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<p>Cytochrome P450s are a super-family of heme containing proteins that are found in all domains of life and are involved in the synthesis and breakdown of steroids, xenobiotics, and pharmaceuticals. Using five heterologously expressed zebrafish (Danio rerio) CYP1s, an assay was developed for CYP activity in order to monitor the consumption of the cofactor NADPH, providing a label-free screening tool to determine function of novel CYP genes. Using well-established fluorogenic substrates, NADPH and NADP+ were separated by capillary electrophoresis (CE) from stopped CYP1 reactions and measured with UV absorbance detection as a surrogate to assess the rate of substrate metabolism. Product formation was confirmed by fluorometric detection of metabolites, giving rates of enzyme activity which could be compared to the rates of cofactor turn-over measured by CE. 17β-estradiol, four alkoxyresorufin and two coumarin based synthetic fluorogenic CYP substrates were screened for activity with recombinant zebrafish CYP1A, 1B1, 1C2, 1C2 and 1D1. Cofactor consumption was generally much larger than product formation for the majority of substrates and CYP1 isoforms, suggesting that the majority of metabolic events were uncoupled. Large uncoupling was seen in CYP1 when metabolizing estradiol, showing that endogenous compounds can also show severe uncoupling. Reactive oxygen species (ROS), a product of uncoupled events, were detected with 2,7- dichorofluorescein. Attempts for concomitant detection of ROS production and cofactor consumption with CE-UV detection were investigated, however, detection limits for 2,7-dichlorofluorescein were not adequate for detection of hydrogen peroxide production from CYP1 mediated reactions. Future work will be required to develop a single assay to quantitatively measure CYP activity by CE for functional determination of CYPs with unknown function.</p> / Master of Science (MSc)

<p>Research in this thesis has focused on development and application of novel methodologies that enhance sample throughput and data fidelity when performing untargeted metabolome profiling by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Metabolomics is a valuable tool in functional genomics research to investigate underlying molecular mechanisms associated with human health since metabolites are “real-world” end-products of gene expression. CE-MS is well-suited for metabolomics because it is a high efficiency microseparation technique that can be used to resolve complex mixtures of polar metabolites in human biofluids without complicated sample workup. In this thesis, a novel CE-MS assay for estrogens and their intact ionic conjugates has been described (<em>Chapter II</em>) to expand metabolome coverage that enables resolution of positional isomers with high selectivity. This is critical for better understanding of underlying perturbations in estrogen metabolism since the biological activity of estrogens are dependent on specific primary and secondary metabolic transformations. MSI-CE-MS has been introduced as a high-throughput approach for large-scale metabolomic studies based on serial injection of multiple segments of sample within a single fused-silica capillary (<em>Chapter III</em>). It reduces analysis times while increasing data quality and confidence in peak assignment together with better quality assurance. An accelerated workflow for metabolomics has also been developed when using MSI-CE-MS, where a dilution trend filter is used as a primary screen to authenticate reproducible sample-derived metabolites from a pooled sample while eliminating spurious artifact and background signals. In this way, complicated time alignment and peak picking algorithms are avoided when processing data in metabolomics to reduce false discoveries. This strategy was subsequently used in two metabolomics applications (<em>Chapters IV</em> and <em>V</em>) to identify plasma markers associated with strenuous exercise and adaptive training responses following a six-week high intensity interval training. The impact of exercise intervention to improve the glucose tolerance of a cohort of overweight/obese yet non-diabetic women was investigated on an individual level when using a cross-over design. Personalized interventions are critical in designing more effective therapies to prevent metabolic diseases due to inter-subject variations in treatment responses, including potential adverse effects. MSI-CE-MS offers a revolutionary approach for biomarker discovery in metabolomics with high sample throughput and high data fidelity, which is critical for validation of safe yet effective lifestyle interventions that promote human health and reduce risk for chronic diseases.</p> / Doctor of Philosophy (PhD)

