Oligofructose, 2'fucosyllactose and ß-glucan in combination induce specific changes in microbial composition and short-chain fatty acid production compared to sole supplementation.

AIMS: In this study, we explored the effects that the prebiotic inulin-type fructans, and prebiotic candidates: 2'fucosyllactose and ß-glucan from barley, singular and in combination had on microbial load, microbiome profile, and short-chain fatty acid production. This was carried out as a prescreening tool to determine combinations that could be taken forward for use in a human intervention trial. METHODS AND RESULTS: Effects of inulin-type fructans, 2'fucosyllactose and ß-glucan from barley in singular and combination on microbial load and profile and short-chain fatty acid production (SCFA) was conducted using in vitro batch culture fermentation over 48 h. Changes in microbial load and profile were assessed by fluorescence in situ hybridization flow cytometry (FISH-FLOW) and 16S rRNA sequencing, and changes in SCFA via gas chromatography. All substrates generated changes in microbial load and profile, achieving peak microbial load at 8 h fermentation with the largest changes in profile across all substrates in Bifidobacterium (Q < 0.05). This coincided with significant increases in acetate observed throughout fermentation (Q < 0.05). In comparison to sole supplementation combinations of oligofructose, ß-glucan and 2'fuscosyllactose induced significant increases in both propionate and butyrate producing bacteria (Roseburia and Faecalibacterium praunitzii), and concentrations of propionate and butyrate, the latter being maintained until the end of fermentation (all Q < 0.05). CONCLUSIONS: Combinations of oligofructose, with ß-glucan and 2'fucosyllactose induced selective changes in microbial combination and SCFA namely Roseburia, F. praunitzii, propionate and butyrate compared to sole supplementation.

Gamma aminobutyric acid production by commercially available probiotic strains.

AIMS: Certain bacteria can produce gamma aminobutyric acid (GABA) from glutamate in the human intestinal tract, leading to the possibility of altering GABA levels through diet. To this end, we assessed the ability of seven commercially available probiotic supplements to produce GABA. METHOD AND RESULTS: Probiotic strains were compared for GABA production in pure culture. The bacteria were inoculated at a concentration of 107 CFU ml-1 in 10 ml MRS supplemented with monosodium glutamate (1% w/v), both with and without oligofructose-enriched inulin (OFI) (1% w/v). Two strains with the highest production of GABA were further assessed for 48 h in pH-controlled anaerobic batch cultures inoculated with faecal bacteria. Liquid chromatography-mass spectrometry (LC-MS) was used for quantification of GABA and microbiota composition was determined through 16S rRNA gene sequencing. Levilactobacillus brevis LB01 (CGMCC 16921) and Lactiplantibacillus plantarum 299v (DSM 9843) were the most efficient producers of GABA. High GABA levels (28.32 mmol l-1 ± 0.29) were produced by the probiotic strain L. brevis LB01 at pH 5.4-5.6. This was significantly higher than the levels of GABA produced by L. plantarum (4.8 mmol l-1 ± 6.8) and a negative control (2.9 mM ± 3.1). The addition of OFI did not further stimulate GABA production under the conditions tested. The ability of these strains to produce GABA in-vitro was further evaluated in a faecal microbiota environment. Once again, L.brevis LB01 produced the highest levels of GABA (40.24 mmol l-1 ± 20.98). CONCLUSIONS: L. brevis LB01 was found to be the most efficient probiotic strain, of those tested, for GABA production.

Multi-Compartment Profiling of Bacterial and Host Metabolites Identifies Intestinal Dysbiosis and Its Functional Consequences in the Critically Ill Child.

