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The ability to identify and quantify small molecule metabolites derived from gut microbial-mammalian cometabolism is essential for the understanding of the distinct metabolic functions of the microbiome. To date, analytical protocols that quantitatively measure a complete panel of microbial metabolites in biological samples have not been established but are urgently needed by the microbiome research community. Here, we report an automated high-throughput quantitative method using a gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) platform to simultaneously measure over one hundred microbial metabolites in human serum, urine, feces, and Escherichia coli cell samples within 15 min per sample. A reference library was developed consisting of 145 methyl and ethyl chloroformate (MCF and ECF) derivatized compounds with their mass spectral and retention index information for metabolite identification. These compounds encompass different chemical classes including fatty acids, amino acids, carboxylic acids, hydroxylic acids, and phenolic acids as well as benzoyl and phenyl derivatives, indoles, etc., that are involved in a number of important metabolic pathways. Within an optimized range of concentrations and sample volumes, most derivatives of both reference standards and endogenous metabolites in biological samples exhibited satisfactory linearity (R 2 > 0.99), good intrabatch reproducibility, and acceptable stability within 6 days (RSD < 20%). This method was further validated by examination of the analytical variability of 76 paired human serum, urine, and fecal samples as well as quality control samples. Our method involved using high-throughput sample preparation, measurement with automated derivatization, and rapid GC/TOFMS analysis. Both techniques are well suited for microbiome metabolomics studies.

Atherosclerosis resulting from hyperlipidemia causes many serious cardiovascular diseases. To understand the systems changes associated with pathogenesis and progression of atherosclerosis, we comprehensively analyzed the dynamic metabonomic changes in multiple biological matrices of LDLR(-/-) mice using NMR and GC-FID/MS with gene expression, clinical chemistry, and histopathological data as well. We found that 12 week "Western-type" diet (WD) treatment caused obvious aortic lesions, macrophage infiltration, and collagen level elevation in LDLR(-/-) mice accompanied by up-regulation of inflammatory factors including aortic ICAM-1, MCP-1, iNOS, MMP2, and hepatic TNFα and IL-1β. The WD-induced atherosclerosis progression was accompanied by metabonomic changes in multiple matrices including biofluids (plasma, urine) and (liver, kidney, myocardial) tissues involving multiple metabolic pathways. These included disruption of cholesterol homeostasis, disturbance of biosynthesis of amino acids and proteins, altered gut microbiota functions together with metabolisms of vitamin-B3, choline, purines, and pyrimidines. WD treatment caused down-regulation of SCD1 and promoted oxidative stress reflected by urinary allantoin elevation and decreases in hepatic PUFA-to-MUFA ratio. When switching to normal diet, atherosclerotic LDLR(-/-) mice reprogrammed their metabolisms and reversed the atherosclerosis-associated metabonomic changes to a large extent, although aortic lesions, inflammation parameters, macrophage infiltration, and collagen content were only partially alleviated. We concluded that metabolisms of fatty acids and vitamin-B3 together with gut microbiota played crucially important roles in atherosclerosis development. These findings offered essential biochemistry details of the diet-induced atherosclerosis and demonstrated effectiveness of the integrated metabonomic analysis of multiple biological matrices for understanding the molecular aspects of cardiovascular diseases.

The interplay between genetic mutation and environmental factors is believed to contribute to the etiology of inflammatory bowel disease (IBD). While focused attention has been paid to the aforementioned research, time-specific and organ-specific metabolic changes associated with IBD are still lacking. Here, we induced acute ulcerative colitis in mice by providing water containing 3% dextran sulfate sodium (DSS) for 7 days and investigated the metabolic changes of plasma, urine, and a range of biological tissues by employing a (1)H nuclear magnetic resonance (NMR)-based metabonomics approach with complementary information on serum clinical chemistry and histopathology. We found that DSS-induced acute ulcerative colitis leads to significant elevations in the levels of amino acids in plasma and decreased levels in the membrane-related metabolites and a range of nucleotides, nucleobases, and nucleosides in the colon. In addition, acute-colitis-induced elevations in the levels of nucleotides in the liver were observed, accompanied by reduced levels of glucose. DSS-induced acute colitis also resulted in increased levels of oxidized glutathione and attenuated levels of taurine in the spleen. Furthermore, acute colitis resulted in depletion in the levels of gut microbial cometabolites in urine along with an increase in citric acid cycle intermediates. These findings suggest that DSS-induced acute colitis causes a disturbance of lipid and energy metabolism, damage to the colon and liver, a promoted antioxidative and anti-inflammatory response, and perturbed gut microbiotal communities. The information obtained here provided details of the time-dependent and holistic metabolic changes in the development of the DSS-induced acute ulcerative colitis, which could be useful in discovery of novel therapeutic targets for management of IBD.