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Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts that has been associated with diverse metabolic carboxylic acids. Mass spectrometric techniques are the method of choice for their analysis. However, the broad investigation of this metabolite class remains challenging. Derivatization of carboxylic acids represents a strategy to overcome these limitations but available methods suffer from diverse analytical challenges. Herein, we have designed a novel strategy introducing 4-nitrophenyl-2H-azirine as a new chemoselective moiety for the first time for carboxylic acid metabolites. This moiety was selected as it rapidly forms a stable amide bond and also generates a new ketone, which can be analyzed by our recently developed quant-SCHEMA method specific for carbonyl metabolites. Optimization of this new method revealed a high reproducibility and robustness, which was utilized to validate 102 metabolic carboxylic acids using authentic synthetic standard conjugates in human plasma samples including nine metabolites that were newly detected. Using this sequential analysis of the carbonyl- and carboxylic acid-metabolomes revealed alterations of the ketogenesis pathway, which demonstrates the vast benefit of our unique methodology. We anticipate that the developed azirine moiety with rapid functional group transformation will find broad application in diverse chemical biology research fields.

Ketamine is a common anesthetic used in human and veterinary medicine. This drug has recently received increased medical and scientific attention due to its indications for neurological diseases. Despite being applied for decades, ketamine's entire metabolism and pharmacological profile have not been elucidated yet. Therefore, insights into the metabolism and brain distribution are important toward identification of neurological effects. Herein, we have investigated ketamine and its metabolites in the pig brain, cerebrospinal fluid, and plasma using mass spectrometric and metabolomics analysis. We discovered previously unknown metabolites and validated their chemical structures. Our comprehensive analysis of the brain distribution of ketamine and 30 metabolites describes significant regional differences detected mainly for phase II metabolites. Elevated levels of these metabolites were identified in brain regions linked to clearance through the cerebrospinal fluid. This study provides the foundation for multidisciplinary studies of ketamine metabolism and the elucidation of neurological effects by ketamine.

Multidrug-resistant microorganisms have become a major public health concern around the world. The gut microbiome is a gold mine for bioactive compounds that protect the human body from pathogens. We used a multi-omics approach that integrated whole-genome sequencing (WGS) of 74 commensal gut microbiome isolates with metabolome analysis to discover their metabolic interaction with Salmonella and other antibiotic-resistant pathogens. We evaluated differences in the functional potential of these selected isolates based on WGS annotation profiles. Furthermore, the top altered metabolites in co-culture supernatants of selected commensal gut microbiome isolates were identified including a series of dipeptides and examined for their ability to prevent the growth of various antibiotic-resistant bacteria. Our results provide compelling evidence that the gut microbiome produces metabolites, including the compound class of dipeptides that can potentially be applied for anti-infection medication, especially against antibiotic-resistant pathogens. Our established pipeline for the discovery and validation of bioactive metabolites from the gut microbiome as novel candidates for multidrug-resistant infections represents a new avenue for the discovery of antimicrobial lead structures.

Background and Aims To advance the understanding of inflammatory bowel disease [IBD] pathophysiology, we compared the mucosal and plasma metabolomes between new-onset paediatric IBD patients and symptomatic non-IBD controls, and correlated plasma inflammation markers and disease characteristics with the altered metabolites. Methods Paired colonic and ileal biopsies and plasma from 67 treatment-naïve children with incident Crohn’s disease [CD; n = 47], ulcerative colitis [UC; n = 9], and non-IBD controls [n = 11] were analysed using ultra-performance liquid chromatography-mass spectrometry [UPLC-MS/MS]. Inflammatory plasma proteins [n = 92] were assessed. Results The metabolomes in inflamed mucosal biopsies differed between IBD patients and controls. In CD, mucosal levels of several lysophospholipids [lysophosphatidylcholines, lysophosphatidyletanolamines, lysophosphatidylinositols, and lysophosphatidylserines] were decreased, correlating with various plasma metabolites including amino acid analogues and N-acetylated compounds. In both CD and UC, mucosal sphingolipids, including ceramide [d18:2/24:1, d18:1/24:2], lactosyl-N-palmitoyl-sphingosine [d18:1/16:0], behenoyl sphingomyelin [d18:1/22:0], lignoceroyl sphingomyelin [d18:1/24:0], and/or sphingomyelin [d18:1/24:1, d18:2/24:0] were increased, correlating with sphingolipids, bile acids, and/or N-acetylated metabolites in plasma. Among proteins associated with CD, interleukin-24 correlated with plasma metabolites, including lactosyl-N-palmitoyl sphingosine [d18:1/16:0] and phosphatidyletanolamine [18:1/18:1], haemoglobin, and faecal calprotectin. In UC, interleukin-24, interleukin-17A, and C-C motif chemokine 11 correlated with several plasma metabolites, including N-acetyltryptophan, tryptophan, glycerate, and threonate, and with the Paediatric Ulcerative Colitis Activity Index, C-reactive protein, and faecal calprotectin. Conclusions Mucosal perturbations of lysophospholipids and sphingolipids characterised the me

