Browsing by Subject "Metabolomics"
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Item MS Based Approaches for the Analysis of Small Molecules such as Sugar Nucleotides and Gangliosides(2013-05) Garcia, Aldo; Mechref, Yehia; Thompson, Jonathan E.; Pare, PaulThe field of metabolomics has become a hot spot of analytical investigations since the discovery of the encompassing significance of the metabolome in complex eukaryotic systems. Most notably, emphasis in this field is currently being placed in the discovery of potential biomarkers through development of highly sensitive methods that are capable of identifying and quantifying small metabolites of interest in complex samples. In this thesis, a concise review is described in chapter 1 over the field of metabolomics as it pertains to the analytical developments of sugar nucleotides and ganglioside analysis. In addition, targeted mass spectrometric approaches are presented in which analytical methodologies are developed for sugar nucleotide and ganglioside profiling for the analysis of diseased biological samples (such as cancer) , with interest focused on the establishment of metabolic profiles of diseased states. Chapter 2 details the analytical method for targeted mass spectrometric analysis of sugar nucleotides. The method described permitted the establishment of a sugar nucleotide linear dynamic range of quantification expanding over almost three orders of magnitude with an extremely reproducible chromatographic separation in less than six minutes. Viability of this method was then tested by successfully quantifying the sugar nucleotide content of human cells from 3 different cancer cell lines. The developed short chromatographic method potentially permits for high throughputs analysis for its integration into population studies for the search for potential biomarkers. Chapter 3 describes the developed methodology for targeted ganglioside analysis. A chromatographic method was developed based on the use of a mixture of bovine brain gangliosides that allowed for complete separation in less than 7.5 minutes. Quantificationof gangliosides was also achieved by the construction of a calibration curve from ganglioside standards. The established method was then successfully employed for the ganglioside profiling of 3 diseased samples of human blood serum along with one disease free control. As a result, ganglioside profiles were quantitatively established at high sensitivity for the different ganglioside samples.Item Untargeted metabolomics analysis of Rheumatoid arthritis patient sera before and after rituximab treatment(2015-08) Sweeney, Shannon Renee; Tiziani, Stefano; Guma, MonicaBackground: Rheumatoid arthritis (RA) is an autoimmune disease with no known cure that affects approximately 1.3 million Americans. RA patients suffer from chronic pain and inflammation and are faced with probable disability, reduced life expectancy, and increased risk of several other diseases. In the last decade, biological therapies have revolutionized RA treatment. Although administration of a tumor necrosis factor (TNF) neutralizing agent is the first-line biological therapy, many RA patients show only partial or no clinical response to treatment. Subsequently, anti-B cell, anti-T cell, or anti-IL6 therapies can be evaluated. Streamlining of treatment protocols is necessary to improve patient outcomes. Methods: Serum was collected from 23 active, seropositive RA patients on concomitant methotrexate, at baseline and six months after treatment with rituximab. Based on the American College of Rheumatology improvement criteria, at a level of 20% (ACR20), patients were categorized as either responders or non-responders. An untargeted metabolomics approach was used to characterize the serum metabolome of patients. High resolution one-dimensional ¹H-NMR spectra were acquired using a Bruker Avance 700 MHz spectrometer. In addition, A Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer was used for UPLC-MS/MS of serum lipids. Data processing, statistical analysis, and pathway mapping were performed in MATLAB in conjunction with several metabolomics software packages including, NMRLab, MetaboLab, Chenomx, MetaboAnalyst, MetaboSearch, VANTED, Xcalibur, and Sieve. Results: Based on the ACR20 criteria, at baseline, 14 patients were characterized as responders and 9 patients were considered non-responders. Similarly, 20 patients followed-up at six months, 13 responders and 7 non-responders. Seven polar metabolites and 15 unique lipid species achieved a p-value of less than 0.05 for a two sample t-test prior to treatment with rituximab. Following rituximab therapy, five polar metabolites and 37 lipid species were statistically significant between groups. Pathway analysis of both polar and apolar metabolites revealed metabolic differences between responder and non-responders before and after treatment with rituximab. Conclusion: A clear relationship between blood metabolic profiles and clinical response to rituximab therapy suggests that ¹H-NMR and UPLC-MS/MS are promising tools for RA therapy optimization and acceleration of treatment protocols to improve patient outcomes.