DMPO-OH Radical Formation from 5,5-Dimethyl-1-pyrroline N-Oxide (DMPO) in Hot Water

Abstract

The spin-trapping method with 5,5-dimethyl-1-pyrroline Noxide (DMPO) has been widely accepted as an assay method to measure hydroxyl radical formation, and to detect the hydroxyl radical scavenging activity of a compound. Although unstability of DMPO is claimed because it turns yellow with time even at -20˚C in a sealed tube under vacuum, 1 the basic chemistry of DMPO and DMPO-OH has not been well understood.In a previous work, 2 we noticed very weak DMPO-OH signals in the baseline of a negative control spectrum which was obtained after standing DMPO solution at 37˚C for 24 h. We suspected that in a DMPO aqueous solution at a higher concentration and at a higher temperature, an appropriate amount of DMPO-OH might generate. And we considered that this reaction should provide a practical preparation method to obtain DMPO-OH radical as a simple aqueous solution which contains no other materials except unreacted DMPO. Also this DMPO-OH aqueous solution may be utilized in the investigation of the chemical or physicochemical properties of DMPO-OH radicals.When DMPO was dissolved in purified water and the solution was heated at 70˚C for 30 min, a sufficient amount of DMPO-OH radical formation was observed by ESR ( Fig. 1). Under an argon atmosphere, DMPO-OH formation was minimized to 1/4, and this slightly increased under a dioxygen atmosphere. This indicates that dioxygen participates in the reaction. Previously, Makino et al. observed DMPO-OH formation in an aqueous mixture of DMPO and 1 mM FeCl3. Based on this observation of the formation of iron chelate with DMPO at 77 K, and DMPO-OCH3 formation in the presence of CH3OH, researchers elucidated that nucleophilic attachment of water to DMPO should occur in the presence of Fe 3+ ion. 3 To study the effect of a small number of metal ions in purified water, we replaced the purified water with ultra-pure water for ICP-MS measurements; the latter should contain iron of no more than 1 ppb. In ultrapure water, and under an argon atmosphere, DMPO-OH signal was still observed after heating, although its intensity decreased to about 1/20. In this communication, we describe DMPO-OH radical formation in hot water, in which dissolved dioxygen participates in the reaction and metal ions such as Fe 3+ might catalyze DMPO-OH formation. University, Japan *3 Tokyo Metropolitan Industrial Technology Research Institute, Itabashi, Japan *4 Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuho, Japan When an aqueous solution of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was heated at 70˚C for 30 min, formation of DMPO-OH was observed by ESR. This DMPO-OH radical formation was suppressed under an argon atmosphere. When water was replaced with ultra-pure water for ICP-MS experiments, DMPO-OH radical formation was also diminished. Under an argon atmosphere in ultra-pure water, the intensity of the DMPO-OH signal decreased to about 1/20 of that observed under aerobic conditions with regular purified water. The addition of hydroxyl radical scavengers such as mannitol did not a...

Temperature-dependent free radical reaction in water

“…12 A and B). This observation did not coincide with a previous report by Shoji et al (15) who reported that the formation of DMPO-OH was suppressed under an argon atmosphere, using ultra-pure water, or the addition of EDTA; however, the addition of α-mannitol or DMSO did not affect the formation of DMPO-OH. In contrast to the previous report, the formation of DMPO-OH was suppressed by the relatively high concentration of • OH scavengers in this paper, in which the experimental system contained 0.05 mM DTPA.…”

Synergistic Effect of Thermal Energy on Bactericidal Action of Photolysis of H 2 O 2 in Relation to Acceleration of Hydroxyl Radical Generation

“…Although the yield of DMPO-OH was formed dependently on the temperature under the H(Ϫ)L(ϩ) and H(Ϫ)L(Ϫ) conditions, the level of DMPO-OH in the absence of H 2 O 2 was much lower than that of in the presence of H 2 O 2 . A trace level of DMPO-OH detected under the H(Ϫ)L(ϩ) and H(Ϫ)L(Ϫ) conditions might be formed by the reaction of DMPO and dissolved oxygen at high water temperatures, as reported in a previous study (19). These findings suggest that photolysis and autolysis of H 2 O 2 are endothermal reactions, so that the yield of hydroxyl radical generated by photolysis of H 2 O 2 can be controlled by the temperature of the H 2 O 2 as well as the by the concentration of the H 2 O 2 .…”

Evaluation of Superoxide Anion Radicals Generated from an Aqueous Extract of Particulate Phase Cigarette Smoke by Electron Spin Resonance Using 5,5-Dimethyl-1-pyrroline-N-oxide

“…The obtained spectra agreed with those of DMPO-OH. 15,16,19) The ESR spectra obtained from the HPX-XOD system are shown as B in Fig. 1.…”

“…An Mn 2þ marker built into the ESR spectrometer, which gave reversed-phase signals of Mn 2þ , was simultaneously analyzed as an external reference. 15) The parameters for operating the ESR spectrometer were as follows, unless otherwise stated: microwave power, 4.0 mW; scan field, 335:3 AE 5:0 mT; modulation frequency, 100 kHz; modulation width, 0.1 mT; scan time, 2 min; time constant, 0.1 s; amplitude, 250; dial number of the marker, 650. All spectra were acquired at room temperature.…”