Strategies to Monitor Oxidative Stress
The overall objective of this project is to investigate oxidative stress arising from ischemia-reperfusion in vivo. Oxidative stress occurs when an excess of oxygen free radicals (OFRs), such as superoxide (O2-·) and the hydroxyl radical (·OH), are present in the body. OFRs have been associated with events such as myocardial infarctions and strokes. To investigate mechanisms of oxidative damage, our group is interested in studying the levels of spin-trapped OFRs (measure of free radicals themselves), antioxidants (measure of scavenger depletion, glutathione), and 8-hydroxy-2’-deoxyguanosine (measure of DNA damage).
In particular, we will investigate various proposed pathways of OFR formation in vivo using microdialysis sampling. Microdialysis sampling provides a means to continuously monitor several biochemical components involved in oxidative stress at specific tissue sites in an experimental animal. Microdialysis experiments can, therefore, provide both temporal and spatial information about OFR formation and the physiological response to it.
Microdialysis can also be used to deliver compounds, such as enzyme inhibitors and radical trapping agents, in high concentrations to well defined tissue regions in vivo. These abilities will allow us to resolve questions that have remained unclear using plasma and urine sampling and standard tissue sampling techniques.
Oxygen Free Radicals
Difficulties arise in the analysis of OFRs due to their short half-lives. For example, the estimated half-life of the hydroxyl radical in cells is reported to be 10-9 s. The indirect detection of these species has been achieved using trapping agents which form stable radical adducts with the OFRs, and enables their detection by electron paramagenetic resonance spectroscopy (EPR). However, EPR detection suffers from hampered sensitivity due to water in biological systems absorbing microwave radiation, increased amounts of free radicals resulting from necessary sample handling, and the occurrence of artifacts and spurious signals. Currently, our group is investigating the analysis of the hydroxyl radical, using 4-hydroxybenzoic acid (4-HBA) as the trapping agent. The in vivo formation of the hydroxyl radical was monitored through the generation of the radical adduct, 3,4-dihydroxybenzoic acid (3,4 DHBA) using capillary electrophoresis. Capillary techniques are more compatible with the small sample volumes produced by microdialysis than are conventional separation techniques.
Glutathione (GSH) is an endogenous compound found to play a key role in the defense against oxidative stress through two main mechanisms: scavenging of free radicals and acting as a cofactor for oxygen damage-protecting enzymes. There are several unresolved issues relating to GSH and its role during an ischemic event in the body. It is unclear whether GSH levels increase inside the cell or only in extracellular fluid, and whether this accumulation occurs during the onset of ischemia or during reperfusion. Our group has found initial evidence that during ischemia, unidirectional efflux of GSH from cells occurs.
In addition, most experiments only determine reduced glutathione (GSH) levels. Since the ratio of reduced GSH to oxidized GSSG is commonly used as an index of oxidative stress in the body, it is necessary to develop methods that can accurately and simultaneously measure the levels of both forms of GSH to determine if this ratio is indeed a reliable indicator of oxidative stress. Microdialysis coupled to capillary electrophoresis with electrochemical detection (CE-EC) provides the direct detection of both GSH and GSSG. It would be of interest to know how the change in GSH levels corresponds to the change in other damage-protecting agents or free radicals during ischemia.
The most common type of damage caused by reactive oxygen species (ROS) in the body is oxidative DNA damage, resulting in cell mutation and cell death. 8-hydroxydeoxyguanosine (8-OHdG), a product of this type of DNA damage, is used as a biomarker for oxidative stress. Many studies rely on tissue samples to correlate 8-OHdG levels with events thought to cause oxidative stress such as ischemia-reperfusion, a model for stroke or heart attack. Microdialysis sampling offers several advantages over tissue sampling, including site-specific, highly time-resolved information and a much simpler sample matrix. The small sample volume associated with microdialysis makes CE well suited for analysis of dialysate samples. However, the high conductivity of such samples can cause significant band broadening, or sample “destacking.” Base-mediated sample stacking was used to enhance the sensitivity in the detection of 8-OHdG in high-ionic strength samples using CE. Advantages to base-stacking also include increased sample-loading capacity in the capillary and no sample pretreatment. Detection limits in the nanomolar range have been achieved for both EC and UV detection, and peak efficiencies of 4 million plates can be routinely achieved using base-stacking.
Mass Spectrometry Analysis of 8-OHdG
Capillary electrophoresis-mass spectrometry (CE/MS) is used in the Lunte group for the analysis 8-OHdG to study oxidative stress. Mass spectrometry provides sensitive and selective detection, and valuable structural information. Coupling CE to mass spectrometry can enhance the performance of both techniques, making it possible to separate, selectively detect, and identify 8-OHdG in urine. Furthermore, other nucleosides of interest may be identified with mass spectrometric detection. The Lunte group uses a sheath-flow interface to couple CE to mass spectrometry. This interface relies on a ‘make-up’ flow liquid in order to make the CE effluent more amenable to electrospray ionization and also serves as a means to effectively ground the cathodic end of the separation capillary. A Micromass Platform LC single quadrupole mass spectrometer is used for method development. In addition, the CE/MS interface can be easily attached to the Chemistry Department’s other mass spectrometers including the quadrupole-time-of-flight (Q-TOF), which has the advantage of higher sensitivity and faster scan rates relative to the single quadrupole.