OBJECTIVES: Adverse physiology and antibiotic exposure devastate the intestinal microbiome in critical illness. Time and cost implications limit the immediate clinical potential of microbial sequencing to identify or treat intestinal dysbiosis. Here, we examined whether metabolic profiling is a feasible method of monitoring intestinal dysbiosis in critically ill children. DESIGN: Prospective multicenter cohort study. SETTING: Three U.K.-based PICUs. PATIENTS: Mechanically ventilated critically ill (n = 60) and age-matched healthy children (n = 55). INTERVENTIONS: Collection of urine and fecal samples in children admitted to the PICU. A single fecal and urine sample was collected in healthy controls. MEASUREMENTS AND MAIN RESULTS: Untargeted and targeted metabolic profiling using 1H-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry or urine and fecal samples. This was integrated with analysis of fecal bacterial 16S ribosomal RNA profiles and clinical disease severity indicators. We observed separation of global urinary and fecal metabolic profiles in critically ill compared with healthy children. Urinary excretion of mammalian-microbial co-metabolites hippurate, 4-cresol sulphate, and formate were reduced in critical illness compared with healthy children. Reduced fecal excretion of short-chain fatty acids (including butyrate, propionate, and acetate) were observed in the patient cohort, demonstrating that these metabolites also distinguished between critical illness and health. Dysregulation of intestinal bile metabolism was evidenced by increased primary and reduced secondary fecal bile acid excretion. Fecal butyrate correlated with days free of intensive care at 30 days (r = 0.38; p = 0.03), while urinary formate correlated inversely with vasopressor requirement (r = -0.2; p = 0.037). CONCLUSIONS: Disruption to the functional activity of the intestinal microbiome may result in worsening organ failure in the critically ill child. Profiling of bacterial metabolites in fecal and urine samples may support identification and treatment of intestinal dysbiosis in critical illness.

Optimization and Application of Direct Infusion Nanoelectrospray HRMS Method for Large-Scale Urinary Metabolic Phenotyping in Molecular Epidemiology.

Large-scale metabolic profiling requires the development of novel economical high-throughput analytical methods to facilitate characterization of systemic metabolic variation in population phenotypes. We report a fit-for-purpose direct infusion nanoelectrospray high-resolution mass spectrometry (DI-nESI-HRMS) method with time-of-flight detection for rapid targeted parallel analysis of over 40 urinary metabolites. The newly developed 2 min infusion method requires <10 µL of urine sample and generates high-resolution MS profiles in both positive and negative polarities, enabling further data mining and relative quantification of hundreds of metabolites. Here we present optimization of the DI-nESI-HRMS method in a detailed step-by-step guide and provide a workflow with rigorous quality assessment for large-scale studies. We demonstrate for the first time the application of the method for urinary metabolic profiling in human epidemiological investigations. Implementation of the presented DI-nESI-HRMS method enabled cost-efficient analysis of >10 000 24 h urine samples from the INTERMAP study in 12 weeks and >2200 spot urine samples from the ARIC study in <3 weeks with the required sensitivity and accuracy. We illustrate the application of the technique by characterizing the differences in metabolic phenotypes of the USA and Japanese population from the INTERMAP study.

Mistargeting of peroxisomal EHHADH and inherited renal Fanconi's syndrome.

BACKGROUND: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. METHODS: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).

Development of a Pipeline for Exploratory Metabolic Profiling of Infant Urine.

Numerous metabolic profiling pipelines have been developed to characterize the composition of human biofluids and tissues, the vast majority of these being for studies in adults. To accommodate limited sample volume and to take into account the compositional differences between adult and infant biofluids, we developed and optimized sample handling and analytical procedures for studying urine from newborns. A robust pipeline for metabolic profiling using NMR spectroscopy was established, encompassing sample collection, preparation, spectroscopic measurement, and computational analysis. Longitudinal samples were collected from five infants from birth until 14 months of age. Methods of extraction and effects of freezing and sample dilution were assessed, and urinary contaminants from breakdown of polymers in a range of diapers and cotton wool balls were identified and compared, including propylene glycol, acrylic acid, and tert-butanol. Finally, assessment of urinary profiles obtained over the first few weeks of life revealed a dramatic change in composition, with concentrations of phenols, amino acids, and betaine altering systematically over the first few months of life. Therefore, neonatal samples require more stringent standardization of experimental design, sample handling, and analysis compared to that of adult samples to accommodate the variability and limited sample volume.

