DMPO-OH radical formation from 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in hot water.
ABSTRACT When an aqueous solution of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was heated at 70 degrees 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 affect the formation of DMPO-OH, but the signal turned faint in the presence of EDTA. We suggest that DMPO reacted with dissolved oxygen to form DMPO-OH.
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ABSTRACT: The reactive oxygen species generated by an aqueous extract of the particulate phase of cigarette smoke were evaluated by an electron spin resonance (ESR) analysis, using spin-trapping agents, and by comparing with model reaction systems. The ESR signals of DMPO-OH were detected from the extract by using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). These signals were eliminated by adding superoxide dismutase, but hardly by catalase. These responses of the ESR signals to the scavengers were similar to those of a hypoxanthine-xanthine oxidase system. The results indicate that the signals of DMPO-OH from the extract were derived from a reaction product of DMPO with superoxide anion radicals and clarify the mechanism by which the extract generated superoxide anion radicals.Bioscience Biotechnology and Biochemistry 01/2011; 75(1):34-9. · 1.27 Impact Factor
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ABSTRACT: Temperature-dependent free radical reactions were investigated using nitroxyl radicals as redox probes. Reactions of two types of nitroxyl radicals, TEMPOL (4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl) and carbamoyl-PROXYL (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl), were tested in this paper. Heating a solution containing a nitroxyl radical and a reduced form of glutathione (GSH) caused temperature-dependent decay of electron paramagnetic resonance (EPR) signal of the nitroxyl radical. Heating a solution of the corresponding hydroxylamine form of the nitroxyl radical showed EPR signal recovery. The GSH-dependent reduction of nitroxyl radicals at 70°C was suppressed by antioxidants, spin trapping agents, and/or bubbling N(2) gas, although heating carbamoyl-PROXYL with GSH showed temporarily enhanced signal decay by bubbling N(2) gas. Since SOD could restrict the GSH-dependent EPR signal decay of TEMPOL, O(2) (•-) is related with this reaction. O(2) (•-) was probably generated from dissolved oxygen in the reaction mixture. Oxidation of the hydroxylamines at 70°C was also suppressed by bubbling N(2) gas. Heating a solution of spin trapping agent, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) showed a temperature-dependent increase of the EPR signal of the hydroxyl radical adduct of DMPO. Synthesis of hydroxyl radical adduct of DMPO at 70°C was suppressed by antioxidants and/or bubbling N(2) gas. The results suggested that heating an aqueous solution containing oxygen can generate O(2) (•-).Journal of Clinical Biochemistry and Nutrition 01/2012; 50(1):40-6. · 2.25 Impact Factor
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ABSTRACT: The purpose of the present study is to evaluate the effect of thermal energy on the yield of and the bactericidal action of hydroxyl radical generated by photolysis of H(2)O(2). Different concentrations of H(2)O(2) (250, 500, 750, and 1,000 mM) were irradiated with light-emitting diodes (LEDs) at a wavelength of 400 ± 20 nm at 25°C to generate hydroxyl radical. The 500 mM H(2)O(2) was irradiated with the LEDs at different temperatures (25, 35, 45, and 55°C). Electron spin resonance spin trapping analysis showed that the yield of hydroxyl radicals increased with the temperature, as well as the concentration of H(2)O(2). Streptococcus mutans and Enterococcus faecalis were used in the bactericidal assay. The LED-light irradiation of the bacterial suspensions in 500 mM H(2)O(2) at 25°C could hardly kill the bacteria within 3 min, while the bactericidal effect was markedly enhanced with the temperature rise. For instance, a temperature increase to 55°C resulted in >99.999% reduction of viable counts of both bacterial species only within 1 min. The photolysis of 500 mM H(2)O(2) at 55°C could reduce the viable counts of bacteria more efficiently than did the photolysis of 1,000 mM H(2)O(2) at 25°C, although the yields of hydroxyl radical were almost the same under the both conditions. These findings suggest that the thermal energy accelerates the generation of hydroxyl radical by photolysis of H(2)O(2), which in turn results in a synergistic bactericidal effect of hydroxyl radical and thermal energy.Antimicrobial Agents and Chemotherapy 01/2012; 56(1):295-301. · 4.57 Impact Factor
The spin-trapping method with 5,5-dimethyl-1-pyrroline N-
oxide (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,1the basic chemistry of
DMPO and DMPO-OH has not been well understood.
In a previous work,2we 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-OCH3formation in the presence of CH3OH, researchers
elucidated that nucleophilic attachment of water to DMPO
should occur in the presence of Fe3+ion.3To 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 ultra-
pure 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 Fe3+might
catalyze DMPO-OH formation.
5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was obtained from
219ANALYTICAL SCIENCES FEBRUARY 2007, VOL. 23
2007 © The Japan Society for Analytical Chemistry
DMPO-OH Radical Formation from 5,5-Dimethyl-1-pyrroline N-Oxide
(DMPO) in Hot Water
Tomoko SHOJI,*1Linxiang LI,*1Yoshihiro ABE,*1†Masahiro OGATA,*2Yoshihisa ISHIMOTO,*1
Ryoko GONDA,*1Tadahiko MASHINO,*1Masataka MOCHIZUKI,*1Michihisa UEMOTO,*3and
*1 Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato, Tokyo 105–8512, Japan
*2 Faculty of Pharmaceutical Sciences, Aomori University, 2-3-1 Koubata, Aomori 030–0943, Japan
*3 Tokyo Metropolitan Industrial Technology Research Institute, 3-13-10 Nishigaoka, Itabashi,
Tokyo 115–8586, Japan
*4 Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho,
Nagoya 467–8603, 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 affect the formation of DMPO-OH, but the signal turned faint in the presence of EDTA. We
suggest that DMPO reacted with dissolved oxygen to form DMPO-OH.
