Development Of Metabolomics Platforms For Gut Microbial-host Co-metabolism

Mammals are considered as superorganisms as a result of theirclose symbiotic associations with the gut microbiota. Gut microbesexert strong control over the mammalian host and are involved in thematuration and development of the host’s immune system,maintenance of host energy, and metabolic homeostasis. Thecomposition and activities of gut microbiota play important roles inhost health and the imbalance of dysbiosis of the microbiota inducesdifferent diseases. Mammalian metabolism involves integration ofmultiple indigenous metabolic processes which were encoded by thehost genome with those of the microbiome. Thus, the symbiotic gutmicrobiome exerts a strong influence on the metabolic phenotype ofmammalian host and participates in extensive microbial-mammlianco-metabolism.To gain better insight into the activity and functionality of gutmicrotiota, metabolomics is uniquely suited to assess the highlycomplex metabolic exchanges, opening a direct biochemical windowinto the metabolome. Metabolomics, as an integral part of thesystems biology, is defined as “the quantitative measurement ofmultiparametric time-related metabolic responses of a complexsystem to genetic modification or a pathophysiological intervention.”This platform offers a well-established high-throughput “omics”technology for analysis of the metabolome using an array ofspectroscopic and spectrometric techniques. Integration of gutmicrobial profiling with high-thourghput metabolic phenotyping promises to delineate the microbiome and the host metabolicphenotypes at a global level to uncover their inherent associations.Such studies help to imporve our understanding of the mechanismsunderlying complex host-microbe interactions.In this dissertation, the untargeted metabolomics platform as wellas targeted analysis of short-chain fatty acids, branched-chain aminoacid and bile acids were developed and validated, characterizing apanel of urinary and fecal metabolites related to microbialmammalian co-metabolism. These approaches were applied in theinvestigations of topographical metabolic signatures of ratgastrointestinal contents and the impact of gut microbiota onmelamine-induced renal toxicity. Main methods and results:A combined GC/MS and LC/MS untargeted metabolomicsapproach was applied to profile the urinary and fecal metabolitesfrom Wistar rats with gut microbiota suppressed by a broad spectrumantibiotic imipenem/cilastatin sodium. A panel of202urinary and223fecal metabolites were significantly altered as a readout of a gutmicrobial-mammalian co-metabolism, many of which have not beenpreviously reported. This study shows extensive gut microbiotamodulation of host systemic metabolism involving tryptophan,tyrosine and phenylalanine metabolism, short-chain fatty acids,medium and long chain fatty acids, bile acids, oligopeptidestransportation, and possibly a compensatory mechanism ofindole-melatonin production. It appeared that the recovery of theglobal metabolomic changes takes about two-weeks. Given theintegral nature of the mammalian genome and metagenome, thispanel of metabolites will provide a new platform for potentialtherapeutic markers and mechanistic solutions to complex problemscommonly encountered in pathology, toxicology or drug metabolismstudies.A targeted metabolomic protocol was developed to determineshort-chain fatty acids and branched-chain amino acids using propylchloroformate derivatization followed by GC/MS analysis. Aone-step derivatization using100μL of propyl chloroformate in a reaction system of water, propanol, and pyridine (v/v/v=8:3:2) at pH8provided the optimal derivatization efficiency. The best extractionefficiency of the derivatized products was achieved by a two-stepextraction with hexane. The method exhibited good derivatizationefficiency and recovery for a wide range of concentrations with a lowlimit of detection for each compound and can be applied in differentbiological samples. This is a complementary assay for the untargetedmetabolomics approach, providing a comprehensive metabolicsignature of gut micribiota and host co-metabolism.A UPLC/TQMS method was established for targeted bile acidsprofiling in biological samples, allowing the simultaneousquantification of27bile acids including14unconjugated,6glycine-conjugated and7taurine-conjugated bile acids. This methodprovides good results in terms of intra-and interday precision,accuracy and linearity. It is also a complementary method forunbiased metabolomics analysis, providing the capability todistinguish isomers with identical m/z values by different daughterions. This method has great values in the investigations of gutmicrobiota metabolism and can readily be extended to clinicalstudies.Integrating above three metabolomics platforms, the metabolitecomposition of contents in different regions of the intestine of normalrats were analyzed, investigating spatially the metabolism of gutmicrobiota in different regions of the intestine. Statistical analysiswere applied to differentiate metabolomic profiles of different regionsand revealed that the metabolite composition in gut contents weregreatly altered along different parts of the intestine, especiallybetween small intestine and large intestine.Based on urinary metabilomics approach, the mechanism ofmelamine-induced renal toxicity was investigated. Cyanuric acidserving as an integral component of the kidney stones is producd inthe gut by microbial transformation of melamine. We demonstratethat melamine-induced toxicity in Wistar rats was attenuated after gutmicrobiota suppression, along with increased melamine excretion. We further demonstrated that melamine can be converted to cyanuric acidin vitro by the cultured bacteria from normal rat feces and Klebsiellawas identified in cultivation of fecal samples by16S rDNAsequencing analysis. In addition, the cultures of Klebsiella terrigena,a species of Klebsiella genus, were able to convert melamine tocyanuric acid. Melamine-induced toxicity in kidneys was exacerbatedwhen rats were colonized with K. terrigena. Cyanuric acid wasdetected in kidneys of rats administered melamine alone and theconcentration was significantly increased after Klebsiellacolonization.