated but propofol downregulated the GLUT1, MPC1, and GLUD1 expressions of ovarian cancer cells. • Sevoflurane enhanced but propofol inhibited ovarian cancer cellular glucose. metabolism and glutaminolysis. • Sevoflurane downregulated PEDF but upregulated the Erk pathway and HIF-1α, while propofol had the adverse effects on ovarian cancer cells.

Duodenal-Jejunal Bypass Liner for the management of Type 2 Diabetes Mellitus and Obesity: A Multicenter Randomized Controlled Trial.

OBJECTIVE: The aim of this study was to examine the clinical efficacy and safety of the duodenal-jejunal bypass liner (DJBL) while in situ for 12 months and for 12 months after explantation. SUMMARY BACKGROUND DATA: This is the largest randomized controlled trial (RCT) of the DJBL, a medical device used for the treatment of people with type 2 diabetes mellitus (T2DM) and obesity. Endoscopic interventions have been developed as potential alternatives to those not eligible or fearful of the risks of metabolic surgery. METHODS: In this multicenter open-label RCT, 170 adults with inadequately controlled T2DM and obesity were randomized to intensive medical care with or without the DJBL. Primary outcome was the percentage of participants achieving a glycated hemoglobin reduction of ≥20% at 12 months. Secondary outcomes included weight loss and cardiometabolic risk factors at 12 and 24 months. RESULTS: There were no significant differences in the percentage of patients achieving the primary outcome between both groups at 12 months [DJBL 54.6% (n = 30) vs control 55.2% (n = 32); odds ratio (OR) 0.93, 95% confidence interval (CI): 0.44-2.0; P = 0.85]. Twenty-four percent (n = 16) patients achieved ≥15% weight loss in the DJBL group compared to 4% (n = 2) in the controls at 12 months (OR 8.3, 95% CI: 1.8-39; P = .007). The DJBL group experienced superior reductions in systolic blood pressure, serum cholesterol, and alanine transaminase at 12 months. There were more adverse events in the DJBL group. CONCLUSIONS: The addition of the DJBL to intensive medical care was associated with superior weight loss, improvements in cardiometabolic risk factors, and fatty liver disease markers, but not glycemia, only while the device was in situ. The benefits of the devices need to be balanced against the higher rate of adverse events when making clinical decisions. TRIAL REGISTRATION: ISRCTN30845205. isrctn.org; Efficacy and Mechanism Evaluation Programme, a Medical Research Council and National Institute for Health Research (NIHR) partnership reference 12/10/04.

Strategy for improved characterization of human metabolic phenotypes using a COmbined Multi-block Principal components Analysis with Statistical Spectroscopy (COMPASS).

MOTIVATION: Large-scale population omics data can provide insight into associations between gene-environment interactions and disease. However, existing dimension reduction modelling techniques are often inefficient for extracting detailed information from these complex datasets. RESULTS: Here, we present an interactive software pipeline for exploratory analyses of population-based nuclear magnetic resonance spectral data using a COmbined Multi-block Principal components Analysis with Statistical Spectroscopy (COMPASS) within the R-library hastaLaVista framework. Principal component analysis models are generated for a sequential series of spectral regions (blocks) to provide more granular detail defining sub-populations within the dataset. Molecular identification of key differentiating signals is subsequently achieved by implementing Statistical TOtal Correlation SpectroscopY on the full spectral data to define feature patterns. Finally, the distributions of cross-correlation of the reference patterns across the spectral dataset are used to provide population statistics for identifying underlying features arising from drug intake, latent diseases and diet. The COMPASS method thus provides an efficient semi-automated approach for screening population datasets. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/cheminfo/COMPASS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Opisthorchis viverrini Infection Induces Metabolic and Fecal Microbial Disturbances in Association with Liver and Kidney Pathologies in Hamsters.