New technologies - new insights into the pathogenesis of hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome which frequently accompanies acute or chronic liver disease. It is characterized by a variety of symptoms of different severity such as cognitive deficits and impaired motor functions. Currently, HE is seen as a consequence of a low grade cerebral oedema associated with the formation of cerebral oxidative stress and deranged cerebral oscillatory networks. However, the pathogenesis of HE is still incompletely understood as liver dysfunction triggers exceptionally complex metabolic derangements in the body which need to be investigated by appropriate technologies. This review summarizes technological approaches presented at the ISHEN conference 2014 in London which may help to gain new insights into the pathogenesis of HE. Dynamic in vivo 13C nuclear magnetic resonance spectroscopy was performed to analyse effects of chronic liver failure in rats on brain energy metabolism. By using a genomics approach, microRNA expression changes were identified in plasma of animals with acute liver failure which may be involved in interorgan interactions and which may serve as organ-specific biomarkers for tissue damage during acute liver failure. Genomics were also applied to analyse glutaminase gene polymorphisms in patients with liver cirrhosis indicating that haplotype-dependent glutaminase activity is an important pathogenic factor in HE. Metabonomics represents a promising approach to better understand HE, by capturing the systems level metabolic changes associated with disease in individuals, and enabling monitoring of metabolic phenotypes in real time, over a time course and in response to treatment, to better inform clinical decision making. Targeted fluxomics allow the determination of metabolic reaction rates thereby discriminating metabolite level changes in HE in terms of production, consumption and clearance.

(1)H NMR Spectroscopy of Fecal Extracts Enables Detection of Advanced Colorectal Neoplasia.

Colorectal cancer (CRC) is a growing cause of mortality in developing countries, warranting investigation into its etiopathogenesis and earlier diagnosis. Here, we investigated the fecal metabolic phenotype of patients with advanced colorectal neoplasia and controls using (1)H-nuclear magnetic resonance (NMR) spectroscopy and multivariate modeling. The fecal microbiota composition was assessed by quantitative real-time PCR as well as Wif-1 methylation levels in stools, serum, and urine and correlated to the metabolic profile of each patient. The predictivity of the model was 0.507 (Q(2)Y), and the explained variance was 0.755 (R(2)Y). Patients with advanced colorectal neoplasia demonstrated increased fecal concentrations of four short-chain fatty acids (valerate, acetate, propionate, and butyrate) and decreased signals relating to ß-glucose, glutamine, and glutamate. The predictive accuracy of the multivariate (1)H NMR model was higher than that of the guaiac-fecal occult blood test and the Wif-1 methylation test for predicting advanced colorectal neoplasia. Correlation analysis between fecal metabolites and bacterial profiles revealed strong associations between Faecalibacterium prausnitzii and Clostridium leptum species with short-chain fatty acids concentration and inverse correlation between Faecalibacterium prausnitzii and glucose. These preliminary results suggest that fecal metabonomics may potentially have a future role in a noninvasive colorectal screening program and may contribute to our understanding of the role of these dysregulated molecules in the cross-talk between the host and its bacterial microbiota.

Metabolic Profiling of Children Undergoing Surgery for Congenital Heart Disease.

