Aspartame sensitivity? : a double blind randomised crossover study

Background Aspartame is a commonly used intense artificial sweetener, being approximately 200 times sweeter than sucrose. There have been concerns over aspartame since approval in the 1980s including a large anecdotal database reporting severe symptoms. The objective of this study was to compare the acute symptom effects of aspartame to a control preparation. Methods This was a double-blind randomized cross over study conducted in a clinical research unit in United Kingdom. Forty-eight individual who has self reported sensitivity to aspartame were compared to 48 age and gender matched aspartame non-sensitive individuals. They were given aspartame (100mg)-containing or control snack bars randomly at least 7 days apart. The main outcome measures were acute effects of aspartame measured using repeated ratings of 14 symptoms, biochemistry and metabonomics. Results Aspartame sensitive and non-sensitive participants differed psychologically at baseline in handling feelings and perceived stress. Sensitive participants had higher triglycerides (2.05 ± 1.44 vs. 1.26 ± 0.84mmol/L; p value 0.008) and lower HDL-C (1.16 ± 0.34 vs. 1.35 ± 0.54 mmol/L; p value 0.04), reflected in 1H NMR serum analysis that showed differences in the baseline lipid content between the two groups. Urine metabonomic studies showed no significant differences. None of the rated symptoms differed between aspartame and control bars, or between sensitive and control participants. However, aspartame sensitive participants rated more symptoms particularly in the first test session, whether this was placebo or control. Aspartame and control bars affected GLP-1, GIP, tyrosine and phenylalanine levels equally in both aspartame sensitive and non-sensitive subjects. Conclusion Using a comprehensive battery of psychological tests, biochemistry and state of the art metabonomics there was no evidence of any acute adverse responses to aspartame. This independent study gives reassurance to both regulatory bodies and the public that acute ingestion of aspartame does not have any detectable psychological or metabolic effects in humans

Autotaxin, bile acid profile and effect of ileal bile acid transporter inhibition in primary biliary cholangitis patients with pruritus

Background and Aims Pruritus is a common symptom in patients with primary biliary cholangitis (PBC) for which ileal bile acid transporter (IBAT) inhibition is emerging as a potential therapy. We explored the serum metabonome and gut microbiota profile in PBC patients with pruritus and investigated the effect of GSK2330672, an IBAT inhibitor. Methods We studied fasting serum bile acids (BAs), autotaxin and faecal microbiota in 22 PBC patients with pruritus at baseline and after 2 weeks of GSK2330672 treatment. Control group included 31 asymptomatic PBC patients and 18 healthy volunteers. BA profiling was done by ultra performance liquid chromatography coupled to a mass spectrometry (UPLC‐MS). Faecal microbiomes were analysed by 16S ribosomal RNA gene sequencing. Results \ud In PBC patients with pruritus, serum levels of total and glyco‐conjugated primary BAs and autotaxin were significantly elevated. Autotaxin activity correlated significantly with tauro‐ and glyco‐conjugated cholic acid (CA) and chenodeoxycholic acid (CDCA), both at baseline and after GSK2330672. GSK2330672 significantly reduced autotaxin and all tauro‐ and glyco‐ conjugated BAs and increased faecal levels of CA (P = 0.048) and CDCA (P = 0.027). Gut microbiota of PBC patients with pruritus was similar to control groups. GSK2330672 increased the relative abundance of Firmicutes (P = 0.033) and Clostridia (P = 0.04) and decreased Bacteroidetes (P = 0.033) and Bacteroidia (P = 0.04). Conclusions Pruritus in PBC does not show a distinct gut bacterial profile but is associated with elevated serum bile acid and autotaxin levels which decrease after IBAT inhibition. In cholestatic pruritus, a complex interplay between BAs and autotaxin is likely and may be modified by IBAT inhibition

Bacterial and metabolic phenotypes associated with inadequate response to ursodeoxycholic acid treatment in primary biliary cholangitis