Opisthorchis viverrini (Ov) infection causes hepatobiliary diseases and is a major risk factor for cholangiocarcinoma. While several omics approaches have been employed to understand the pathogenesis of opisthorchiasis, effects of Ov infection on the host systemic metabolism and fecal microbiota have not been fully explored. Here, we used a 1H NMR spectroscopy-based metabolic phenotyping approach to investigate Ov infection-induced metabolic disturbances at both the acute (1 month postinfection, 1 mpi) and chronic (4 mpi) stages in hamsters. A total of 22, 3, and 4 metabolites were found to be significantly different in the liver, serum, and urine, respectively, between Ov+ and Ov- groups. Elevated levels of hepatic amino acids and tricarboxylic acid (TCA)-cycle intermediates (fumarate and malate) were co-observed with liver injury in acute infection, whereas fibrosis-associated metabolites (e.g., glycine and glutamate) increased at the chronic infection stage. Lower levels of lipid signals ((CH2)n and CH2CH2CO) and higher levels of lysine and scyllo-inositol were observed in serum from Ov+ hamsters at 1 mpi compared to Ov- controls. Urinary levels of phenylacetylglycine (a host-bacterial cometabolite) and tauro-ß-muricholic acid were higher in the Ov+ group, which coexisted with hepatic and mild kidney fibrosis. Furthermore, Ov+ animals showed higher relative abundances of fecal Methanobrevibacter (Archaea), Akkermansia, and Burkholderia-Paraburkholderia compared to the noninfected controls. In conclusion, along with liver and kidney pathologies, O. viverrini infection resulted in hepatic and mild renal pathologies, disturbed hepatic amino acid metabolism and the TCA cycle, and induced changes in the fecal microbial composition and urinary host-microbial cometabolism. This study provides the initial step toward an understanding of local and systemic metabolic responses of the host to O. viverrini infection.

Differential effects of L- and D-phenylalanine on pancreatic and gastrointestinal hormone release in humans: A randomized crossover study.

AIM: To investigate the effects of L-phenylalanine on gastroenteropancreatic hormone release, glucose levels, subjective appetite and energy intake in humans, and to determine whether these effects were stereoisomer-specific by comparing them with D-phenylalanine. MATERIALS AND METHODS: A dose-finding, non-randomized, unblinded, crossover study was conducted during October-December 2017 at the NIHR Imperial Clinical Research Facility in five participants, in which the tolerability of escalating doses of oral L-phenylalanine was assessed (0, 3, 6 and 10 g). Also, an acute, randomized, double-blind, placebo-controlled crossover study was conducted during January-May 2018 at the NIHR Imperial Clinical Research Facility in 11 participants, in which the effects of oral 10 g L-phenylalanine relative to D-phenylalanine and placebo on gastroenteropancreatic hormone (insulin, glucagon, glucose-dependent insulinotropic polypeptide [GIP], peptide tyrosine tyrosine [PYY], glucagon-like peptide-1) and glucose concentrations, visual analogue scales for subjective appetite and energy intake at an ad libitum meal served 70 minutes postingestion, were investigated. RESULTS: L-phenylalanine was well-tolerated and increased insulin and glucagon concentrations prior to meal ingestion at several time points relative to placebo and D-phenylalanine (P < .05). L-phenylalanine also increased GIP concentrations relative to D-phenylalanine (P = .0420) and placebo (P = .0249) 70 minutes following ingestion. L-phenylalanine reduced postprandial glucose area under the curve (AUC)70-150mins relative to placebo (P = .0317) but did not affect subjective appetite or energy intake (P > .05). D-phenylalanine increased postprandial PYY AUC70-150mins concentrations relative to placebo (P = .0002). CONCLUSIONS: Ingestion of L-phenylalanine, but not D-phenylalanine, increases insulin, glucagon and GIP concentrations without appearing to have a marked effect on appetite.

Longitudinal metabolic and gut bacterial profiling of pregnant women with previous bariatric surgery.

OBJECTIVE: Due to the global increase in obesity rates and success of bariatric surgery in weight reduction, an increasing number of women now present pregnant with a previous bariatric procedure. This study investigates the extent of bariatric-associated metabolic and gut microbial alterations during pregnancy and their impact on fetal development. DESIGN: A parallel metabonomic (molecular phenotyping based on proton nuclear magnetic resonance spectroscopy) and gut bacterial (16S ribosomal RNA gene amplicon sequencing) profiling approach was used to determine maternal longitudinal phenotypes associated with malabsorptive/mixed (n=25) or restrictive (n=16) procedures, compared with women with similar early pregnancy body mass index but without bariatric surgery (n=70). Metabolic profiles of offspring at birth were also analysed. RESULTS: Previous malabsorptive, but not restrictive, procedures induced significant changes in maternal metabolic pathways involving branched-chain and aromatic amino acids with decreased circulation of leucine, isoleucine and isobutyrate, increased excretion of microbial-associated metabolites of protein putrefaction (phenylacetlyglutamine, p-cresol sulfate, indoxyl sulfate and p-hydroxyphenylacetate), and a shift in the gut microbiota. The urinary concentration of phenylacetylglutamine was significantly elevated in malabsorptive patients relative to controls (p=0.001) and was also elevated in urine of neonates born from these mothers (p=0.021). Furthermore, the maternal metabolic changes induced by malabsorptive surgery were associated with reduced maternal insulin resistance and fetal/birth weight. CONCLUSION: Metabolism is altered in pregnant women with a previous malabsorptive bariatric surgery. These alterations may be beneficial for maternal outcomes, but the effect of elevated levels of phenolic and indolic compounds on fetal and infant health should be investigated further.