OBJECTIVE: Inflammation and metabolism are closely interlinked. Both undergo significant dysregulation following surgery for congenital heart disease, contributing to organ failure and morbidity. In this study, we combined cytokine and metabolic profiling to examine the effect of postoperative tight glycemic control compared with conventional blood glucose management on metabolic and inflammatory outcomes in children undergoing congenital heart surgery. The aim was to evaluate changes in key metabolites following congenital heart surgery and to examine the potential of metabolic profiling for stratifying patients in terms of expected clinical outcomes. DESIGN: Laboratory and clinical study. SETTING: University Hospital and Laboratory. PATIENTS: Of 28 children undergoing surgery for congenital heart disease, 15 underwent tight glycemic control postoperatively and 13 were treated conventionally. INTERVENTIONS: Metabolic profiling of blood plasma was undertaken using proton nuclear magnetic resonance spectroscopy. A panel of metabolites was measured using a curve-fitting algorithm. Inflammatory cytokines were measured by enzyme-linked immunosorbent assay. The data were assessed with respect to clinical markers of disease severity (Risk Adjusted Congenital heart surgery score-1, Pediatric Logistic Organ Dysfunction, inotrope score, duration of ventilation and pediatric ICU-free days). MEASUREMENTS AND MAIN RESULTS: Changes in metabolic and inflammatory profiles were seen over the time course from surgery to recovery, compared with the preoperative state. Tight glycemic control did not significantly alter the response profile. We identified eight metabolites (3-D-hydroxybutyrate, acetone, acetoacetate, citrate, lactate, creatine, creatinine, and alanine) associated with surgical and disease severity. The strength of proinflammatory response, particularly interleukin-8 and interleukin-6 concentrations, inversely correlated with PICU-free days at 28 days. The interleukin-6/interleukin-10 ratio directly correlated with plasma lactate. CONCLUSIONS: This is the first report on the metabolic response to cardiac surgery in children. Using nuclear magnetic resonance to monitor the patient journey, we identified metabolites whose concentrations and trajectory appeared to be associated with clinical outcome. Metabolic profiling could be useful for patient stratification and directing investigations of clinical interventions.

Discovery and validation of urinary metabotypes for the diagnosis of hepatocellular carcinoma in West Africans.

UNLABELLED: There is no clinically applicable biomarker for surveillance of hepatocellular carcinoma (HCC), because the sensitivity of serum alpha-fetoprotein (AFP) is too low for this purpose. Here, we determined the diagnostic performance of a panel of urinary metabolites of HCC patients from West Africa. Urine samples were collected from Nigerian and Gambian patients recruited on the case-control platform of the Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) program. Urinary proton nuclear magnetic resonance ((1) H-NMR) spectroscopy was used to metabolically phenotype 290 subjects: 63 with HCC; 32 with cirrhosis (Cir); 107 with noncirrhotic liver disease (DC); and 88 normal control (NC) healthy volunteers. Urine samples from a further cohort of 463 subjects (141 HCC, 56 Cir, 178 DC, and 88 NC) were analyzed, the results of which validated the initial cohort. The urinary metabotype of patients with HCC was distinct from those with Cir, DC, and NC with areas under the receiver operating characteristic (AUROC) curves of 0.86 (0.78-0.94), 0.93 (0.89-0.97), and 0.89 (0.80-0.98) in the training set and 0.81 (0.73-0.89), 0.96 (0.94-0.99), and 0.90 (0.85-0.96), respectively, in the validation cohort. A urinary metabolite panel, comprising inosine, indole-3-acetate, galactose, and an N-acetylated amino acid (NAA), showed a high sensitivity (86.9% [75.8-94.2]) and specificity (90.3% [74.2-98.0]) in the discrimination of HCC from cirrhosis, a finding that was corroborated in a validation cohort (AUROC: urinary panel = 0.72; AFP = 0.58). Metabolites that were significantly increased in urine of HCC patients, and which correlated with clinical stage of HCC, were NAA, dimethylglycine, 1-methylnicotinamide, methionine, acetylcarnitine, 2-oxoglutarate, choline, and creatine. CONCLUSION: The urinary metabotyping of this West African cohort identified and validated a metabolite panel that diagnostically outperforms serum AFP.

Multiplatform characterization of dynamic changes in breast milk during lactation.