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease with ursodeoxycholic acid (UDCA) as first-line treatment. Poor response to UDCA is associated with a higher risk of progressing to cirrhosis, but the underlying mechanisms are unclear. UDCA modulates the composition of primary and bacterial-derived bile acids (BAs). We characterized the phenotypic response to UDCA based on BA and bacterial profiles of PBC patients treated with UDCA. Patients from the UK-PBC cohort (n = 419) treated with UDCA for a minimum of 12-months were assessed using the Barcelona dynamic response criteria. BAs from serum, urine, and feces were analyzed using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry and fecal bacterial composition measured using 16S rRNA gene sequencing. We identified 191 non-responders, 212 responders, and a subgroup of responders with persistently elevated liver biomarkers (n = 16). Responders had higher fecal secondary and tertiary BAs than non-responders and lower urinary bile acid abundances, with the exception of 12-dehydrocholic acid, which was higher in responders. The sub-group of responders with poor liver function showed lower alpha-diversity evenness, lower abundance of fecal secondary and tertiary BAs than the other groups and lower levels of phyla with BA-deconjugation capacity (Actinobacteriota/Actinomycetota, Desulfobacterota, Verrucomicrobiota) compared to responders. UDCA dynamic response was associated with an increased capacity to generate oxo-/epimerized secondary BAs. 12-dehydrocholic acid is a potential biomarker of treatment response. Lower alpha-diversity and lower abundance of bacteria with BA deconjugation capacity might be associated with an incomplete response to treatment in some patients

A two-way interaction between methotrexate and the gut microbiota of male Sprague Dawley rats

Methotrexate (MTX) is a chemotherapeutic agent that can cause a range of toxic side effects including gastrointestinal damage, hepatotoxicity, myelosuppression, and nephrotoxicity and has potentially complex interactions with the gut microbiome. Following untargeted UPLC-qtof-MS analysis of urine and fecal samples from male Sprague–Dawley rats administered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependent changes in the endogenous metabolite profiles were detected. Semiquantitative targeted UPLC-MS detected MTX excreted in urine as well as MTX and two metabolites, 2,4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxy-MTX, in the feces. DAMPA is produced by the bacterial enzyme carboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling (16S rRNA gene amplicon sequencing) of fecal samples showed an increase in the relative abundance of Firmicutes over the Bacteroidetes at low doses of MTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h, which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to induce community and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thus may delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinal toxicity

Inflammatory bowel disease outcomes following fecal microbiota transplantation for recurrent C. difficile infection

Background Recurrent Clostridioides difficile infection (CDI) in patients with inflammatory bowel disease (IBD) is a clinical challenge. Fecal microbiota transplantation (FMT) has emerged as a recurrent CDI therapy. Anecdotal concerns exist regarding worsening of IBD activity; however, prospective data among IBD patients are limited. Methods Secondary analysis from an open-label, prospective, multicenter cohort study among IBD patients with 2 or more CDI episodes was performed. Participants underwent a single FMT by colonoscopy (250 mL, healthy universal donor). Secondary IBD-related outcomes included rate of de novo IBD flares, worsening IBD, and IBD improvement—all based on Mayo or Harvey-Bradshaw index (HBI) scores. Stool samples were collected for microbiome and targeted metabolomic profiling. Results Fifty patients enrolled in the study, among which 15 had Crohn’s disease (mean HBI, 5.8 ± 3.4) and 35 had ulcerative colitis (mean partial Mayo score, 4.2 ± 2.1). Overall, 49 patients received treatment. Among the Crohn’s disease cohort, 73.3% (11 of 15) had IBD improvement, and 4 (26.6%) had no disease activity change. Among the ulcerative colitis cohort, 62% (22 of 34) had IBD improvement, 29.4% (11 of 34) had no change, and 4% (1 of 34) experienced a de novo flare. Alpha diversity significantly increased post-FMT, and ulcerative colitis patients became more similar to the donor than Crohn’s disease patients (P = 0.04). Conclusion This prospective trial assessing FMT in IBD-CDI patients suggests IBD outcomes are better than reported in retrospective studies

Fecal microbiota and bile acid interactions with systemic and adipose tissue metabolism in diet-induced weight loss of obese postmenopausal women

