Alanyl-glutamine supplementation for Clostridioides difficile infection treatment (ACT): a double-blind randomised controlled trial study protocol.

INTRODUCTION: Clostridioides difficile is the leading cause of healthcare-associated infections in the USA, with an estimated 1 billion dollars in excess cost to the healthcare system annually. C. difficile infection (CDI) has high recurrence rate, up to 25% after first episode and up to 60% for succeeding episodes. Preliminary in vitro and in vivo studies indicate that alanyl-glutamine (AQ) may be beneficial in treating CDI by its effect on restoring intestinal integrity in the epithelial barrier, ameliorating inflammation and decreasing relapse. METHODS AND ANALYSIS: This study is a randomised, placebo-controlled, double-blind, phase II clinical trial. The trial is designed to determine optimal dose and safety of oral AQ at 4, 24 and 44 g doses administered daily for 10 days concurrent with standard treatment of non-severe or severe uncomplicated CDI in persons age 18 and older. The primary outcome of interest is CDI recurrence during 60 days post-treatment follow-up, with the secondary outcome of mortality during 60 days post-treatment follow-up. Exploratory analysis will be done to determine the impact of AQ supplementation on intestinal and systemic inflammation, as well as intestinal microbial and metabolic profiles. ETHICS AND DISSEMINATION: The study has received University of Virginia Institutional Review Board approval (HSR200046, Protocol v9, April 2023). Findings will be disseminated via conference presentations, lectures and peer-reviewed publications. TRIAL REGISTRATION NUMBER: NCT04305769.

Gut Microbial and Metabolic Profiling Reveal the Lingering Effects of Infantile Iron Deficiency Unless Treated with Iron.

SCOPE: Iron deficiency (ID) compromises the health of infants worldwide. Although readily treated with iron, concerns remain about the persistence of some effects. Metabolic and gut microbial consequences of infantile ID were investigated in juvenile monkeys after natural recovery (pID) from iron deficiency or post-treatment with iron dextran and B vitamins (pID+Fe). METHODS AND RESULTS: Metabolomic profiling of urine and plasma is conducted with 1 H nuclear magnetic resonance (NMR) spectroscopy. Gut microbiota are characterized from rectal swabs by amplicon sequencing of the 16S rRNA gene. Urinary metabolic profiles of pID monkeys significantly differed from pID+Fe and continuously iron-sufficient controls (IS) with higher maltose and lower amounts of microbial-derived metabolites. Persistent differences in energy metabolism are apparent from the plasma metabolic phenotypes with greater reliance on anaerobic glycolysis in pID monkeys. Microbial profiling indicated higher abundances of Methanobrevibacter, Lachnobacterium, and Ruminococcus in pID monkeys and any history of ID resulted in a lower Prevotella abundance compared to the IS controls. CONCLUSIONS: Lingering metabolic and microbial effects are found after natural recovery from ID. These long-term biochemical derangements are not present in the pID+Fe animals emphasizing the importance of the early detection and treatment of early-life ID to ameliorate its chronic metabolic effects.

Microbiota Supplementation with Bifidobacterium and Lactobacillus Modifies the Preterm Infant Gut Microbiota and Metabolome: An Observational Study.

Supplementation with members of the early-life microbiota as "probiotics" is increasingly used in attempts to beneficially manipulate the preterm infant gut microbiota. We performed a large observational longitudinal study comprising two preterm groups: 101 infants orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 infants non-supplemented (control) matched by age, sex, and delivery method. 16S rRNA gene profiling on fecal samples (n = 592) showed a predominance of Bifidobacterium and a lower abundance of pathobionts in the Bif/Lacto group. Metabolomic analysis showed higher fecal acetate and lactate and a lower fecal pH in the Bif/Lacto group compared to the control group. Fecal acetate positively correlated with relative abundance of Bifidobacterium, consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides into acetate. This study demonstrates that microbiota supplementation is associated with a Bifidobacterium-dominated preterm microbiota and gastrointestinal environment more closely resembling that of full-term infants.

Chemotherapy-induced cachexia dysregulates hypothalamic and systemic lipoamines and is attenuated by cannabigerol.