The multicomponent analysis of human breast milk (BM) by metabolic profiling is a new area of study applied to determining milk composition, and is capable of associating BM composition with maternal characteristics, and subsequent infant health outcomes. A multiplatform approach combining HPLC-MS and ultra-performance LC-MS, GC-MS, CE-MS, and 1 H NMR spectroscopy was used to comprehensively characterize metabolic profiles from seventy BM samples. A total of 710 metabolites spanning multiple molecular classes were defined. The utility of the individual and combined analytical platforms was explored in relation to numbers of metabolites identified, as well as the reproducibility of the methods. The greatest number of metabolites was identified by the single phase HPLC-MS method, while CE-MS uniquely profiled amino acids in detail and NMR was the most reproducible, whereas GC-MS targeted volatile compounds and short chain fatty acids. Dynamic changes in BM composition were characterized over the first 3 months of lactation. Metabolites identified as altering in abundance over lactation included fucose, di- and triacylglycerols, and short chain fatty acids, known to be important for infant immunological, neurological, and gastrointestinal development, as well as being an important source of energy. This extensive metabolic coverage of the dynamic BM metabolome provides a baseline for investigating the impact of maternal characteristics, as well as establishing the impact of environmental and dietary factors on the composition of BM, with a focus on the downstream health consequences this may have for infants.

Oligofructose alone and in combination with 2'fucosyllactose induces physiologically relevant changes in γ-aminobutyric acid and organic acid production compared to sole 2'fucosyllactose supplementation: an in vitro study.

We explored the potential for the prebiotic oligofructose and prebiotic candidate 2'fucosyllactose, alone and in combination (50:50 blend) to induce physiologically relevant increases in neurotransmitter (γ-aminobutyric acid, serotonin, tryptophan, and dopamine) and organic acid (acetate, propionate, butyrate, lactate, and succinate) production as well as microbiome changes using anaerobic pH-controlled in vitro batch culture fermentations over 48 h. Changes in organic acid and neurotransmitter production were assessed by gas chromatography and liquid chromatography and, bacterial enumeration using fluorescence in situ hybridization, respectively. Both oligofructose and oligofructose/2'fucosyllactose combination fermentations induced physiologically relevant concentrations of γ-aminobutyric acid, acetate, propionate, butyrate, and succinate at completion (all P ≤ .05). A high degree of heterogeneity was seen amongst donors in both neurotransmitter and organic acid production in sole 2'FL fermentations suggesting a large responder/nonresponder status exists. Large increases in Bifidobacterium, Lactobacillus, and Bacteroides numbers were detected in oligofructose fermentation, smallest increases being detected in 2'fucosyllactose fermentation. Bacterial numbers in the combined oligofructose/2'fucosyllactose fermentation were closer to that of sole oligofructose. Our results indicate that oligofructose and oligofructose/2'fucosyllactose in combination have the potential to induce physiologically relevant increases in γ-aminobutyric and organic acid production along with offsetting the heterogenicity seen in response to sole 2'fucosyllactose supplementation.

Inulin-type fructans and short-chain fructooligosaccharides-their role within the food industry as fat and sugar replacers and texture modifiers-what needs to be considered!

Inulin and oligofructose are classes of prebiotics belonging to a group of nondigestible carbohydrates referred to as inulin-type fructans. While short-chain fructooligosaccharides are enzymatically synthesized from the hydrolysis and transglycosylation of sucrose. Inulin-type fructans and short-chain fructooligosaccharides act as carbon sources for selective pathways supporting digestive health including altering the composition of the gut microbiota along with improving transit time. Due to their physicochemical properties, inulin-type fructans and short-chain fructooligosaccharides have been widely used in the food industry as partial replacements for both fat and sugar. Yet, levels of replacement need to be carefully considered as it may result in changes to physical and sensory properties that could be detected by consumers. Furthermore, it has been reported depending on the processing parameters used during production that inulin-type fructans and short-chain fructooligosaccharides may or may not undergo structural alterations. Therefore, this paper reviews the role of inulin-type fructans and short-chain fructooligosaccharides within the food industry as fat and sugar replacers and texture modifiers, their impact on final sensory properties, and to what degree processing parameters are likely to impact their functional properties.

