Synthesis, Characterization, and Potential Usefulness in Liver Function Assessment of Novel Bile Acid Derivatives with Near-Infrared Fluorescence (NIRBAD).

Conventional serum markers often fail to accurately detect cholestasis accompanying many liver diseases. Although elevation in serum bile acid (BA) levels sensitively reflects impaired hepatobiliary function, other factors altering BA pool size and enterohepatic circulation can affect these levels. To develop fluorescent probes for extracorporeal noninvasive hepatobiliary function assessment by real-time monitoring methods, 1,3-dipolar cycloaddition reactions were used to conjugate near-infrared (NIR) fluorochromes with azide-functionalized BA derivatives (BAD). The resulting compounds (NIRBADs) were chromatographically (FC and PTLC) purified (>95%) and characterized by fluorimetry, 1H NMR, and HRMS using ESI ionization coupled to quadrupole TOF mass analysis. Transport studies using CHO cells stably expressing the BA carrier NTCP were performed by flow cytometry. Extracorporeal fluorescence was detected in anesthetized rats by high-resolution imaging analysis. Three NIRBADs were synthesized by conjugating alkynocyanine 718 with cholic acid (CA) at the COOH group via an ester (NIRBAD-1) or amide (NIRBAD-3) spacer, or at the 3α-position by a triazole link (NIRBAD-2). NIRBADs were efficiently taken up by cells expressing NTCP, which was inhibited by taurocholic acid (TCA). Following i.v. administration of NIRBAD-3 to rats, liver uptake and consequent release of NIR fluorescence could be extracorporeally monitored. This transient organ-specific handling contrasted with the absence of release to the intestine of alkynocyanine 718 and the lack of hepatotropism observed with other probes, such as indocyanine green. NIRBAD-3 administration did not alter serum biomarkers of hepatic and renal toxicity. NIRBADs can serve as probes to evaluate hepatobiliary function by noninvasive extracorporeal methods.

A novel chemically engineered multifunctional statin conjugate as self-assembled nanoparticles inhibiting bile acid transporters.

Statins are widely used to treat hyperlipidemia; however, their mechanism-inhibiting cholesterol production without promoting its utilization-causes problems, such as inducing diabetes. In our research, we develop, for the first time, a chemically engineered statin conjugate that not only inhibits cholesterol production but also enhances its consumption through its multifunctional properties. The novel rosuvastatin (RO) and ursodeoxycholic acid (UDCA) conjugate (ROUA) is designed to bind to and inhibit the core of the apical sodium-dependent bile acid transporter (ASBT), effectively blocking ASBT's function in the small intestine, maintaining the effect of rosuvastatin. Consequently, ROUA not only preserves the cholesterol-lowering function of statins but also prevents the reabsorption of bile acids, thereby increasing cholesterol consumption. Additionally, ROUA's ability to self-assemble into nanoparticles in saline-attributable to its multiple hydroxyl groups and hydrophobic nature-suggests its potential for a prolonged presence in the body. The oral administration of ROUA nanoparticles in animal models using a high-fat or high-fat/high-fructose diet shows remarkable therapeutic efficacy in fatty liver, with low systemic toxicity. This innovative self-assembling multifunctional molecule design approach, which boosts a variety of therapeutic effects while minimizing toxicity, offers a significant contribution to the advancement of drug development.

Drug solubilization in dog intestinal fluids with and without administration of lipid-based formulations.

The use of animal experiments can be minimized with computational models capable of reflecting the simulated environments. One such environment is intestinal fluid and the colloids formed in it. In this study we used molecular dynamics simulations to investigate solubilization patterns for three model drugs (carvedilol, felodipine and probucol) in dog intestinal fluid, a lipid-based formulation, and a mixture of both. We observed morphological transformations that lipids undergo due to the digestion process in the intestinal environment. Further, we evaluated the effect of bile salt concentration and observed the importance of interindividual variability. We applied two methods of estimating solubility enhancement based on the simulated data, of which one was in good qualitative agreement with the experimentally observed solubility enhancement. In addition to the computational simulations, we also measured solubility in i) aspirated dog intestinal fluid samples and ii) simulated canine intestinal fluid in the fasted state, and found there was no statistical difference between the two. Hence, a simplified dissolution medium suitable for in vitro studies provided physiologically relevant data for the systems explored. The computational protocol used in this study, coupled with in vitro studies using simulated intestinal fluids, can serve as a useful prescreening tool in the process of drug delivery strategies development.