BACKGROUND: Muscle wasting, anorexia, and metabolic dysregulation are common side-effects of cytotoxic chemotherapy, having a dose-limiting effect on treatment efficacy, and compromising quality of life and mortality. Extracts of Cannabis sativa, and analogues of the major phytocannabinoid Δ9-tetrahydrocannabinol, have been used to ameliorate chemotherapy-induced appetite loss and nausea for decades. However, psychoactive side-effects limit their clinical utility, and they have little efficacy against weight loss. We recently established that the non-psychoactive phytocannabinoid cannabigerol (CBG) stimulates appetite in healthy rats, without neuromotor side-effects. The present study assessed whether CBG attenuates anorexia and/or other cachectic effects induced by the broad-spectrum chemotherapy agent cisplatin. METHODS: An acute cachectic phenotype was induced in adult male Lister-hooded rats by 6 mg/kg (i.p.) cisplatin. In total 66 rats were randomly allocated to groups receiving vehicle only, cisplatin only, or cisplatin and 60 or 120 mg/kg CBG (po, b.i.d.). Feeding behavior, bodyweight and locomotor activity were recorded for 72 hours, at which point rats were sacrificed for post-mortem analyses. Myofibre atrophy, protein synthesis and autophagy dysregulation were assessed in skeletal muscle, plasma metabolic profiles were obtained by untargeted 1H-NMR metabonomics, and levels of endocannabinoid-like lipoamines quantified in plasma and hypothalami by targeted HPLC-MS/MS lipidomics. RESULTS: CBG (120 mg/kg) modestly increased food intake, predominantly at 36-60hrs (p<0.05), and robustly attenuated cisplatin-induced weight loss from 6.3% to 2.6% at 72hrs (p<0.01). Cisplatin-induced skeletal muscle atrophy was associated with elevated plasma corticosterone (3.7 vs 13.1ng/ml, p<0.01), observed selectively in MHC type IIx (p<0.05) and IIb (p<0.0005) fibres, and was reversed by pharmacological rescue of dysregulated Akt/S6-mediated protein synthesis and autophagy processes. Plasma metabonomic analysis revealed cisplatin administration produced a wide-ranging aberrant metabolic phenotype (Q2Y=0.5380, p=0.001), involving alterations to glucose, amino acid, choline and lipid metabolism, citrate cycle, gut microbiome function, and nephrotoxicity, which were partially normalized by CBG treatment (Q2Y=0.2345, p=0.01). Lipidomic analysis of hypothalami and plasma revealed extensive cisplatin-induced dysregulation of central and peripheral lipoamines (29/79 and 11/26 screened, respectively), including reversible elevations in systemic N-acyl glycine concentrations which were negatively associated with the anti-cachectic effects of CBG treatment. CONCLUSIONS: Endocannabinoid-like lipoamines may have hitherto unrecognized roles in the metabolic side-effects associated with chemotherapy, with the N-acyl glycine subfamily in particular identified as a potential therapeutic target and/or biomarker of anabolic interventions. CBG-based treatments may represent a novel therapeutic option for chemotherapy-induced cachexia, warranting investigation in tumour-bearing cachexia models.

A murine model of diarrhea, growth impairment and metabolic disturbances with Shigella flexneri infection and the role of zinc deficiency.

Shigella is one of the major enteric pathogens worldwide. We present a murine model of S. flexneri infection and investigate the role of zinc deficiency (ZD). C57BL/6 mice fed either standard chow (HC) or ZD diets were pretreated with an antibiotic cocktail and received S. flexneri strain 2457T orally. Antibiotic pre-treated ZD mice showed higher S. flexneri colonization than non-treated mice. ZD mice showed persistent colonization for at least 50 days post-infection (pi). S. flexneri-infected mice showed significant weight loss, diarrhea and increased levels of fecal MPO and LCN in both HC and ZD fed mice. S. flexneri preferentially colonized the colon, caused epithelial disruption and inflammatory cell infiltrate, and promoted cytokine production which correlated with weight loss and histopathological changes. Infection with S. flexneri ΔmxiG (critical for type 3 secretion system) did not cause weight loss or diarrhea, and had decreased stool shedding duration and tissue burden. Several biochemical changes related to energy, inflammation and gut-microbial metabolism were observed. Zinc supplementation increased weight gains and reduced intestinal inflammation and stool shedding in ZD infected mice. In conclusion, young antibiotic-treated mice provide a new model of oral S. flexneri infection, with ZD promoting prolonged infection outcomes.

L-rhamnose as a source of colonic propionate inhibits insulin secretion but does not influence measures of appetite or food intake.

Activation of free fatty acid receptor (FFAR)2 and FFAR3 via colonic short-chain fatty acids, particularly propionate, are postulated to explain observed inverse associations between dietary fiber intake and body weight. Propionate is reported as the predominant colonic fermentation product from l-rhamnose, a natural monosaccharide that resists digestion and absorption reaching the colon intact, while effects of long-chain inulin on appetite have not been extensively investigated. In this single-blind randomized crossover study, healthy unrestrained eaters (n = 13) ingested 25.5 g/d l-rhamnose, 22.4 g/d inulin or no supplement (control) alongside a standardized breakfast and lunch, following a 6-d run-in to investigate if appetite was inhibited. Postprandial qualitative appetite, breath hydrogen, and plasma glucose, insulin, triglycerides and non-esterified fatty acids were assessed for 420 min, then an ad libitum meal was provided. Significant treatment x time effects were found for postprandial insulin (P = 0.009) and non-esterified fatty acids (P = 0.046) with a significantly lower insulin response for l-rhamnose (P = 0.023) than control. No differences between treatments were found for quantitative and qualitative appetite measures, although significant treatment x time effects for meal desire (P = 0.008) and desire to eat sweet (P = 0.036) were found. Breath hydrogen was significantly higher with inulin (P = 0.001) and l-rhamnose (P = 0.009) than control, indicating colonic fermentation. These findings suggest l-rhamnose may inhibit postprandial insulin secretion, however neither l-rhamnose or inulin influenced appetite.

The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism.

Increased intake of dietary carbohydrate that is fermented in the colon by the microbiota has been reported to decrease body weight, although the mechanism remains unclear. Here we use in vivo(11)C-acetate and PET-CT scanning to show that colonic acetate crosses the blood-brain barrier and is taken up by the brain. Intraperitoneal acetate results in appetite suppression and hypothalamic neuronal activation patterning. We also show that acetate administration is associated with activation of acetyl-CoA carboxylase and changes in the expression profiles of regulatory neuropeptides that favour appetite suppression. Furthermore, we demonstrate through (13)C high-resolution magic-angle-spinning that (13)C acetate from fermentation of (13)C-labelled carbohydrate in the colon increases hypothalamic (13)C acetate above baseline levels. Hypothalamic (13)C acetate regionally increases the (13)C labelling of the glutamate-glutamine and GABA neuroglial cycles, with hypothalamic (13)C lactate reaching higher levels than the 'remaining brain'. These observations suggest that acetate has a direct role in central appetite regulation.

The impact of oligofructose on stimulation of gut hormones, appetite regulation and adiposity.

OBJECTIVE: To investigate the effect of nutrient stimulation of gut hormones by oligofructose supplementation on appetite, energy intake (EI), body weight (BW) and adiposity in overweight and obese volunteers. METHODS: In a parallel, single-blind and placebo-controlled study, 22 healthy overweight and obese volunteers were randomly allocated to receive 30 g day(-1) oligofructose or cellulose for 6 weeks following a 2-week run-in. Subjective appetite and side effect scores, breath hydrogen, serum short chain fatty acids (SCFAs), plasma gut hormones, glucose and insulin concentrations, EI, BW and adiposity were quantified at baseline and post-supplementation. RESULTS: Oligofructose increased breath hydrogen (P < 0.0001), late acetate concentrations (P = 0.024), tended to increase total area under the curve (tAUC)420 mins peptide YY (PYY) (P = 0.056) and reduced tAUC450 mins hunger (P = 0.034) and motivation to eat (P = 0.013) when compared with cellulose. However, there was no significant difference between the groups in other parameters although within group analyses showed an increase in glucagon-like peptide 1 (GLP-1) (P = 0.006) in the cellulose group and a decrease in EI during ad libitum meal in both groups. CONCLUSIONS: Oligofructose increased plasma PYY concentrations and suppressed appetite, while cellulose increased GLP-1 concentrations. EI decreased in both groups. However, these positive effects did not translate into changes in BW or adiposity.

Hydrophilic interaction chromatography-mass spectrometry for anionic metabolic profiling of urine from antibiotic-treated rats.

Hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) was used for anionic metabolic profiling of urine from antibiotic-treated rats to study microbial-host co-metabolism. Rats were treated with the antibiotics penicillin G and streptomycin sulfate for four or eight days and compared to a control group. Urine samples were collected at day zero, four and eight, and analyzed by HILIC-MS. Multivariate data analysis was applied to the urinary metabolic profiles to identify biochemical variation between the treatment groups. Principal component analysis found a clear distinction between those animals receiving antibiotics and the control animals, with twenty-nine discriminatory compounds of which twenty were down-regulated and nine up-regulated upon treatment. In the treatment group receiving antibiotics for four days, a recovery effect was observed for seven compounds after cessation of antibiotic administration. Thirteen discriminatory compounds could be putatively identified based on their accurate mass, including aconitic acid, benzenediol sulfate, ferulic acid sulfate, hippuric acid, indoxyl sulfate, penicillin G, phenol and vanillin 4-sulfate. The rat urine samples had previously been analyzed by capillary electrophoresis (CE) with MS detection and proton nuclear magnetic resonance ((1)H NMR) spectroscopy. Using CE-MS and (1)H NMR spectroscopy seventeen and twenty-five discriminatory compounds were found, respectively. Both hippuric acid and indoxyl sulfate were detected across all three platforms. Additionally, eight compounds were observed with both HILIC-MS and CE-MS. Overall, HILIC-MS appears to be highly complementary to CE-MS and (1)H NMR spectroscopy, identifying additional compounds that discriminate the urine samples from antibiotic-treated and control rats.

Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS.

The first application of high field NMR spectroscopy (800 MHz for (1)H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz (1)H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional (1)H-(1)H TOCSY and (1)H-(13)C HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids (cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% (glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.