Spin trap depmpo

Chemicals used in the synthesis and spectroscopic EPR measurements were obtained from Merck (Darmstadt, Germany), except for spin-trap DEPMPO, which was purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Chemicals used in the experiments were obtained from Merck (Darmstadt, Germany) except for spin-trap DEPMPO which was purchased from Enzo Life Sciences (Farmingdale, NY, USA). All of the chemicals were of the analytical grade. The IR vibrational spectra were recorded on the Perkin-Elmer Spectrum (Bruker, Germany. Model: 197). One FT-IR spectrometer using the KBr pellet technique. The NMR spectra were obtained on the Varian Gemini spectrometer (Varian, Palo Alto, CA, 200 MHz for ¹H and 50 MHz for ¹³C) in DMSO-d6. The elemental microanalysis (C, H, N) was performed on the Elemental analysis system VARIO EL III CHNOS, model—Elementar Analysensysteme (GmbH, VarioEL, Germany), 2003 in the Centre for Instrumental Analysis, at the Faculty of Chemistry, University of Belgrade.

Radical-generating systems were prepared as described previously (Miljkovic et al., 2015 (link)). The ability of benfotiamine to scavenge hydroxyl radical (^·OH) was tested using the Fenton system (Fe²⁺ + H₂O₂ → Fe³⁺ + OH⁻ + ^·OH). The Fenton reaction was performed in PBS (pH = 7.4) by combining 1 mM of H₂O₂ (Renal, Budapest, Hungary) and 0.2 mM of FeSO₄(Merck, Darmstadt, Germany). Spin trap DEPMPO (5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide; Enzo Life Sciences International, Plymouth Meeting, PA, USA; 10 mM final concentration) was added prior to H₂O₂. The time period between the initiation of reaction and EPR measurements was 2 min. Benfotiamine was supplemented before the initiation of reaction at the final concentration of 1 mM.
Superoxide was generated using ^·O⁻₂ thermal source SOTS-1 (Cayman Chemicals, Ann Arbor, MI, USA). Immediately prior to the start of any experiment the SOTS-1 was dissolved in DMSO, and was further supplemented to PBS solution containing DEPMPO (10 mM) and DTPA (1 mM; chelating agent, which is added in order to suppress the redox activity of transition metals impurities in ^·OH-generating Haber-Weiss reaction), to a final concentration of 0.2 mM. This system was incubated for 5 min at 37°C. The time period between the end of incubation and EPR measurements was 2 min.