Quantification of gut microbiome metabolites using chemical isotope derivatization strategy combined with LC-MS/MS: Application in neonatal hypoxic-ischemic encephalopathy rat model.

The gut microbiome plays pivotal roles in various physiological and pathological processes, with key metabolites including short chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) derivatives gaining significant attention for their diverse physiological roles. However, quantifying these metabolites presents challenges due to structural similarity, low abundance, and inherent technical limitations in traditional detection methods. In this study, we developed a precise and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing a chemical isotope derivatization technique employing 4-(aminomethyl)-N,N-dimethylaniline-d0/d6 (4-AND-d0/d6) reagents to quantify 37 typical gut microbiome-derived metabolites. This method achieved an impressive 1500-fold enhancement in sensitivity for detecting metabolites, compared to methods using non-derivatized, intact molecules. Moreover, the quantitative accuracy of our chemical isotope derivatization strategy proved comparable to the stable isotope labeled internal standards (SIL-IS) method. Subsequently, we successfully applied this newly developed method to quantify target metabolites in plasma, brain, and fecal samples obtained from a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. The aim was to identify crucial metabolites associated with the progression of HIE. Overall, our sensitive and reliable quantification method holds promise in elucidating the role of gut microbiome metabolites in the pathogenesis of various diseases.

In situ interaction of pea peptides and bile salts under in vitro digestion: Potential impact on lipolysis.

The present work evaluated how a native pea protein isolate (PPI) affects the key roles carried out by bile salts (BS) in lipid digestion by means of the in vitro static INFOGEST protocol. Two gastric residence times were evaluated (10 and 60 min), and then the peptides obtained (GPPP) were mixed with BS at physiological concentration in simulated intestinal fluid to understand how they interact with BS both at the bulk and at the interface. Both GPPP give rise to a film with a predominant viscous character that does not constitute a barrier to the penetration of BS, but interact with BS in the bulk duodenal fluid. When the peptides flushing from the stomach after the different gastric residence times undergo duodenal digestion, it was found that for the longer gastric residence time the percentage of soluble fraction in the duodenal phase, that perform synergistically with BS micelles, was twice that of the lower residence time, leading to an increase in the solubilization of oleic acid. These results finally lead to a greater extent of lipolysis of olive oil emulsions. This work demonstrates the usefulness of in vitro models as a starting point to study the influence of gastric residence time of pea protein on its interaction with BS, affecting lipolysis. Pea proteins were shown to be effective emulsifiers that synergistically perform with BS improving the release and bioaccessibility of bioactive lipids as olive oil.

Tailoring FXR Modulators for Intestinal Specificity: Recent Progress and Insights.

While FXR has shown promise in regulating bile acid synthesis and maintaining glucose and lipid homeostasis, undesired side effects have been observed in clinical trials. To address this issue, the development of intestinally restricted FXR modulators has gained attention as a new avenue for drug design with the potential for safer systematic effects. Our review examines all currently known intestinally restricted FXR ligands and provides insights into the steps taken to enhance intestinal selectivity.

Bile acid/fatty acid integrated nanoemulsomes for nonalcoholic fatty liver targeted lovastatin delivery: stability, in-vitro, ex-vivo, and in-vivo analyses.

BACKGROUND: Controlled and targeted drug delivery to treat nonalcoholic fatty liver disease (NAFLD) can benefit from additive attributes of natural formulation ingredients incorporated into the drug delivery vehicles. METHODS: Lovastatin (LVN) loaded, bile acid (BA) and fatty acid (FA) integrated nanoemulsomes (NES) were formulated by thin layer hydration technique for synergistic and targeted delivery of LVN to treat NAFLD. Organic phase NES was comprised of stearic acid with garlic (GL) and ginger (GR) oils, separately. Ursodeoxycholic acid and linoleic acid were individually incorporated as targeting moieties. RESULTS: Stability studies over 90 days showed average NES particle size, surface charge, polydispersity index, and entrapment efficiency values of 270 ± 27.4 nm, -23.8 ± 3.5 mV, 0.2 ± 0.04 and 81.36 ± 3.4%, respectively. Spherical NES were observed under a transmission electron microscope. In-vitro LVN release depicted non-fickian release mechanisms from GL and GR oils-based NES. Ex-vivo permeation of BA/FA integrated NES through isolated rat intestines showed greater flux than non-integrated ones. CONCLUSION: Liver histopathology of experimental rats together with in-vivo lipid profiles and liver function tests illustrated that these NES possess the clinical potential to be promising drug carriers for NAFLD.