Sulfur is an essential element of life. Thiol-containing metabolites in all organisms are involved in the regulation of diverse biological processes. Especially, the microbiome produces bioactive metabolites or biological intermediates of this compound class. The analysis of thiol-containing metabolites is challenging due to the lack of specific tools, making these compounds difficult to investigate selectively. We have now developed a new methodology comprising bicyclobutane for chemoselective and irreversible capturing of this metabolite class. We utilized this new chemical biology tool immobilized onto magnetic beads for the investigation of human plasma, fecal samples, and bacterial cultures. Our mass spectrometric investigation detected a broad range of human, dietary and bacterial thiol-containing metabolites and we even captured the reactive sulfur species cysteine persulfide in both fecal and bacterial samples. The described comprehensive methodology represents a new mass spectrometric strategy for the discovery of bioactive thiol-containing metabolites in humans and the microbiome.

The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin., De två första författarna delar förstaförfattarskapet.

The human body hasevolved to remove xenobiotics through a multistepclearance process. Non-endogenous metabolites are converted througha series of phase I and different phase II enzymes into compoundswith higher hydrophilicity. These compounds are important for diverseresearch fields such as toxicology, nutrition, biomarker discovery,doping control, and microbiome metabolism. One of the challenges inthese research fields has been the investigation of the two majorphase II modifications, sulfation and glucuronidation, and the correspondingunconjugated aglycon independently. We have now developed a new methodologyutilizing an immobilized arylsulfatase and an immobilized & beta;-glucuronidaseto magnetic beads for treatment of human urine samples. The enzymeactivities remained the same compared to the enzyme in solution. Theseparate mass spectrometric investigation of each metabolite classin a single sample was successfully applied to obtain the dietaryglucuronidation and sulfation profile of 116 compounds. Our new chemicalbiology strategy provides a new tool for the investigation of metabolitesin biological samples with the potential for broad-scale applicationin metabolomics, nutrition, and microbiome studies.

The microbiome has been identified to have a key role for the physiology of their human host. One of the major impacts is the clearance of bacterial pathogens. We have now developed a chemoselective probe methodology for the absolute quantification of short-chain fatty acids at low nM concentrations, with high reproducibility and spiked isotope labelled internal standards. Immobilization to magnetic beads allows for separation from the matrix and the tagged metabolites upon bioorthogonal cleavage can be analyzed via UHPLC-MS. The major advantage of our sensitive method is the simple combination with global metabolomics analysis as only a small sample volume is required. We have applied this chemical metabolomics strategy for targeted SCFA analysis combined with global metabolomics on gut microbiome co-cultures with Salmonella and investigated the effect of antibiotic treatment.

Sleep is a state in which important restorative and anabolic processes occur. Understanding changes of these metabolic processes during the circadian rhythm in the brain is crucial to elucidate neurophysiological mechanisms important for sleep function. Investigation of amino acid modifications and dipeptides has recently emerged as a valuable approach in the metabolic profiling of the central nervous system. Nonetheless, very little is known about the effects of sleep on the brain levels of amino acid analogues. In the present study, we examined brain regional sleep-induced alterations selective for modified amino acids and dipeptides using Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) based metabolomics. Our approach enabled the detection and identification of numerous amino acid-containing metabolites in the cortex, the hippocampus, the midbrain, and the cerebellum. In particular, analogues of the aromatic amino acids phenylalanine, tyrosine and tryptophan were significantly altered during sleep in the investigated brain regions. Cortical levels of medium and long chain N-acyl glycines were higher during sleep. Regional specific changes were also detected, especially related to tyrosine analogues in the hippocampus and the cerebellum. Our findings demonstrate a strong correlation between circadian rhythms and amino acid metabolism specific for different brain regions that provide previously unknown insights in brain metabolism.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and adults in endemic areas. Gene regulation of ETEC during growth in vitro and in vivo needs to be further evaluated, and here we describe the full transcriptome and metabolome of ETEC during growth from mid-logarithmic growth to early stationary phase in rich medium (LB medium). We identified specific genes and pathways subjected to rapid transient alterations in gene expression and metabolite production during the transition from logarithmic to stationary growth. The transient phase was found to be different from the subsequent induction of early stationary phase-induced genes. The transient phase was characterized by the repression of genes and metabolites involved in organic substance transport. Genes involved in fucose and putrescine metabolism were upregulated, and genes involved in iron transport were repressed. Expression of toxins and colonization factors were not changed, suggesting retained virulence from mid-logarithmic to the start of the stationary phase. Metabolomic analyses showed that the transient phase was characterized by a drop of intracellular amino acids, e.g., l-tyrosine, l-tryptophan, l-phenylalanine, l-leucine, and l-glutamic acid, followed by increased levels at induction of stationary phase. A pathway enrichment analysis of the entire combined transcriptome and metabolome revealed that significant pathways during progression from logarithmic to early stationary phase are involved in the degradation of neurotransmitters aminobutyrate (GABA) and precursors of 5-hydroxytryptamine (serotonin). This work provides a comprehensive framework for further studies on transcriptional and metabolic regulation in pathogenic E. coli. IMPORTANCE We show that E. coli, exemplified by the pathogenic subspecies enterotoxigenic E. coli (ETEC), undergoes a stepwise transcriptional and metabolic transition into the stationary phase. At a specific entry point, E. coli induces activ