Inulin-type fructans and 2'fucosyllactose alter both microbial composition and appear to alleviate stress-induced mood state in a working population compared to placebo (maltodextrin): the EFFICAD Trial, a randomized, controlled trial.

BACKGROUND: There is increasing interest in the bidirectional relationship existing between the gut and brain and the effects of both oligofructose and 2'fucosyllactose to alter microbial composition and mood state. Yet, much remains unknown about the ability of oligofructose and 2'fucosyllactose to improve mood state via targeted manipulation of the gut microbiota. OBJECTIVES: We aimed to compare the effects of oligofructose and 2'fucosyllactose alone and in combination against maltodextrin (comparator) on microbial composition and mood state in a working population. METHODS: We conducted a 5-wk, 4-arm, parallel, double-blind, randomized, placebo-controlled trial in 92 healthy adults with mild-to-moderate levels of anxiety and depression. Subjects were randomized to oligofructose 8 g/d (plus 2 g/d maltodextrin); maltodextrin 10 g/d; oligofructose 8 g/d plus 2'fucosyllactose (2 g/d) or 2'fucosyllactose 2 g/d (plus 8 g/d maltodextrin). Changes in microbial load (fluorescence in situ hybridization-flow cytometry) and composition (16S ribosomal RNA sequencing) were the primary outcomes. Secondary outcomes included gastrointestinal sensations, bowel habits, and mood state parameters. RESULTS: There were significant increases in several bacterial taxa including Bifidobacterium, Bacteroides, Roseburia, and Faecalibacterium prausnitzii in both the oligofructose and oligofructose/2'fucosyllactose interventions (all P ≤ 0.05). Changes in bacterial taxa were highly heterogenous upon 2'fuscoyllactose supplementation. Significant improvements in Beck Depression Inventory, State Trait Anxiety Inventory Y1 and Y2, and Positive and Negative Affect Schedule scores and cortisol awakening response were detected across oligofructose, 2'fucosyllactose, and oligofructose/2'fucosyllactose combination interventions (all P ≤ 0.05). Both sole oligofructose and oligofructose/2'fuscosyllactose combination interventions outperformed both sole 2'fucosyllactose and maltodextrin in improvements in several mood state parameters (all P ≤ 0.05). CONCLUSION: The results of this study indicate that oligofructose and combination of oligofructose/2'fucosyllactose can beneficially alter microbial composition along with improving mood state parameters. Future work is needed to understand key microbial differences separating individual responses to 2'fucosyllactose supplementation. This trial was registered at clinicaltrials.gov as NCT05212545.

Impact of inorganic iron and haem on the human gut microbiota; An in vitro batch-culture approach.

Although iron is an essential nutrient for humans, as well as for almost all other organisms, it is poorly absorbed (~15%) from the diet such that most passes through the upper gut into the large intestine. The colonic microbiota is thus exposed to, and potentially influenced by, such residual iron which could have an impact on human health. The aim of the research described here is to determine how the major forms of dietary iron (inorganic iron and haem) influence metabolic activity and composition of the human gut microbiota by utilizing an in vitro parallel, pH-controlled anaerobic batch culture approach. Controlled iron provision was enabled by the design of a 'modified' low-iron gut-model medium whereby background iron content was reduced from 28 to 5 µM. Thus, the impact of both low and high levels of inorganic and haem iron (18-180 µM and 7.7-77 µM, respectively) could be explored. Gut-microbiota composition was determined using next generation sequencing (NGS) based community profiling (16S rRNA gene sequencing) and flow-fluorescent in situ hybridization (FISH). Metabolic-end products (organic acids) were quantified using gas chromatography (GC) and iron incorporation was estimated by inductively coupled plasma optical emission spectroscopy (ICP-OES). Results showed that differences in iron regime induced significant changes in microbiota composition when low (0.1% w/v) fecal inoculation levels were employed. An increase in haem levels from 7.7 to 77 µM (standard levels employed in gut culture studies) resulted in reduced microbial diversity, a significant increase in Enterobacteriaceae and lower short chain fatty acid (SCFA) production. These effects were countered when 18 µM inorganic iron was also included into the growth medium. The results therefore suggest that high-dietary haem may have a detrimental effect on health since the resulting changes in microbiota composition and SCFA production are indicators of an unhealthy gut. The results also demonstrate that employing a low inoculum together with a low-iron gut-model medium facilitated in vitro investigation of the relationship between iron and the gut microbiota.