Improved Spatial Resolution of Metabolites in Tissue Biopsies Using High-Resolution Magic-Angle-Spinning Slice Localization NMR Spectroscopy.

High-resolution magic-angle-spinning 1H NMR spectroscopy (HR-MAS NMR) is a well-established technique for assessing the biochemical composition of intact tissue samples. In this study, we utilized a method based on HR-MAS NMR spectroscopy with slice localization (SLS) to achieve spatial resolution of metabolites. The obtained 7 slice spectra from each of the model samples (i.e., chicken thigh muscle with skin and murine renal biopsy including medulla (M) and cortex (C)) showed distinct metabolite compositions. Furthermore, we analyzed previously acquired 1H HR-MAS NMR spectra of separated cortex and medulla samples using multivariate statistical methods. Concentrations of glycerophosphocholine (GPC) were found to be significantly higher in the renal medulla compared to the cortex. Using GPC as a biomarker, we identified the tissue slices that were predominantly the cortex or medulla. This study demonstrates that HR-MAS SLS combined with multivariate statistics has the potential for identifying tissue heterogeneity and detailed biochemical characterization of complex tissue samples.

Inhibiting Growth of Clostridioides difficile by Restoring Valerate, Produced by the Intestinal Microbiota.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is effective for treating recurrent Clostridioides difficile infection (CDI), but there are concerns about its long-term safety. Understanding the mechanisms of the effects of FMT could help us design safer, targeted therapies. We aimed to identify microbial metabolites that are important for C difficile growth. METHODS: We used a CDI chemostat model as a tool to study the effects of FMT in vitro. The following analyses were performed: C difficile plate counts, 16S rRNA gene sequencing, proton nuclear magnetic resonance spectroscopy, and ultra-performance liquid chromatography and mass spectrometry bile acid profiling. FMT mixtures were prepared using fresh fecal samples provided by donors enrolled in an FMT program in the United Kingdom. Results from chemostat experiments were validated using human stool samples, C difficile batch cultures, and C57BL/6 mice with CDI. Human stool samples were collected from 16 patients with recurrent CDI and healthy donors (n = 5) participating in an FMT trial in Canada. RESULTS: In the CDI chemostat model, clindamycin decreased valerate and deoxycholic acid concentrations and increased C difficile total viable counts and valerate precursors, taurocholic acid, and succinate concentrations. After we stopped adding clindamycin, levels of bile acids and succinate recovered, whereas levels of valerate and valerate precursors did not. In the CDI chemostat model, FMT increased valerate concentrations and decreased C difficile total viable counts (94% decrease), spore counts (86% decrease), and valerate precursor concentrations; concentrations of bile acids were unchanged. In stool samples from patients with CDI, valerate was depleted before FMT but restored after FMT. Clostridioides difficile batch cultures confirmed that valerate decreased vegetative growth, and that taurocholic acid was required for germination but had no effect on vegetative growth. Clostridioides difficile total viable counts were decreased by 95% in mice with CDI given glycerol trivalerate compared with phosphate buffered saline. CONCLUSIONS: We identified valerate as a metabolite that is depleted with clindamycin and only recovered with FMT. Valerate is a target for a rationally designed recurrent CDI therapy.

Evaluation of Direct from Sample Metabolomics of Human Feces Using Rapid Evaporative Ionization Mass Spectrometry.