Enzyme-Driven LC-HRMS Approach for Specific Recognition of 12α-Hydroxy Bile Acids.

The synthesis of 12α-hydroxylated bile acids (12HBAs) and non-12α-hydroxylated bile acids (non-12HBAs) occurs via classical and alternative pathways, respectively. The composition of these BAs is a crucial index for pathophysiologic assessment. However, accurately differentiating 12HBAs and non-12HBAs is highly challenging due to the limited standard substances. Here, we innovatively introduce 12α-hydroxysteroid dehydrogenase (12α-HSDH) as an enzymatic probe synthesized by heterologous expression in Escherichia coli, which can specifically and efficiently convert 12HBAs in vitro under mild conditions. Coupled to the conversion rate determined by liquid chromatography-high resolution mass spectrometry (LC-HRMS), this enzymatic probe allows for the straightforward distinguishing of 210 12HBAs and 312 non-12HBAs from complex biological matrices, resulting in a BAs profile with a well-defined hydroxyl feature at the C12 site. Notably, this enzyme-driven LC-HRMS approach can be extended to any molecule with explicit knowledge of enzymatic transformation. We demonstrate the practicality of this BAs profile in terms of both revealing cross-species BAs heterogeneity and monitoring the alterations of 12HBAs and non-12HBAs under asthma disease. We envisage that this work will provide a novel pattern to recognize the shift of BA metabolism from classical to alternative synthesis pathways in different pathophysiological states, thereby offering valuable insights into the management of related diseases.

Multistimuli responsive microcapsules produced by the prilling/vibration technique for targeted colonic delivery of probiotics.

This study aimed to microencapsulate the probiotic strain Lactiplantibacillus plantarum 4S6R (basonym Lactobacillus plantarum) in both microcapsules and microspheres by prilling/vibration technique. A specific polymeric mixture, selected for its responsiveness to parallel colonic stimuli, was individuated as a carrier of microparticles. Although the microspheres were consistent with some critical quality parameters, they showed a low encapsulation efficiency and were discarded. The microcapsules produced demonstrated high yields (97.52%) and encapsulation efficiencies (90.06%), with dimensional analysis and SEM studies confirming the desired size morphology and structure. The results of thermal stress tests indicate the ability of the microcapsules to protect the probiotic. Stability studies showed a significant advantage of the microcapsules over non-encapsulated probiotics, with greater stability over time. The release study under simulated gastrointestinal conditions demonstrated the ability of the microcapsules to protect the probiotics from gastric acid and bile salts, ensuring their viability. Examination in a simulated faecal medium revealed the ability of the microcapsules to release the bacteria into the colon, enhancing their beneficial impact on gut health. This research suggests that the selected mixture of reactive polymers holds promise for improving the survival and efficacy of probiotics in the gastrointestinal tract, paving the way for the development of advanced probiotic products.

Analysis of 15 bile acids in human plasma based on C18 functionalized magnetic organic polymer nanocomposite coupled with liquid chromatography-tandem mass spectrometry.

Because of the "enterohepatic circulation" of bile acid, liver damage can be reflected by monitoring the content of bile acid in the serum of the organism. To monitor the concentration of 15 bile acids in plasma samples, a new technique of PRiME (process, ruggedness, improvement, matrix effect, ease of use) pass-through cleanup procedure combined with high performance liquid chromatography-tandem quadrupole mass spectrometry (HPLC-MS/MS) was developed. The sorbent used in the PRiME pass-through cleanup procedure is a new type of magnetic organic resin composite nano-material modified by C18 (C18-PS-DVB-GMA-Fe3O4), which has high cleanup efficiency of plasma samples. It also shows good performance in the separation and analysis of 15 kinds of bile acids. Under the optimal conditions, the results show higher cleanup efficiency of C18-PS-DVB-GMA-Fe3O4 with recoveries in the range of 82.1-115 %. The limit of quantitative (LOQs) of 15 bile acids were in the range of 0.033 µg/L-0.19 µg/L, and the RSD values of 15 bile acids were in the range of 3.00-11.9 %. Validation results on linearity, specificity, accuracy and precision, as well as on the application to analysis of 15 bile acids in 100 human plasma samples demonstrate the applicability to clinical studies.

