DEVELOPMENT OF MICROCHIP ELECTROPHORESIS BASED METHODS TO PROFILE CELLULAR NITROSATIVE STRESS

Abstract

Nitric oxide (NO) is the main reactive nitrogen species (RNS) produced by immune cells. NO reacts with superoxide to produce peroxynitrite (ONOO-). These two RNS are capable of nitration, nitrosylation and oxidation of intracellular biomolecules that can alter various biochemical processes. To help maintain cellular redox homeostasis in nitrosative and oxidative stress, antioxidant molecules are present in the cells. However, excessive nitrosative stress can alter the balance between antioxidants and prooxidants and therefore it plays an important role in cancer, cardiovascular and neurodegenerative diseases. A method for the simultaneous detection of prooxidants and antioxidants associated with nitrosative stress in biological samples would be beneficial for better understanding of their role in disease states. Therefore, in this dissertation a separation-based approach is described that makes it possible to detect and quantify cellular antioxidants and prooxidants such as NO, ONOO-, glutathione and ascorbic acid. Microchip electrophoresis (ME) was selected as the separation method due to its fast analysis times, compatibility with low sample volumes and potential future application to chemical cytometry. Most prooxidants and antioxidants are electrochemically active and therefore, electrochemical detection was used as the primary detection mode. First, a ME method with in-channel amperometric detection that employed an isolated potentiostat was developed for the detection of NO, ONOO-, and other biologically important molecules associated nitrosative stress. Separation of these species was achieved in less than 35 s, which made it possible to detect prooxidants before they significantly degraded. Following this, two dual electrode configurations were developed and evaluated for better identification of reactive species in standard mixtures and cell lysates using voltammetric characterization. The ME-amperometric method was then used for detection and quantification of NO2- and NO in macrophage cells under native and LPS stimulated conditions. Glutathione, a cellular antioxidant, was also measured in these studies and compared with the prooxidant levels in the cells. For further confirmation of NO production in these cells, ME with laser induced fluorescence detection was used for the determination of NO using diaminofluorofluorescein. This same probe and separation was also used to investigate the heterogeneity of NO production in single cells using a cytometric device in collaboration with the Culbertson group. The main future goal of this project is to monitor macrophage cellular heterogeneity during nitrosative stress using an electrochemical cytometric device.