Mass spectrometry is a powerful tool in the investigation of the human fecal metabolome. However, current approaches require time-consuming sample preparation, chromatographic separations, and consequently long analytical run times. Rapid evaporative ionization mass spectrometry (REIMS) is a method of ambient ionization mass spectrometry and has been utilized in the metabolic profiling of a diverse range of biological materials, including human tissue, cell culture lines, and microorganisms. Here, we describe the use of an automated, high-throughput REIMS robotic platform for direct analysis of human feces. Through the analysis of fecal samples from five healthy male participants, REIMS analytical parameters were optimized and used to assess the chemical information obtainable using REIMS. Within the fecal samples analyzed, bile acids, including primary, secondary, and conjugate species, were identified, and phospholipids of possible bacterial origin were detected. In addition, the effect of storage conditions and consecutive freeze/thaw cycles was determined. Within the REIMS mass spectra, the lower molecular weight metabolites, such as fatty acids, were shown to be significantly affected by storage conditions for prolonged periods at temperatures above -80 °C and consecutive freeze/thaw cycles. However, the complex lipid region was shown to be unaffected by these conditions. A further cohort of 50 fecal samples, collected from patients undergoing bariatric surgery, were analyzed using the optimized REIMS parameters and the complex lipid region mass spectra used for multivariate modeling. This analysis showed a predicted separation between pre- and post-surgery specimens, suggesting that REIMS analysis can detect biological differences, such as microbiome-level differences, which have traditionally been reliant upon methods utilizing extensive sample preparations and chromatographic separations and/or DNA sequencing.

Post-operative delirium associated with metabolic alterations following hemi-arthroplasty in older patients.

BACKGROUND: post-operative delirium (POD) is a common complication in older patients, though a possible link between metabolic changes and POD development has yet to be investigated. METHODS: older patients with hip fracture who underwent hemi-arthroplasty were recruited, and delirious states were assessed for 3 days after surgery using the confusion assessment method-Chinese revision. Simultaneously, fasting blood samples were collected on the morning of surgery and on the first post-operative day. Ultimately, 244 older patients who met the inclusion and exclusion criteria were assessed. Blood samples from 60 patients with POD and 60 matched controls were analysed using metabolomics platforms. RESULTS: sixty patients (24.6%) developed POD. Principal component analysis scores plot and cross-validated scores plots from orthogonal partial least squares-discriminant analysis were implemented to visualise the differences in metabolites between the two groups before and after surgery (P < 0.05). Our data indicate that levels of ω3 and ω6 fatty acids were lower in the POD group than in the NPOD (non-POD) group both before and after surgery; tricarboxylic cycle intermediate levels were lower in the POD group than in the NPOD group, but glycolysis products were higher in the POD group than in the NPOD group after surgery. Furthermore, the branched-chain amino acid (BCAA)/aromatic amino acid ratio was lower in the POD group than in the NPOD group after surgery. CONCLUSIONS: metabolic abnormalities, including deficiencies in ω3 and ω6 fatty acids, perturbations in tricarboxylic cycle and oxidative stress and metabolic imbalances in BCAA and AAA might contribute to POD development.

Effects of Vancomycin and Ciprofloxacin on the NMRI Mouse Metabolism.

The reduction in gut microbiota diversity is associated with a range of human diseases. Overuse of antibiotics has been associated with a diminished gut-microbial diversity in humans and may promote microbiota-associated negative effects to physical health, such as the metabolic syndrome-cluster of diseases and mental illnesses. There is a pressing need to deepen the understanding of the effects of antibiotics at the biochemical level. The current study investigated metabolic effects of two widely prescribed antibiotics-vancomycin and ciprofloxacin-on biofluids and brain tissue samples of NMRI female mice using a 1H nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling approach. While both antibiotics significantly affected the host metabolic signatures of urine and feces, only ciprofloxacin induced metabolic changes in plasma. Metabolic perturbations were pronounced 1 day post-treatment, reverting back to baseline at day 20 post-treatment. Both antibiotics induced changes in the choline metabolism, host-microbial cometabolites, short chain fatty acid production, and protein/purine degradation. The metabolic profiles of brain tissue aqueous extracts did not show any antibiotics-related changes by day 20 post-treatment. The data suggest that the metabolic disruptions in biofluids caused by antibiotics are reversed by day 20 post-treatment when compared to the pre-treatment profiles.

Long-term multi-species Lactobacillus and Bifidobacterium dietary supplement enhances memory and changes regional brain metabolites in middle-aged rats.