The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

The investigation of microbiome-derived metabolites is important to understand metabolic interactions with their human host. New methodologies for mass spectrometric discovery of undetected metabolites with unknown bioactivity are required. Herein, we introduce squaric acid as a new chemoselective moiety for amine metabolite analysis in human fecal samples.

The gut microbiome converts dietary compounds that are absorbed in the gastrointestinal tract and further metabolized by the human host. Sulfated metabolites are a major compound class derived from this co-metabolism and have been linked to disease development. In the present multidisciplinary study, we have investigated human urine samples from a dietary intervention study with 22 individuals collected before and after consumption of a polyphenol rich breakfast. These samples were analyzed utilizing our method combining enzymatic metabolite hydrolysis using an arylsulfatase and mass spectrometric metabolomics. Key to this study is the validation of 235 structurally diverse sulfated metabolites. We have identified 48 significantly upregulated metabolites upon dietary intervention including 11 previously unknown sulfated metabolites for this diet. We observed a large variation in subjects based on their potential to sulfate metabolites, which may be the foundation for classification of subjects as high and low sulfate metabolizers in future large cohort studies. The reported sulfatase-based method is a robust tool for the discovery of unknown microbiota-derived metabolites in human samples.

Metabolomics analysis of biological samples is widely applied in medical and natural sciences. Assigning the correct chemical structure in the metabolite identification process is required to draw the correct biological conclusions and still remains a major challenge in this research field. Several metabolite tandem mass spectrometry (MS/MS) fragmentation spectra libraries have been developed that are either based on computational methods or authentic libraries. These libraries are limited due to the high number of structurally diverse metabolites, low commercial availability of these compounds, and the increasing number of newly discovered metabolites. Phase II modification of xenobiotics is a compound class that is underrepresented in these databases despite their importance in diet, drug, or microbiome metabolism. The O-sulfated metabolites have been described as a signature for the co-metabolism of bacteria and their human host. Herein, we have developed a straightforward chemical synthesis method for rapid preparation of sulfated metabolite standards to obtain mass spectrometric fragmentation pattern and retention time information. We report the preparation of 38 O-sulfated alcohols and phenols for the determination of their MS/MS fragmentation pattern and chromatographic properties. Many of these metabolites are regioisomers that cannot be distinguished solely by their fragmentation pattern. We demonstrate that the versatility of this method is comparable to standard chemical synthesis. This comprehensive metabolite library can be applied for co-injection experiments to validate metabolites in different human sample types to explore microbiota-host co-metabolism, xenobiotic, and diet metabolism.

N-Acetyltransferases play critical roles in the deactivation and clearance of xenobiotics, including clinical drugs. NAT2 has been classified as an arylamineN-acetyltransferase that mainly converts aromatic amines, hydroxylamines, and hydrazines. Herein, we demonstrate that the human arylamineN-acetyltransferase NAT2 also acetylates aliphatic endogenous amines. Metabolomic analysis and chemical synthesis revealed increased intracellular concentrations of mono- and diacetylated spermidine in human cell lines expressing the rapid compared to the slow acetylator NAT2 phenotype. The regioselectiveN(8)-acetylation of monoacetylated spermidine by NAT2 answers the long-standing question of the source of diacetylspermidine. We also identified selective acetylation of structurally diverse alkylamine-containing drugs by NAT2, which may contribute to variations in patient responses. The results demonstrate a previously unknown functionality and potential regulatory role for NAT2, and we suggest that this enzyme should be considered for re-classification.

Fossils of extinct species allow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today. The origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown. The most prominent idea in the field posits that biology was preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication. This RNA world concept stands against other hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles. The question whether RNA or peptides were first is addressed by the RNA-peptide world concept, which postulates a parallel existence of both molecular species. A plausible experimental model of how such an RNA-peptide world may have looked like, however, is absent. Here we report the synthesis and physicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodon stem-loop of tRNAs from all three kingdoms of life. We show that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA-peptide world.