Successful methods for the separation of biomolecules like amino acids, proteins, peptides, and DNA have been developed previously using HPLC, GC, GC-MS, and CE. Recently CE has become a routine laboratory technique in the analysis of biological molecules. Even though high-resolution separations with small sample volumes is the main advantage, CE is limited by lower sensitivity detection of analytes when universal detectors like UV absorption or refractive index detectors are used. Therefore, sensitivity enhancement can be obtained by either using different detection schemes or electrophoretically based pre- or on-line concentration methods. These can be grouped into two categories. The first category includes IEF, CGF or TGF where sensitivity is achieved through equilibrium electrofocusing. In these methods, electrophoresis and bulk solution is combined in the capillary or separation column to form a null velocity point, a point at which the net velocity of the analyte is zero. Using these methods 10-10,000 fold sensitivity enhancement is achieved. The second category uses velocity gradients but not the nul velocity for the enrichment of samples. These methods include FASS, LVSS, NSM, etc., which are applied for the analysis of small molecules, and 10-10,000 fold sensitivity enhancement is reported by using these methods. In this work, first GEITP an on-line preconcentration technique is applied for the detection of amino acids (using Trp and Tyr as model analytes). This work also established the effects of different parameters on enrichment. The parameters studied include effect of current flow acceleration across capillary inner diameter, the effect of leading electrolyte (LE) concentration on current density, and the effect of applied electric fields on the current density. To explore the application of GEITP in biological fluids, optimized parameters were developed for the detection and separation of Trp and Tyr in artificial cerebrospinal fluid (aCSF). Next, GEITP was applied for enrichment and separation of physiologically relevant concentrations of chromophore-derivatized Asp and Glu in high conductivity samples like artificial cerebrospinal fluid (aCSF). It was concluded from this work that the major factors which influence the enrichment is the ratio of current density to sample conductivity. Finally, GEITP is applied as a prior step before CZE to increase the resolution between analytes without using ampholyte mixtures. In this method GEITP was combined to CZE to achieve resolution adjustment between amino acids mixture using low pressure hydrodynamic flow during CZE without changing the separation column, field strength, or electrolyte system. In this work, a rapid CE method for extraction and analysis of amino acids in planarians, labeled with 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), was developed. This method was applied to detect the changes in the levels of amino acids when planarians were fed and starved. This method can be applied to study pharmacological effects in planaria, as it can monitor different amino acid levels with respect to feeding. Finally, ssDNA photoproducts of different lengths (11-mer and 63-mer) were separated using two different matrices, a traditional C18 and a PV/DBS (PLRP-S) matrix. A faster separation (within ~ 10 mins) was achieved for a 11-mer by the PLRP-S column. A separation was achieved in the PLRP-S column for the 63-mer while there was no separation in C18 column. Baseline resolution was not achieved. Therefore, C18 can best be used for small length DNA while PLRP-S can be applied for longer length DNA, as it is more hydrophobic than C18 column. Parameters can still be optimized for a baseline separation. / Chemistry

The metabolism of amino acids produces important chemical signaling molecules called neurotransmitters, which are responsible for carrying out important actions within the human body. There are approximately one hundred identified neurotransmitters. Neurotransmitter study is important due to their involvement in biological, physiological, pharmacological, and pathological functions. Commonly employed methods for neurotransmitter detection are mainly based upon microdialysis. However, the methods suffer from disadvantages. Microdialysis fails to determine the absolute concentration of analytes and therefore requires it to be tied in with an analytical technique such as high performance liquid chromatography or capillary electrophoresis. Although high performance liquid chromatography is the most powerful analytical technique to date, it necessitates high maintenance and suffers from poor temporal resolution. While capillary electrophoresis affords more rapid separations than high performance liquid chromatography, it suffers from poor concentration limits of detection and requires large sample dilutions of highly conductive samples, such as biological fluids. Consequently, research is focused on detection of various amino acids and neurotransmitters employing novel analytical techniques along with traditional capillary electrophoresis. First, a method was developed using traditional capillary electrophoresis with laser induced fluorescence detection to detect two major excitatory neurotransmitters, glutamate and aspartate in planaria. The method was later applied to detect several biogenic amines using micellar electrokinetic chromatography with laser induced fluorescence detection in planaria to study the effect of feeding on the levels of biogenic amines within individual planaria homogenates. The concentration sensitivity issue of capillary electrophoresis led to the use of a new method for sensitive neurotransmitter measurements, gradient elution isotachophoresis. Gradient elution isotachophoresis is an efficient capillary-based enrichment and separation technique based on balancing hydrodynamic counter-flow against electrophoresis. Enrichment is achieved with the aid of high concentrations of leading electrolyte in the counter-flow solution that creates an ionic interface near the capillary inlet. Discrete electrolyte spacers or carrier ampholyte mixtures are used to separate analyte zones. The method was applied to the enrichment and separation of physiologically relevant concentrations of aspartate and glutamate labeled with dansyl chloride, phenyl isothiocyanate, or carboxyfluorescein, succinimidyl ester in artificial cerebrospinal fluid using ultraviolet absorbance detection. Finally, gradient elution isotachophoresis was combined with capillary zone electrophoresis to eliminate the use of spacers and provide rapid separations and enrichment. The technique was applied for the detection of biogenic amines in a glass microfluidic device. / Chemistry