Ageing is associated with changes in the gut microbiome that may contribute to age-related changes in cognition. Previous work has shown that dietary supplements with multi-species live microorganisms can influence brain function, including induction of hippocampal synaptic plasticity and production of brain derived neurotrophic factor, in both young and aged rodents. However, the effect of such dietary supplements on memory processes has been less well documented, particularly in the context of aging. The main aim of the present study was to examine the impact of a long-term dietary supplement with a multi-species live Lactobacillus and Bifidobacteria mixture (Lactobacillus acidophilus CUL60, L. acidophilus CUL21, Bifidobacterium bifidum CUL20 and B. lactis CUL34) on tests of memory and behavioural flexibility in 15-17-month-old male rats. Following behavioural testing, the hippocampus and prefrontal cortex was extracted and analysed ex vivo using 1H nuclear magnetic resonance (1H NMR) spectroscopy to examine brain metabolites. The results showed a small beneficial effect of the dietary supplement on watermaze spatial navigation and robust improvements in long-term object recognition memory and short-term memory for object-in-place associations. Short-term object novelty and object temporal order memory was not influenced by the dietary supplement in aging rats. 1H NMR analysis revealed diet-related regional-specific changes in brain metabolites; which indicated changes in several pathways contributing to modulation of neural signaling. These data suggest that chronic dietary supplement with multi-species live microorganisms can alter brain metabolites in aging rats and have beneficial effects on memory.

Systemic Characterization of an Obese Phenotype in the Zucker Rat Model Defining Metabolic Axes of Energy Metabolism and Host-Microbial Interactions.

The Zucker (fa/fa) rat is a valuable and extensively utilized model for obesity research. However, the metabolic networks underlying the systemic response in the obese Zucker rats remain to be elucidated. This information is important to further our understanding of the circulation of the microbial or host-microbial metabolites and their impact on host metabolism. (1)H nuclear magnetic resonance spectroscopy-based metabolic profiling was used to probe global metabolic differences in portal vein and peripheral blood plasma, urine and fecal water between obese (fa/fa, n = 12) and lean (fa/+, n = 12) Zucker rats. Urinary concentrations of host-microbial co-metabolites were found to be significantly higher in lean Zucker rats. Higher concentrations of fecal lactate, short chain fatty acids (SCFAs), 3-hydroxyphenyl propionic acid and glycerol, and lower levels of valine and glycine were observed in obese rats compared with lean animals. Regardless of phenotype, concentrations of SCFAs, tricarboxylic acid cycle intermediates, and choline metabolites were higher in portal vein blood compared to peripheral blood. However, higher levels of succinate, phenylalanine and tyrosine were observed in portal vein blood compared with peripheral blood from lean rats but not in obese rats. Our findings indicate that the absorption of propionate, acetate, choline, and trimethylamine is independent of the Zucker rat phenotypes. However, urinary host-microbial co-metabolites were highly associated with phenotypes, suggesting distinct gut microbial metabolic activities in lean and obese Zucker rats. This work advances our understanding of metabolic processes associated with obesity, particularly the metabolic functionality of the gut microbiota in the context of obesity.

Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces.

Fecal metabolites are being increasingly studied to unravel the host-gut microbial metabolic interactions. However, there are currently no guidelines for fecal sample collection and storage based on a systematic evaluation of the effect of time, storage temperature, storage duration, and sampling strategy. Here we derive an optimized protocol for fecal sample handling with the aim of maximizing metabolic stability and minimizing sample degradation. Samples obtained from five healthy individuals were analyzed to assess topographical homogeneity of feces and to evaluate storage duration-, temperature-, and freeze-thaw cycle-induced metabolic changes in crude stool and fecal water using a (1)H NMR spectroscopy-based metabolic profiling approach. Interindividual variation was much greater than that attributable to storage conditions. Individual stool samples were found to be heterogeneous and spot sampling resulted in a high degree of metabolic variation. Crude fecal samples were remarkably unstable over time and exhibited distinct metabolic profiles at different storage temperatures. Microbial fermentation was the dominant driver in time-related changes observed in fecal samples stored at room temperature and this fermentative process was reduced when stored at 4 °C. Crude fecal samples frozen at -20 °C manifested elevated amino acids and nicotinate and depleted short chain fatty acids compared to crude fecal control samples. The relative concentrations of branched-chain and aromatic amino acids significantly increased in the freeze-thawed crude fecal samples, suggesting a release of microbial intracellular contents. The metabolic profiles of fecal water samples were more stable compared to crude samples. Our recommendation is that intact fecal samples should be collected, kept at 4 °C or on ice during transportation, and extracted ideally within 1 h of collection, or a maximum of 24 h. Fecal water samples should be extracted from a representative amount (∼15 g) of homogenized stool sample, aliquoted, and stored at <-20 °C, avoiding further freeze-thaw cycles.

