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(Unpublished)
This is the latest version of this item.
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.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
18 October 2013 |
| Date Type: |
Publication |
| Defense Date: |
25 June 2013 |
| Approval Date: |
18 October 2013 |
| Submission Date: |
13 August 2013 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
235 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
microfluidics, thiols and disulfides, metabolism, electroosmotic sampling, extracellular, organotypic hippocampal slice cultures |
| Date Deposited: |
18 Oct 2013 16:22 |
| Last Modified: |
15 Nov 2016 14:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/19758 |
Available Versions of this Item
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MONITORING THE METABOLISM OF THIOLS/DISULFIDES IN THE EXTRACELLULAR SPACE OF ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES BY ONLINE MICROFLUIDIC ANALYSIS COUPLED WITH ELECTROOSMOTIC SAMPLING. (deposited 18 Oct 2013 16:22)
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