Metabotyping of long-lived mice using 1H NMR spectroscopy.

Significant advances in understanding aging have been achieved through studying model organisms with extended healthy lifespans. Employing 1H NMR spectroscopy, we characterized the plasma metabolic phenotype (metabotype) of three long-lived murine models: 30% dietary restricted (DR), insulin receptor substrate 1 null (Irs1-/-), and Ames dwarf (Prop1df/df). A panel of metabolic differences were generated for each model relative to their controls, and subsequently, the three long-lived models were compared to one another. Concentrations of mobile very low density lipoproteins, trimethylamine, and choline were significantly decreased in the plasma of all three models. Metabolites including glucose, choline, glycerophosphocholine, and various lipids were significantly reduced, while acetoacetate, d-3-hydroxybutyrate and trimethylamine-N-oxide levels were increased in DR compared to ad libitum fed controls. Plasma lipids and glycerophosphocholine were also decreased in Irs1-/- mice compared to controls, as were methionine and citrate. In contrast, high density lipoproteins and glycerophosphocholine were increased in Ames dwarf mice, as were methionine and citrate. Pairwise comparisons indicated that differences existed between the metabotypes of the different long-lived mice models. Irs1-/- mice, for example, had elevated glucose, acetate, acetone, and creatine but lower methionine relative to DR mice and Ames dwarfs. Our study identified several potential candidate biomarkers directionally altered across all three models that may be predictive of longevity but also identified differences in the metabolic signatures. This comparative approach suggests that the metabolic networks underlying lifespan extension may not be exactly the same for each model of longevity and is consistent with multifactorial control of the aging process.

Urinary metabolic phenotyping the slc26a6 (chloride-oxalate exchanger) null mouse model.

The prevalence of renal stone disease is increasing, although it remains higher in men than in women when matched for age. While still somewhat controversial, several studies have reported an association between renal stone disease and hypertension, but this may be confounded by a shared link with obesity. However, independent of obesity, hyperoxaluria has been shown to be associated with hypertension in stone-formers, and the most common type of renal stone is composed of calcium oxalate. The chloride-oxalate exchanger slc26a6 (also known as CFEX or PAT-1), located in the renal proximal tubule, was originally thought to have an important role in sodium homeostasis and thereby blood pressure control, but it has recently been shown to have a key function in oxalate balance by mediating oxalate secretion in the gut. We have applied two orthogonal analytical platforms (NMR spectroscopy and capillary electrophoresis with UV detection) in parallel to characterize the urinary metabolic signatures related to the loss of the renal chloride-oxalate exchanger in slc26a6 null mice. Clear metabolic differentiation between the urinary profiles of the slc26a6 null and the wild type mice were observed using both methods, with the combination of NMR and CE-UV providing extensive coverage of the urinary metabolome. Key discriminating metabolites included oxalate, m-hydroxyphenylpropionylsulfate (m-HPPS), trimethylamine-N-oxide, glycolate and scyllo-inositol (higher in slc26a6 null mice) and hippurate, taurine, trimethylamine, and citrate (lower in slc26a6 null mice). In addition to the reduced efficiency of anion transport, several of these metabolites (hippurate, m-HPPS, methylamines) reflect alteration in gut microbial cometabolic activities. Gender-related metabotypes were also observed in both wild type and slc26a6 null groups. Urinary metabolites that showed a sex-specific pattern included trimethylamine, trimethylamine-N-oxide, citrate, spermidine, guanidinoacetate, and 2-oxoisocaproate. The gender-dependent metabolic expression of the consequences of slc26a6 deletion might have relevance to the difference in prevalence of renal stone formation in men and women. The different composition of microbial metabolites in the slc26a6 null mice is consistent with the fact that the slc26a6 transporter is found in a range of tissues, including the kidney and intestine, and provides further evidence for the "long reach" of the microbiota in physiological and pathological processes.

