Development of analytical methodologies for the separation and detection of reactive oxygen and nitrogen species
Advisor: Lunte, SusanSchool: The University of Kansas
School Location: United States -- Kansas
Keyword(s): Separation, Reactive oxygen species, Reactive nitrogen species, Peroxynitrite
Source: DAI-B 67/12, Jun 2007
Source type: Dissertation
Subjects: Pharmacology
Publication Number: AAT 3244383
Document URL: [url]http://proquest.umi.com/pqdweb?did=1232430661&sid=1&Fmt=2&clientId=45596&RQT=309&VName=PQD[/url]
ProQuest document ID: 1232430661
Abstract (Document Summary)
Understanding the role reactive oxygen and nitrogen species play in disease is critical to understanding, and monitoring health. However, because these species are highly reactive, they posses a very short lifetime, and their detection presents an analytical challenge. This dissertation addresses that challenge, and describes the development of methodologies for the separation and sensitive and selective detection of reactive oxygen and nitrogen species.
First a method for the separation and indirect detection of nitric oxide (NO) generated at the blood brain barrier (BBB) is described. The detection of nitrite (NO 2 ¯), a degradation product of NO, was investigated using capillary electrophoresis (CE) with amperometric detection. Next a reaction product of NO, peroxynitrite (ONOO¯), was separated from NO 2 ¯, nitrate (NO 3 ¯), and hydrogen peroxide (H 2 O 2 ), and all were detected directly, using CE-UV. Because these species are so similar in reactivity, it can be difficult to differentiate between them in cellular samples using typical detection methods such as oxidation of fluorescent probes, or electrochemical sensors. However, they can be distinguished through separation using CE.
Then the CE-UV method was transferred to microchip, and electrochemical detection was employed to detect OH¯, NO 2 ¯, ONOO¯, and H 2 O 2 . With the microfluidic format it is possible to achieve very fast separations, an advantage when dealing with highly reactive species. The microchip method was also used to measure the production of NO 2 ¯ and ONOO¯ from the spontaneous degradation of a cardiovascular drug, 3-morpholinosydnonimine (SIN-1).
Next, the interaction of ONOO¯ and glutathione (GSH), and ONOO¯ and tyrosine (tyr) was studied using CE-UV, microchip electrophoresis with amperometric detection, and mass spectrometry (MS). These reactions were important because GSH and tyr are both present in cellular environments in high concentrations and could react with ONOO¯ in cellular extracts at high pH, giving a lower value for ONOO¯. If they had reacted with ONOO¯ under the same conditions as used by the separation and detection method, then the full concentration of ONOO¯ would not be measured.
Finally, NO 2 ¯ and NO 3 ¯ were separated using microchip electrophoresis and detected using simultaneous on-chip amperometric and conductivity detection. NO 3 ¯ is not electroactive, and is not detectable by amperometry. However, it can be detected using conductivity. NO 2 ¯ could be detected by both techniques; however, lower concentrations were detected with amperometry. Therefore it was advantageous to incorporate both techniques onto one device.
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