Microbiota and bile acids in the gastrointestinal tract profoundly alter systemic metabolic processes. In obese subjects, gradual weight loss ameliorates adipose tissue inflammation and related systemic changes. We assessed how rapid weight loss due to a very low calorie diet (VLCD) affects the fecal microbiome and fecal bile acid composition, and their interactions with the plasma metabolome and subcutaneous adipose tissue inflammation in obesity. We performed a prospective cohort study of VLCD-induced weight loss of 10% in ten grades 2-3 obese postmenopausal women in a metabolic unit. Baseline and post weight loss evaluation included fasting plasma analyzed by mass spectrometry, adipose tissue transcription by RNA sequencing, stool 16S rRNA sequencing for fecal microbiota, fecal bile acids by mass spectrometry, and urinary metabolic phenotyping by H-NMR spectroscopy. Outcome measures included mixed model correlations between changes in fecal microbiota and bile acid composition with changes in plasma metabolite and adipose tissue gene expression pathways. Alterations in the urinary metabolic phenotype following VLCD-induced weight loss were consistent with starvation ketosis, protein sparing, and disruptions to the functional status of the gut microbiota. We show that the core microbiome was preserved during VLCD-induced weight loss, but with changes in several groups of bacterial taxa with functional implications. UniFrac analysis showed overall parallel shifts in community structure, corresponding to reduced abundance of the genus Roseburia and increased Christensenellaceae;g__ (unknown genus). Imputed microbial functions showed changes in fat and carbohydrate metabolism. A significant fall in fecal total bile acid concentration and reduced deconjugation and 7-α-dihydroxylation were accompanied by significant changes in several bacterial taxa. Individual bile acids in feces correlated with amino acid, purine, and lipid metabolic pathways in plasma. Furthermore, several fecal bile acids and bacterial species correlated with altered gene expression pathways in adipose tissue. VLCD dietary intervention in obese women changed the composition of several fecal microbial populations while preserving the core fecal microbiome. Changes in individual microbial taxa and their functions correlated with variations in the plasma metabolome, fecal bile acid composition, and adipose tissue transcriptome

Implementation of holistic analytical technologies for the study of the effect of exercise in metabolic profile

Exercise and regular physical activity, being powerful modifiers of animal metabolism, exertnumerous beneficial effects, including reduced risk of diseases, beneficial effects on already existingdiseases and even enhanced longevity. So far, biochemichal studies on the effects of exercisefocused mainly on a small number of molecules in specific, well-characterized biochemical pathways.In this study we tried to incorporate the non-targeted holistic approach of metabonomics in the fieldof exercise biochemistry. Metabonomics, having matured over the last few years are now apowerfull tool in the investigation of the effects of a series of perturbations in a number of biologicalsystems. NMR, LC-MS and GC-MS, along with developments in data extraction and handling, are thehorsepower in the quest for a better view of systems biology through metabonomics.In the first study of the present thesis, the effects of exhaustive exercise, in our case swimming, onrat metabolism were investigated. Blood plasma and liver samples were analyzed through LC-MS,and blood and muscle samples were analyzed through biochemical assays. Nonetheless, due to poorexperimental design, the study was abandoned since the results of the preliminary phases werediscouraging.In the second study, 1H NMR-based metabonomics provided useful information for theunderstanding of metabolic changes induced by specific training schedules. Urine samples obtainedafter exercise protocols differing in as little as the rest interval between repeated sprints, not thesprints themselves, can be classified and safely predicted even when applying unsupervisedstatistical methods of analysis. In this way, important biomolecules involved in exercise biochemistrycan be identified and further studied. Separation of pre- from post-exercise samples was attributedmainly to lactate, pyruvate, hypoxanthine, compounds of the Krebs cycle, amino acids, products ofBCAA catabolism, 2-hydroxybutyrate, and hippurate. Most of these metabolites increased in urinewith exercise and have been described to also increase in muscle with exercise. Separation of the 10s from the 1 min rest interval was attributed mainly to lactate, pyruvate, alanine, compounds of theKrebs cycle, 2-oxoacids of BCAA, and 2-hydroxybutyrate. All of these metabolites increased morewith the short compared to the long interval, thus supporting the hypothesis that the former elicitedgreater metabolic disturbances than the latter as a result of the very limited time available forrecovery. The facts that such methodology can be applied to urine, a biological material readilyavailable and noninvasively obtained, and that urine reflects many of the exercise-induced changesoccurring in muscle are additional advantages. The third study, being an extension of the previous work on urine, reaffirms the power of themetabonomic approach in the study of exercise biochemistry. Serum samples acquired pre- andpost-exercise, as well as pre- and post-training, enabled us to classify and predict the effects ofexercise and training by using multivariate chemometric techniques. Separation of serum samplesaccording to the rest interval employed between repeated sprints was not possible (contrary to ourfindings in urine), possibly because serum is an equilibrium-regulated biofluid where differences inmetabolite concentrations can be mitigated by metabolite transitions between different tissues. Themain metabolites increasing after exercise were lactate, pyruvate, alanine, and an unidentifiedmetabolite resonating at 8.17 ppm. Lower concentrations after exercise were exhibited by leucine,valine, isoleucine, arginine/lysine and glycoprotein acetyls. Training induced increases inmethylguanidine, citrate, glucose, valine, taurine, trimethylamine N-oxide, choline-containingcompounds, histidines (including histidine, 1-methylhistidine, and 3-methylhistidine), andacetoacetate/acetone. Lactate, pyruvate, glycoprotein acetyls, and lipids (in general) decreased aftertraining. These findings provide further evidence for the utility of metabonomics in providing aholistic view of the short- and long-term impact of exercise on human metabolism and in identifyingnovel biomarkers of the body’s response to exercise.The fourth study describes the development of a GC-MS protocol for the untargeted analysis ofblood plasma to serve in the metabolomic study of the effects of exercise and allopurinoladministration. We describe a strategy for data treatment that employs advanced software utilities(GAVIN, Fiehn spectral library), followed by thorough statistical analysis to ensure the validity of thefindings. The current state of the art in untargeted analysis and especially in GC-MS, which employssample derivatisation, necessitates meticulous data treatment and utilization of different validationtools such as the use of QC samples. Following this approach, a number of metabolites were foundto be major differentiators between the different sample groups. Lactic and pyruvic acids, indicatingincreased carbohydrate breakdown, as well as 2-hydroxybutyric and pyroglutamic acids, indicatingincreased glutathione synthesis in response to oxidative stress, were among the majordifferentiators of exercise from the resting state. Inosine, hypoxanthine, xanthine, xanthosine anduric acid, indicating xanthine oxidase inhibition, as well as methionine, proline and leucine, indicatingincreased protein synthesis, were among the major differentiators of allopurinol administration fromplacebo. Finally, the metabolic responses to exercise were not affected by allopurinoladministration.By using holistic metabonomic analysis with LC-MS, we were able to demonstrate the repeatabilityof the metabolic fingerprint of urine before and after two swimming tests at maximum intensity. Our findings are in partial agreement with the findings of a previous work with the same samples, wherebiochemical assays for lactic acid in both the urine and the blood of athletes showed significantrepeatability in the 3 x 100 m test but not the 6 x 50 m test. Another characteristic was the inabilityto separate samples from the two different exercise protocols. This can lead to the conclusion thatthe two protocols did not cause different changes in the metabolic profile. The energy sources usedfor the two protocols are very similar, mainly from the lactic acid energy system. Also the restintervals between successive swims (5 and 10 minutes) is deemed to be sufficient to replenish theenergy systems and so each new effort starts from the same energy level.In the last study we utilized RP-UPLC-MS and NMR to study the effect of acute exercise on the urinemetabolome of young untrained men. The exercise was performed in two identical test sessions,spaced at least 3 days apart. We were able to confirm the reproducibility of these tests and todetect, identify and monitor the concentrations of several metabolites for a period of two hoursafter exercise. With both platforms we were able to identify compounds of the Krebs cycle, BCAAcatabolism, aminoacids and purine metabolism as major discriminators of exercise from rest.Interestingly, hypoxanthine was the only compound showing elevated values two hours after theend of the exercise

