Quantifying ROS Production via EPR

The experimental protocol adopted for ROS detection is shown in Figure 1. For each subject, recruited for exercise procedure, capillary blood was taken from the fingertip before and after (immediately, 10, and 20 minutes) a constant-load exercise.
In the antioxidant treatment, for each subject, capillary blood was drawn from the fingertip before and after R-thioctic acid administration (at 20, 40, 60, 90 minutes, 2, and 3 hours). Control sampling at rest, at the same interval time, was carried out on the same subjects two days before supplementation.
For both experimental procedures, 50 μL of blood, collected in heparinized capillary tubes (Cholestech LDX, Germany), were analyzed (Figure 1(a)). Among spin trapping (otherwise labelled probe) molecules, suitable for biological utilization, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, Noxygen Science Transfer & Diagnostics, Germany) was adopted. A 1 mM CMH solution was prepared in buffer (Krebs-Hepes buffer (KHB) containing 25 μM deferroxamine methane-sulfonate salt (DF) chelating agent and 5 μM sodium diethyldithio-carbamate trihydrate (DETC)) at pH 7.4. Blood was immediately treated with CMH (1 : 1). 50 μL of the obtained solution was put in the glass EPR capillary tube (Noxygen Science Transfer & Diagnostics, Germany), that was placed inside the cavity of the e-scan spectrometer (Bruker, Germany) for data acquisition (Figure 1(b)). The actual amount of solution analyzed was chosen to fill the entire sensitive area of the resonator cavity. Acquisition EPR parameters were: microwave frequency = 9.652 GHz; modulation frequency: 86 kHz; modulation amplitude: 2.28 G; center field: 3456.8 G; sweep width: 60 G; microwave power: 21.90 mW; number of scans: 10; receiver gain: 3.17 · 10¹. Sample temperature was firstly stabilized and then kept at 37°C by the temperature and Gas controller “Bio III” unit, interfaced to the spectrometer. An example of the recorded EPR signal showing the triplet coming from the interaction of the ¹⁴N–OH group of CMH with the ROS oxygen unpaired electron (NOH + O₂^• → NO^• + H₂O₂) is displayed in Figure 1(d). The radicals generated by the reaction of the probe with the blood radicals were acquired and the spectra sequentially recorded for about 5 min in order to calculate the ROS production rate. The EPR signal is proportional to the unpaired electron numbers and could, in turn, be transformed in absolute produced micromoles (μmol · min⁻¹): the stable CP^• (3-Carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) radical signal was recorded in a separate session and used as reference.
The high reproducibility of the EPR measurements is shown up in the plots reported in Figure 2. The data are referred to a couple of EPR measurement data (test I (open squares), test II (closed squares)) performed on blood capillary samples taken from the same healthy subject six hours apart. The data are expressed as arbitrary units and refer to EPR signal double integrals. The regression lines obtained from the collected data show an excellent correlation coefficient (R² = 0.99) resulting in almost superimposable plots: test I (slope: 7.98, intercept: 15.49); test II (slope: 7.95, intercept: 15.95). About 0.5% discrepancy between the ROS absolute production (μmol · min⁻¹) in the two tests was calculated.