Optimization of metabolite extraction of human vein tissue for ultra performance liquid chromatography-mass spectrometry and nuclear magnetic resonance-based untargeted metabolic profiling.

Human vein tissue is an important matrix to examine when investigating vascular diseases with respect to understanding underlying disease mechanisms. Here, we report the development of an extraction protocol for multi-platform metabolic profiling of human vein tissue. For the first stage of the optimization, two different ratios of methanol/water and 5 organic solvents--namely dichloromethane, chloroform, isopropanol, hexane and methyl tert-butyl ether (MTBE) solutions with methanol--were tested for polar and organic compound extraction, respectively. The extraction output was assessed using (1)H Nuclear Magnetic Resonance (NMR) spectroscopy and a panel of Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) methodologies. On the basis of the reproducibility of extraction replicates and metabolic coverage, the optimal aqueous (methanol/water) and organic (MTBE/methanol) solvents identified from the first stage were used in a sequential approach for metabolite extraction, altering the order of solvent-mixture addition. The combination of organic metabolite extraction with MTBE/methanol (3 : 1) followed by extraction of polar compounds with methanol/water (1 : 1) was shown to be the best method for extracting metabolites from human vein tissue in terms of reproducibility and number of signals detected and could be used as a single extraction procedure to serve both NMR and UPLC-MS analyses. Molecular classes such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines, sphingolipids, purines, and pyrimidines were reproducibly extracted. This study enabled an optimal extraction protocol for robust and more comprehensive metabolome coverage for human vein tissue. Many of the physiological and pathological processes affecting the composition of human vein tissue are common to other tissues and hence the extraction method developed in this study can be generically applied.

Systemic responses of BALB/c mice to Salmonella typhimurium infection.

Salmonella typhimurium is a bacterial pathogen that poses a great threat to humans and animals. In order to discover hosts' responses to S. typhimurium infection, we collected and analyzed biofluids and organ tissues from mice which had ingested S. typhimurium. We employed (1)H NMR spectroscopy coupled with multivariate data analysis and immunological techniques. The results indicate that infection leads to a severe impact on mice spleen and ileum, which are characterized by splenomegaly and edematous villi, respectively. We found that increased levels of itaconic acid were correlated with the presence of splenomegaly during infection and may play an important role in Salmonella-containing vacuole acidification. In addition, metabonomic analyses of urine displayed the development of salmonellosis in mice, which is characterized by dynamic changes in energy metabolism. Furthermore, we found that the presence of S. typhimurium activated an anti-oxidative response in infected mice. We also observed changes in the gut microbial co-metabolites (hippurate, TMAO, TMA, methylamine). This investigation sheds much needed light on the host-pathogen interactions of S. typhimurium, providing further information to deepen our understanding of the long co-evolution process between hosts and infective bacteria.

Bariatric surgery modulates circulating and cardiac metabolites.

Bariatric procedures such as the Roux-en-Y gastric bypass (RYGB) operation offer profound metabolic enhancement in addition to their well-recognized weight loss effects. They are associated with significant reduction in cardiovascular disease risk and mortality, which suggests a surgical modification on cardiac metabolism. Metabolic phenotyping of the cardiac tissue and plasma postsurgery may give insight into cardioprotective mechanisms. The aim of the study was to compare the metabolic profiles of plasma and heart tissue extracts from RYGB- and sham-operated Wistar rats to identify the systemic and cardiac signature of metabolic surgery. A total of 27 male Wistar rats were housed individually for a week and subsequently underwent RYGB (n = 13) or sham (n = 14) operation. At week 8 postoperation, a total of 27 plasma samples and 16 heart tissue samples (8 RYGB; 8 Sham) were collected from animals and analyzed using (1)H nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography (UPLC-MS) to characterize the global metabolite perturbation induced by RYGB operation. Plasma bile acids, phosphocholines, amino acids, energy-related metabolites, nucleosides and amine metabolites, and cardiac glycogen and amino acids were found to be altered in the RYGB operated group. Correlation networks were used to identify metabolite association. The metabolic phenotype of this bariatric surgical model inferred systematic change in both myocardial and systemic activity post surgery. The altered metabolic profile following bariatric surgery reflects an enhancement of cardiac energy metabolism through TCA cycle intermediates, cardiorenal protective activity, and biochemical caloric restriction. These surgically induced metabolic shifts identify some of the potential mechanisms that contribute toward bariatric cardioprotection through gut microbiota ecological fluxes and an enterocardiac axis to shield against metabolic syndrome of cardiac dysfunction.