Rapid analysis of the chemical constituents of traditional Tibetan medicine Sbyor-bzo-ghi-wang using ballpoint electrospray ionization technique.

RATIONALE: The chemical constituents of traditional Tibetan medicines (TTM) can be identified using high-performance liquid chromatography and high-resolution mass spectrometry (HPLC-MS/MS) technique. However, the HPLC-MS/MS technique requires the sample to be pretreated and then separated using the specific liquid chromatography method, which is time consuming. This study developed a ballpoint electrospray ionization (BPESI) technique for analyzing the chemical constituents of Sbyor-bzo-ghi-wang. This technique is a simple and inexpensive method for the rapid identification of the chemical constituents of TTMs. METHODS: After the important parameters of the homemade BPESI device were optimized, the chemical constituents of Sbyor-bzo-ghi-wang were quickly identified without sample pretreatment. The raw data were converted to mzML file using MSConvert and then identified using SIRIUS 5 software. RESULTS: The results showed that 30 compounds were identified from Sbyor-bzo-ghi-wang, namely eight bile acids, six flavonoids, four alkaloids, three amino acids, and nine others. Compared to the ultra-high-performance liquid chromatography-Q/Orbitrap and high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS) technique, the BPESI technique identified almost similar types of compounds and also a comparable number of compounds. CONCLUSIONS: Compared with the traditional HPLC-MS/MS methods, the BPESI technique does not require complex sample pretreatment and subsequent chromatographic separation steps; also it consumes a small quantity of samples. Therefore, BPESI can be used for the qualitative analysis of the chemical constituents of Sbyor-bzo-ghi-wang.

Metal-Organic Cage Receptors for Encapsulation and Sensing of Bile Acids.

Developing synthetic supramolecular receptors to solubilize, scavenge, recognize, encapsulate, and sense steroids is challenging. Despite a limited number of receptors having affinity with steroids, none exists to bind steroidal bile acids selectively. Herein, we report a C2-symmetric metal-organic cage [Pd6L24]12+ and an expanded version of the Fujita cage [Pd6L14]12+, built with a conformationally flexible ligand L2, accessed through coordination-driven self-assembly. We examined both cages for steroid recognition in water: both have certain shared characteristics and distinctive features. [Pd6L14]12+ binds hydrophobic bile acids and other steroids by forming a 1:1 complex. In contrast, the expanded [Pd6L24]12+ cage exhibits an affinity for amphiphilic bile acids and selective steroids to encapsulate them as dimers, promoted by cooperative interguest hydrogen bonding. [Pd6L24]12+ has a 5 times stronger solubility enhancement ability for cholic acid compared to [Pd6L14]12+. Further, the expanded [Pd6L24]12+ cage can selectively sense bile acids in nanomolar detection limits through indicator displacement assay by employing sulforhodamine 101 (SR101).

Bile Salt Hydrolase Activity-Based Probes for Monitoring Gut Microbial Bile Acid Metabolism.

Bile acids are bioactive metabolites that are biotransformed into secondary bile acids by the gut microbiota, a vast consortium of microbes that inhabit the intestines. The first step in intestinal secondary bile acid metabolism is carried out by a critical enzyme, bile salt hydrolase (BSH), that catalyzes the gateway reaction that precedes all subsequent microbial metabolism of these important metabolites. As gut microbial metabolic activity is difficult to probe due to the complex nature of the gut microbiome, approaches are needed to profile gut microbiota-associated enzymes such as BSH. Here, we develop a panel of BSH activity-based probes (ABPs) to determine how changes in diurnal rhythmicity of gut microbiota-associated metabolism affects BSH activity and substrate preference. This panel of covalent probes enables determination of BSH activity and substrate specificity from multiple gut anerobic bacteria derived from the human and mouse gut microbiome. We found that both gut microbiota-associated BSH activity and substrate preference is rhythmic, likely due to feeding patterns of the mice. These results indicate that this ABP-based approach can be used to profile changes in BSH activity in physiological and disease states that are regulated by circadian rhythms.

Biotechnological Effects of Advanced Smart-Bile Acid Cyclodextrin-Based Nanogels for Ear Delivery and Treatment of Hearing Loss.