Monitoring the Response of the Human Urinary Metabolome to Brief Maximal Exercise by a Combination of RP-UPLC-MS and ¹H NMR Spectroscopy

The delineation of exercise biochemistry by utilizing metabolic fingerprinting has become an established strategy. We present a combined RP-UPLC-MS and ¹H NMR strategy, supplemented by photometric assays, to monitor the response of the human urinary metabolome to short maximal exercise. Seventeen male volunteers performed two identical sprint sessions on separate days, consisting of three 80 m maximal runs. Using univariate and multivariate analyses, we followed the fluctuation of 37 metabolites at 1, 1.5, and 2 h postexercise. 2-Hydroxyisovalerate, 2-hydroxybutyrate, 2-oxoisocaproate, 3-methyl-2-oxovalerate, 3-hydroxyisobutyrate, 2-oxoisovalerate, 3-hydroxybutyrate, 2-hydroxyisobutyrate, alanine, pyruvate, and fumarate increased 1 h postexercise and then returned toward baseline. Lactate and acetate were higher than baseline at 1 and 1.5 h. Hypoxanthine and inosine remained above baseline throughout the postexercise period. Urate decreased at 1 h and increased at 1.5 h before returning to baseline. Valine, isoleucine, succinate, citrate, trimethylamine, trimethylamine <i>N</i>-oxide, tyrosine, and formate decreased at 1 h and/or 1.5 h postexercise and then returned to baseline. Creatinine gradually decreased over the sampling period. Glycine, 4-aminohippurate, and hippurate remained below baseline throughout the postexercise period. Our findings show that even one-half minute of maximal exercise elicited major perturbations in human metabolism, several of which persisted for at least 2 h