eprintid: 19758 rev_number: 20 userid: 1500 dir: disk0/00/01/97/58 datestamp: 2013-10-18 16:22:45 lastmod: 2016-11-15 14:15:10 status_changed: 2013-10-18 16:22:45 type: thesis_degree succeeds: 19627 metadata_visibility: show contact_email: wujfpitt@gmail.com item_issues_id: duplicate_title_19753 item_issues_id: thesis_degree_versioning item_issues_type: duplicate_title item_issues_type: thesis_degree_versioning item_issues_description: Duplicate title to Wu, Juanfang MONITORING THE METABOLISM OF THIOLS/DISULFIDES IN THE EXTRACELLULAR SPACE OF ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES BY ONLINE MICROFLUIDIC ANALYSIS COUPLED WITH ELECTROOSMOTIC SAMPLING. Doctoral Dissertation, University of Pittsburgh. item_issues_description: ETD 19758 is using versioning. item_issues_timestamp: 2013-09-07 06:20:16 item_issues_timestamp: 2013-09-11 17:55:00 item_issues_status: autoresolved item_issues_status: discovered item_issues_count: 1 eprint_status: archive creators_name: Wu, Juanfang creators_email: juw30@pitt.edu creators_id: JUW30 title: MONITORING THE METABOLISM OF THIOLS/DISULFIDES IN THE EXTRACELLULAR SPACE OF ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES BY ONLINE MICROFLUIDIC ANALYSIS COUPLED WITH ELECTROOSMOTIC SAMPLING ispublished: unpub divisions: sch_as_chemistry full_text_status: public keywords: microfluidics, thiols and disulfides, metabolism, electroosmotic sampling, extracellular, organotypic hippocampal slice cultures abstract: Sulfur-containing compounds, such as glutathione (GSH), cysteamine (CSH), homocysteine (Hcy), cysteine (Cys) and their disulfides, are critical for maintaining various cellular functions. Concentration profiles of these compounds in biological samples are useful for evaluating cellular physiological status and diagnosing diseases. Traditional quantitative methods involve tedious sample pretreatment and are not suited to dealing with submicroliter samples. We have developed a microfluidic system for online analysis of thiol-containing compounds in the extracellular space of rat organotypic hippocampal slices cultures (OHSCs). This system is also capable of monitoring ectoenzymatic activity involved in the metabolism of thiols/disulfides. A microfluidic chip capable of derivatization, multiple injection, separation, and quantitation was designed and coupled to a homemade confocal laser induced fluorescence detector with a multifunctional control program. The microfluidic system was evaluated in an on-chip kinetic study of the reduction of oxidized glutathione (GSSG) catalyzed by glutathione reductase (GR, EC 1.8.1.7) followed by derivatization of GSH with ThioGlo-1. The separation of analytes was successfully achieved within 4.5 s and 9 mm separation length. The linear range of the system is up to 50 µM (GSH). The mass and concentration detection limits are 10-18 mol and 4.2 nM, respectively. Based on GSH growth rates, the apparent Michaelis constants of GR were determined to be 40 ± 11 μM (GSSG) and 4.4 ± 0.6 μM (β-NADPH). Electroosmotic sampling of the extracellular fluid of OHSCs was further coupled to a microfluidic device for in situ quantitation of endogenous aminothiols in OHSCs, by which CSH (10.6 ± 1.0 nM), Hcy (0.18 ± 0.01 μM) and Cys (11.1 ± 1.2 μM) were separated and evaluated simultaneously. Using this method, we also observed the complete biodegradation of coenzyme A (CoA) in the extracellular space of OHSCs and determined the kinetic parameters of this sequential multi-enzyme reaction in in situ. Metabolism of cystamine (CSSC) and pantethine (PSSP) in OHSCs have also been investigated and the percentage yields of CSH from CSSC and PSSP after ~55 s exposure time to OHSCs were 91% ± 4% and 0.01% - 0.03%, respectively, which explains the differences of the drugs in clinical effectiveness and toxicity. date: 2013-10-18 date_type: published pages: 235 institution: University of Pittsburgh refereed: TRUE etdcommittee_type: committee_chair etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_name: Weber, Stephen G etdcommittee_name: Liu, Xinyu etdcommittee_name: Michael, Adrian etdcommittee_name: Kagan, Valerian E etdcommittee_email: sweber@pitt.edu etdcommittee_email: xinyuliu@pitt.edu etdcommittee_email: amichael@pitt.edu etdcommittee_email: kagan@pitt.edu etdcommittee_id: SWEBER etdcommittee_id: XINYULIU etdcommittee_id: AMICHAEL etdcommittee_id: KAGAN etd_defense_date: 2013-06-25 etd_approval_date: 2013-10-18 etd_submission_date: 2013-08-13 etd_release_date: 2013-10-18 etd_access_restriction: 1_year etd_patent_pending: FALSE thesis_type: dissertation degree: PhD citation: Wu, Juanfang (2013) MONITORING THE METABOLISM OF THIOLS/DISULFIDES IN THE EXTRACELLULAR SPACE OF ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES BY ONLINE MICROFLUIDIC ANALYSIS COUPLED WITH ELECTROOSMOTIC SAMPLING. Doctoral Dissertation, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/19758/1/Juanfang_Wu_dissertation.pdf