Several manuscripts have published protocols relevant to these goals, using cell-permeable, cell-impermeable, and mitochondrial targeted spin probes to target different cellular compartments in vitro and process tissue for analysis in mouse models 14, 15. The goal of this project is to demonstrate practical EPR methods for designing experiments and preparing samples to detect superoxide using spin probes in different cellular compartments in vitro and in different tissue compartments in vivo. Applications for spin trapping have been well-documented in biomedical research 9, 10, 11, 12, 13. While spin traps exhibit specificity, with distinct spectral patterns depending on the trapped species, they have slow kinetics for superoxide spin trapping and are prone to biodegradation of the radical adducts. Cyclic hydroxylamines react with superoxide 100 times faster than spin traps, enabling them to compete with cellular antioxidants, but they lack specificity and require the use of appropriate controls and inhibitors to identify the radical species or source responsible for the nitroxide signal. One commonly used class of spin probes are cyclic hydroxylamines, which are EPR-silent and react with short-lived radicals to form a stable nitroxide. Both classes (spin probes and spin traps) have advantages and limitations. Spin traps have also been developed to capture short-lived radicals and form long-living adducts, which facilitates detection by EPR 8.
To facilitate the use of EPR for biological studies, a variety of spin probes have been synthesized that can measure a range of biologically relevant free radical species as well as pO 2, pH, and redox states 2, 3, 4, 5, 6, 7. Though fluorescent probes are relatively inexpensive and easy to use and provide rapid, sensitive detection of ROS, they do have serious limitations due to artifactual signals, an inability to calculate ROS concentrations, and a general lack of specificity 1. Spin probes have advantages over the more commonly used fluorescent probes. An electron paramagnetic resonance (EPR) technique is the most unambiguous method for detecting free radicals. While measures of oxidative stress and reactive oxygen species are important to the study of diverse diseases across all organ systems, the detection of reactive oxygen species (ROS) is challenging due to a short half-life and high reactivity. The samples can be stored in specialized tubing stable at -80 ☌ and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 ☌ and analyzed by EPR at 77 K. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Spin probes and EPR spectroscopy can also be applied to in vivo models. The mitochondrial 1-hydroxy-4-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4- piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide). In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD, allows for the differentiation of extracellular from cytosolic superoxide. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously.
The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings.