Superoxide production by Electron Paramagnetic Resonance Spectroscopy (EPR)

Superoxide production in plasma, bronchoalveolar lavage fluid (BALF) and lung was measured by EPR using compartment-specific EPR spin probes to detect cellular or mitochondrial superoxide [17, 18]. CPH and CMH are both cell-permeable EPR spin probes that react with high sensitivity with superoxide, as well as radicals derived from superoxide including peroxynitrite, but do not react with hydrogen peroxide [19–21]. Mito-TEMPO-H is an EPR spin probe targeted to the mitochondria that reacts with mitochondrial superoxide [22]. Following the oxidation of the spin probes, the resultant nitroxide radicals were detected by EPR using the parameters described below.

Seven-days post bleomycin treatment, mice were injected with CPH, and plasma was collected to determine circulating extracellular superoxide levels. CPH was first dissolved in degassed KHB containing the following ion chelators, 25 μM deferoxamine mesylate salt and 5 μM diethyl dithiocarbamate, for a final stock CPH concentration of 2 mg/ml. CPH was delivered as a 9 mg/kg intraperitoneal bolus followed by a single subcutaneous dose of 13.5 mg/kg as previously described, based on an average weight of 20 g per mouse [23]. One hour after injecting the CPH spin probe, mice were anesthetized, and blood was drawn via right ventricular puncture into a syringe coated with 1000 USP/mL heparin. Plasma was collected by spinning the blood at 3000 rpm for 10 min. 50 μl of plasma was loaded in EPR capillary tube for EPR measurements at room temperature (RT).

Lung tissue was also obtained from the mice injected with CPH as described above to detect lung cellular superoxide. Following collection of blood and euthanasia, the chest was opened, and lungs were flushed with 10 ml cold PBS via the right ventricle to remove blood. 10 mg of lung tissue was cut, weighed and placed in a tissue cell, an accessory used to detect EPR signal from tissues (Bruker BioSpin). EPR measurements were performed at RT. The tissue cell accessory was cleaned with ethanol between samples.

To detect cellular superoxide in BALF cells, a separate cohort of mice were euthanized 7 days post bleomycin and a cannula was surgically placed in the trachea. BALF was collected by slowly instilling and withdrawing via the tracheal cannula five (1 mL) aliquots of PBS containing the metal chelator, DTPA (100 μM). BALF cells were collected by pooling the 5 BALF aliquots and spinning down the cells at 700 g for 7 min. The cells were resuspended in KHB contains 100 μM DTPA and incubated with CMH 0.4 mM final concentration for 50 min at 37C. The samples were loaded in Teflon tubing, flash frozen in liquid nitrogen and measured at 77K [17].

To detect lung mitochondrial superoxide, a separate cohort of mice were euthanized 7 days post bleomycin. Lungs were lavaged, flushed and fresh lung tissue was weighed (100 mg) and homogenized in 300 μl Tris-EDTA buffer containing 0.25 M sucrose using the BEAD RUPTOR (OMANI). The tissue homogenate was immediately placed on ice and homogenate transferred to a 1.5 mL Eppendorf tube. 30 μl homogenate was added to 165 μl KHB + 100 μM DTPA, treated with 5 μl of the mitochondrial specific EPR spin probe, mito-TEMPO-H (9.5 mM stock) and incubated for 1h at 37 °C. After 1h, 150 μl was loaded into PTFE tubing, flash frozen in liquid nitrogen and measured at 77K.

EPR measurements at RT used the following EPR acquisition parameters: microwave frequency = 9.65 GHz; center field = 3432 G; modulation amplitude = 2.0 G; sweep width = 80 G; microwave power = 19.9 mW; total number of scans = 10; sweep time = 12.11 s; and time constant = 20.48 ms.

EPR measurements at 77k used the following EPR acquisition parameters: microwave frequency = 9.65 GHz; center field = 3438 G; modulation amplitude =4.0 G or 6 G; sweep width = 150 G; microwave power = 0.316 mW; total number of scans = 2; sweep time = 60 s; and time constant = 1.28 ms.

The nitroxide radical concentration was obtained by simulating the spectra using the SpinFit module incorporated in the Xenon software of the bench-top Bruker EMXnano EPR spectrometer or by double integration followed by the SpinCount module (Bruker).