The Potential Role of Human Milk Oligosaccharides in Irritable Bowel Syndrome.

Irritable Bowel Syndrome (IBS) is the most common gastrointestinal (GI) disorder in Western populations and therefore a major public health/economic concern. However, despite extensive research, psychological and physiological factors that contribute to the aetiology of IBS remain poorly understood. Consequently, clinical management of IBS is reduced to symptom management through various suboptimal options. Recent evidence has suggested human milk oligosaccharides (HMOs) as a potential therapeutic option for IBS. Here, we review literature concerning the role of HMOs in IBS, including data from intervention and in vitro trials. HMO supplementation shows promising results in altering the gut microbiota and improving IBS symptoms, for instance by stimulating bifidobacteria. Further research in adults is required into HMO mechanisms, to confirm the preliminary results available to date and recommendations of HMO use in IBS.

Profiling gut microbiota and bile acid metabolism in critically ill children.

Broad-spectrum antimicrobial use during the treatment of critical illness influences gastrointestinal fermentation endpoints, host immune response and metabolic activity including the conversion of primary to secondary bile acids. We previously observed reduced fermentation capacity in the faecal microbiota of critically ill children upon hospital admission. Here, we further explore the timecourse of the relationship between the microbiome and bile acid profile in faecal samples collected from critically ill children. The microbiome was assayed by sequencing of the 16S rRNA gene, and faecal water bile acids were measured by liquid chromatography mass spectrometry. In comparison to admission faecal samples, members of the Lachnospiraceae recovered during the late-acute phase (days 8-10) of hospitalisation. Patients with infections had a lower proportion of Lachnospiraceae in their gut microbiota than controls and patients with primary admitting diagnoses. Keystone species linked to ecological recovery were observed to decline with the length of PICU admission. These species were further suppressed in patients with systemic infection, respiratory failure, and undergoing surgery. Bile acid composition recovers quickly after intervention for critical illness which may be aided by the compositional shift in Lachnospiraceae. Our findings suggest gut microbiota recovery can be readily assessed via measurement of faecal bile acids.

Relation of urinary calcium and magnesium excretion to blood pressure: The International Study Of Macro- And Micro-nutrients And Blood Pressure and The International Cooperative Study On Salt, Other Factors, And Blood Pressure.

Data indicate an inverse association between dietary calcium and magnesium intakes and blood pressure (BP); however, much less is known about associations between urinary calcium and magnesium excretion and BP in general populations. The authors assessed the relation of BP to 24-hour excretion of calcium and magnesium in 2 cross-sectional studies. The International Study of Macro- and Micro-Nutrients and Blood Pressure (INTERMAP) comprised 4,679 persons aged 40-59 years from 17 population samples in China, Japan, the United Kingdom, and the United States, and the International Cooperative Study on Salt, Other Factors, and Blood Pressure (INTERSALT) comprised 10,067 persons aged 20-59 years from 52 samples around the world. Timed 24-hour urine collections, BP measurements, and nutrient data from four 24-hour dietary recalls (INTERMAP) were collected. In multiple linear regression analyses, urinary calcium excretion was directly associated with BP. After adjustment for multiple confounders (including weight, height, alcohol intake, calcium intake, urinary sodium level, and urinary potassium intake), systolic BP was 1.9 mm Hg higher per each 4.1 mmol per 24 hours (2 standard deviations) of higher urinary calcium excretion (associations were smaller for diastolic BP) in INTERMAP. Qualitatively similar associations were observed in INTERSALT analyses. Associations between magnesium excretion and BP were small and nonsignificant for most of the models examined. The present data suggest that altered calcium homoeostasis, as exhibited by increased calcium excretion, is associated with higher BP levels.