An X-band EPR instrument (E-Scan—Bruker BioSpin, GmbH, MA USA) was adopted for determinations. The instrument allows us to deal with very low-concentration amounts of paramagnetic species in small (50 μL) samples. As is well known, ROS half-life is too short if compared to the EPR time scale so that they result in being EPR-invisible but become EPR detectable once “trapped” and transformed in a more stable radical species. Among spin trapping or spin probe molecules, suitable for biological utilization, CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) probe was adopted.
For each recruited subject, ROS production rate was determined at rest by means of a recently developed EPR method [16 ] analyzing 50 μL samples immediately treated with CMH solution (1 : 1). 50 μL of the obtained solution was put in a glass EPR capillary tube (Noxygen Science Transfer & Diagnostics, Germany) that was placed inside the cavity of the E-scan spectrometer for data acquisition (Figure 1 ).
Acquisition parameters were microwave frequency 9.652 GHz; modulation frequency 86 kHz; modulation amplitude 2.28 G; sweep width 60 G, microwave power 21.90 mW, number of scans 10; and receiver gain 3.17·101. Sample temperature was firstly stabilized and then kept at 37°C by the Temperature & Gas Controller “Bio III” unit, interfaced to the spectrometer. Spectra were recorded and analyzed by using Win EPR software (2.11 version) standardly supplied by Bruker.
EPR measurements allowed us to attain a relative quantitative determination of ROS production rate in samples. All data were, in turn, converted in absolute concentration levels (μmol·min−1) by adopting CP ∙ (3-Carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) stable radical as external reference.
For each recruited subject, ROS production rate was determined at rest by means of a recently developed EPR method [16 ] analyzing 50 μL samples immediately treated with CMH solution (1 : 1). 50 μL of the obtained solution was put in a glass EPR capillary tube (Noxygen Science Transfer & Diagnostics, Germany) that was placed inside the cavity of the E-scan spectrometer for data acquisition (
Acquisition parameters were microwave frequency 9.652 GHz; modulation frequency 86 kHz; modulation amplitude 2.28 G; sweep width 60 G, microwave power 21.90 mW, number of scans 10; and receiver gain 3.17·101. Sample temperature was firstly stabilized and then kept at 37°C by the Temperature & Gas Controller “Bio III” unit, interfaced to the spectrometer. Spectra were recorded and analyzed by using Win EPR software (2.11 version) standardly supplied by Bruker.
EPR measurements allowed us to attain a relative quantitative determination of ROS production rate in samples. All data were, in turn, converted in absolute concentration levels (μmol·min−1) by adopting CP ∙ (3-Carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) stable radical as external reference.
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