Inner ear delivery requires safe and effective drug delivery vehicles incorporating high-viscosity formulations with permeation enhancers. This study designs novel thermoresponsive-smart polymer-bile acid and cyclodextrin-based nanogels for inner ear delivery. Nanogels are examined for their rheological and physical properties. The biocompatibility studies will be assessed on auditory and macrophage cell lines by investigating the impact of nanogels on cellular viability, mitochondrial respiration, glycolysis, intracellular oxidative stress, inflammatory profile, and macrophage polarization. Novel ther nanogels based on bile acid and beta-cyclodextrin show preserved porous nanogels' inner structure, exhibit non-Newtonian, shear-thinning fluid behavior, have fast gelation at 37 °C and minimal albumin adsorption on the surface. The nanogels have minimal impact on cellular viability, mitochondrial respiration, glycolysis, intracellular oxidative stress, and inflammatory profile of the auditory cell line House Ear Institute-Organ of Corti 1 after 24 h incubation. Nanogel exposure of 24 h to macrophage cell line RAW264.7 leads to decreased viability, mitochondrial dysfunction, and increased intracellular ROS and inflammatory cytokines. However, polarization changes from M2 anti-inflammatory to M1 pro-inflammatory macrophages are minimal, and inflammatory products of RAW264.7 macrophages do not overly disrupt the survivability of HEI-OC1 cells. Based on these results, thermoresponsive bile acid and cyclodextrin nanogels can be potential drug delivery vehicles for inner ear drug delivery.

The underappreciated diversity of bile acid modifications.

The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.

The Use of Global Sensitivity Analysis to Assess the Oral Absorption of Weakly Basic Compounds: A Case Example of Dipyridamole.

OBJECTIVE: To utilize the global system analysis (GSA) in oral absorption modeling to gain a deeper understanding of system behavior, improve model accuracy, and make informed decisions during drug development. METHODS: GSA was utilized to give insight into which drug substance (DS), drug product (DP), and/or physiological parameter would have an impact on peak plasma concentration (Cmax) and area under the curve (AUC) of dipyridamole as a model weakly basic compound. GSA guided the design of in vitro experiments and oral absorption risk assessment using FormulatedProducts v2202.1.0. The solubility and precipitation profiles of dipyridamole in different bile salt concentrations were measured. The results were then used to build a mechanistic oral absorption model. RESULTS: GSA warranted further investigation into the precipitation kinetics and its link to the levels of bile salt concentrations. Mechanistic modeling studies demonstrated that a precipitation-integrated modeling approach appropriately predicted the mean plasma profiles, Cmax, and AUC from the clinical studies. CONCLUSIONS: This work shows the value of GSA utilization in early development to guide in vitro experimentation and build more confidence in identifying the critical parameters for the mathematical models.

Design and development of novel self-assembled catechol-modified bile acid conjugates as pH-responsive apical sodium-dependent bile acid transporter targeting nanoparticles.

Catechol-based biomaterials demonstrate biocompatibility, making them suitable for a wide range of therapeutic applications when integrated into various molecular frameworks. However, the development of orally available catechol-based biomaterials has been hindered by significant pH variations and complex interactions in the gastrointestinal (GI) tract. In this study, we introduce a novel catechol-modified bile acid (CMBA), which is synthesized by anchoring the FDA-approved drug, ursodeoxycholic acid to the neurotransmitter dopamine. This modification could form a new apical sodium-dependent bile acid transporter (ASBT) inhibitor (ASBTi) due to the bile acid moiety. The computational analysis using the TRAnsient Pockets in Proteins (TRAPP) module, coupled with MD simulations, revealed that CMBA exhibits a strong binding affinity at residues 51-55 of ASBT with a low inhibitory constant (Ki) value. Notably, in slightly alkaline biological conditions, CMBA molecules self-assemble into carrier-free nanoparticles with an average size of 240.2 ± 44.2 nm, while maintaining their ability to bind with ASBT. When administered orally, CMBA accumulates in the ileum and liver over 24 h, exhibiting significant therapeutic effects on bile acid (BA) metabolism in a high-fat diet (HFD)-fed mouse model. This study underscores the therapeutic potential of the newly developed catechol-based, pH-responsive ASBT-inhibiting nanoparticles presenting a promising avenue for advancing therapy.

Bile salt hydrolase catalyses formation of amine-conjugated bile acids.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

Bile salt hydrolase acyltransferase activity expands bile acid diversity.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

Reverse metabolomics for the discovery of chemical structures from humans.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.