Urine metabolomics is widely used for biomarker research in the fields of medicine and toxicology. As a consequence, characterization of the variations of the urine metabolome under basal conditions becomes critical in order to avoid confounding effects in cohort studies. Such physiological information is however very scarce in the literature and in metabolomics databases so far. 
Here we studied the influence of age, body mass index (BMI), and gender on metabolite concentrations in a large cohort of 183 adults by using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). We implemented a comprehensive statistical workflow for univariate hypothesis testing and modeling by orthogonal partial least-squares (OPLS), which we made available to the metabolomics community within the online Workflow4Metabolomics.org resource. 
We found 108 urine metabolites displaying concentration variations with either age, BMI, or gender, by integrating the results from univariate p-values and multivariate variable importance in projection (VIP). Several metabolite clusters were further evidenced by correlation analysis, and they allowed stratification of the cohort. In conclusion, our study highlights the impact of gender and age on the urinary metabolome, and thus it indicates that these factors should be taken into account for the design of metabolomics studies.

Analysis of the Human Adult Urinary Metabolome Variations with Age, Body Mass Index, and Gender by Implementing a Comprehensive Workflow for Univariate and OPLS Statistical Analyses

Obtaining comprehensive, untargeted metabolic profiles for complex solid samples, e.g., animal tissues, requires sample preparation and access to information-rich analytical methodologies such as mass spectrometry (MS). Here we describe a practical two-step process for tissue samples that is based on extraction into 'aqueous' and 'organic' phases for polar and nonpolar metabolites. Separation methods such as ultraperformance liquid chromatography (UPLC) in combination with MS are needed to obtain sufficient resolution to create diagnostic metabolic profiles and identify candidate biomarkers. We provide detailed protocols for sample preparation, chromatographic procedures, multivariate analysis and metabolite identification via tandem MS (MS/MS) techniques and high-resolution MS. By using these optimized approaches, analysis of a set of samples using a 96-well plate format would take ∼48 h: 1 h for system setup, 8–10 h for sample preparation, 34 h for UPLC-MS analysis and 2–3 h for preliminary/exploratory data processing, representing a robust method for untargeted metabolic screening of tissue samples.

Global metabolic profiling of animal and human tissues via UPLC-MS

Metabolomics uses advanced analytical chemistry techniques to enable the high-throughput characterization of metabolites from cells, organs, tissues, or biofluids. The rapid growth in metabolomics ...

Metabolomics for Investigating Physiological and Pathophysiological Processes

Untargeted metabolomics provides a comprehensive platform for identifying metabolites whose levels are altered between two or more populations. By using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS), hundreds to thousands of peaks with a unique m/z ratio and retention time are routinely detected from most biological samples in an untargeted profiling experiment. Each peak, termed a metabolomic feature, can be characterized on the basis of its accurate mass, retention time and tandem mass spectral fragmentation pattern. Here a seven-step protocol is suggested for such a characterization by using the METLIN metabolite database. The protocol starts from untargeted metabolomic LC-Q-TOF-MS data that have been analyzed with the bioinformatics program XCMS, and it describes a strategy for selecting interesting features as well as performing subsequent targeted tandem MS. The seven steps described will require 2-4 h to complete per feature, depending on the compound.

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Liquid chromatography quadrupole time-of-flight mass spectrometry characterization of metabolites guided by the METLIN database

New advances in separation science for metabolomics: resolving chemical diversity in a post-genomic era

The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ∼400-600 metabolites in 120 urine samples per week.

Global urinary metabolic profiling procedures using gas chromatography–mass spectrometry

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction.

The cGAS-STING pathway is the major cytosolic DNA sensing pathway to induce downstream innate immune responses against viral infection as well as tumorigenesis. Recently it has been demonstrated that cGAS-STING pathway takes central stage in the immune sensing of tumor. STING activation induces the production of type I interferons (IFNα and β) and pro-inflammatory cytokines that collectively stimulate tumor antigen cross presentation to CD8 T cells and primes the adaptive immune response against tumor. In order to evaluate the potential of STING agonism to activate the anti-tumor immune response, we developed GSK532, a novel cyclic dinucleotide (CDN). Unlike cGAMP, the endogenous CDN ligand for STING, GSK532 potently activates STING both in vitro and in vivo. In vitro functional assays demonstrated the ability of GSK532 to induce cytokine responses in a panel of human PBMC samples with various STING haplotypes. Furthermore, GSK532 was shown to activate STING orthologs from preclinical species including cyno, minipig, dog, rat and mice. GSK532 demonstrated high stability in human whole blood with minimal degradation observed over 52 hrs. When dosed intratumorally, GSK532 induced a strong anti-tumor effect in the CT26 murine syngeneic model in both the injected (primary) and uninjected (distal) tumors. The cured mice were resistant to rechallenge with same tumor cell line indicating the involvement of an adaptive immune response. Further evidence of the development of anti-tumor adaptive immune response comes from the observation that the efficacy of GSK532 was significantly attenuated when CD8+ T cells were depleted in the murine model. Consistent with the anti-tumor efficacy, pharmacokinetic analysis demonstrated high exposure and long retention of GSK532 in both injected and distal tumors and pharmacodynamic studies showed induction of IFNβ and several inflammatory cytokines (TNFα, IL-6, etc) in both tumors. Currently, GSK532 is being progressed towards clinical investigation. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Jingsong Yang, Michael Adam, Jim Clemens, Katrina Creech, Jess Schneck, Kishore Pasikanti, Jean-Luc Tran, Devika Joglekar, Chris Hopson, Scott Pesiridis, Joshi Ramanjulu, Yiqian Lian, Jerry L. Adams, James Smothers, Axel Hoos. Preclinical characterization of GSK532, a novel STING agonist with potent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5554.

Kishore Pasikanti

Papers

Global urinary metabolic profiling procedures using gas chromatography–mass spectrometry

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction.

Abstract 5554: Preclinical characterization of GSK532, a novel STING agonist with potent anti-tumor activity