(Received August 7, 2006; Accepted November 9, 2006; Published February 10, 2007)
†To whom correspondence should be addressed.
DMPO-OH radical formed in a heated aqueous solution of
Labotec Co. (Tokyo, Japan), and used without further purification.
The water used in this study was purified by ion-exchange
resin (18.2 MΩ). Ultra-pure water for ICP-MS was obtained by
two successive distillations of purified water with an all-quartz
apparatus, in which Fe3+ion was not detected (at most 1 ppb).
In experiments under an argon atmosphere, water was deaerated
under reduced pressure using an aspirator, and then stored under
argon by attaching an argon balloon.
An electron spin resonance spectrometer, JES-RE1X (JEOL,
Tokyo, Japan), and a JEOL flat quartz cell were used. The
conditions were: field, 336 ± 5 mT width; power, 4 mW; field
modulation, 0.200 mT; time constant, 0.1; and amplitude, 500.
A manganese signal was used for the external standard. Pyrex
glassware was washed with 1 M nitric acid by sonication, rinsed
well with purified water, and air dried.
DMPO-OH radical formation in hot water
Neat DMPO was dissolved in purified water to make a 2.5%
aqueous solution. In cases of pH controlled experiments, 2.5%
DMPO solution in an acetate buffer or a phosphate buffer was
used. A 2-mL volume of sample was heated in a test tube for
30 min in a water bath at 70˚C. Under an argon atmosphere or a
dioxygen atmosphere, 10 mL of 2.5% DMPO aqueous solution
was introduced into a 25 mL round-bottomed flask, and a three-
way stopcock with an argon (or dioxygen) balloon was attached.
The solution was deaerated under reduced pressure (aspirator),
and argon (or dioxygen) was introduced from the balloon. After
heating at 70˚C for 30 min, an aliquot was removed and was
measured by ESR just 1 min after the tube was taken out of the
Results and Discussion
After heating the aqueous solution of DMPO at 70˚C for 30
min, we observed typical ESR signals of DMPO-OH radical
with an intensity ratio of 1:2:2:1 (AH1.50 mT, AN1.50 mT; Ref.
4, AH1.53 mT, AN1.53 mT) (Figs. 1 and 2).
It was reported that Fe3+ions catalyze the addition of water to
DMPO to produce DMPO-OH radical.3In Fig. 2, however, its
formation decreased under Ar atmosphere (Figs. 2(b) and (d)),
also in ultra-pure water under aerobic conditions (Fig. 2(c)). In
all spectra in Fig. 2, unknown weak signals can be seen.
DMPO-OH radical formation by heating was not suppressed by
the addition of a hydroxyl radical scavenger, mannitol (1 – 100
mM) or DMSO (1 – 100 mM). This indicates that DMPO-OH
radical should not be formed via hydroxyl radical attachment to
DMPO. However, DMPO-OH signal intensity was decreased to
about 1/6 in the presence of 1 mM of EDTA. Thus, it was
suspected that dissolved dioxygen, which is a biradical
molecule, should be added directly to DMPO, where catalytic
participation of metal ions might exist (Eq. (1)). Biradical
might produce a DMPO-OOH radical, and this would develop
into a DMPO-OH radical quickly at higher temperatures.
DMPO-OH signals were also observed in acetate buffer (pH
5.0 – 6.8), phosphate buffer (pH 7.0 – 10.0) or disodium
phosphate solution of which the pH was adjusted with sodium
hydroxide solution (pH 13.0) (Fig. 3). The half-lives of DMPO-
OH5or DMPO-OOH6,7at basic pH are much shorter than those
at neutral or acidic conditions, and the decrease of the intensity
of DMPO-OH signal can be considered as the unstability of
these radical species. The optimum pH was found to be around
pH 8.5, thus, the catalytic action of H+may not be plausible.
DMPO + O2⎯ →•[DMPO-OO]•
•[DMPO-O2]•+ H2O ⎯ → [DMPO-OOH]•+ •OH (2)
[DMPO-OOH]•⎯ → [DMPO-OH]•
Even at low concentrations, transition metal ions such as Fe3+
may catalyze the first step (Eq. (1)), or catalyze the reaction of
[DMPO-O2] biradical with water to [DMPO-OOH]•(Eq. (2)),
although we could not detect [DMPO-OOH] signals nor OH
radical formation by the addition of DMSO. DMPO-OOH
radical is known to be labile so as to decompose to DMPO-OH
with a half-time of 50 s at pH 76,7(Eq. (3)), and the
decomposition mechanisms of DMPO-OOH adducts to DMPO-
OH are discussed by Villamena et al.8
The formation of DMPO-OH with time was traced at 70˚C
(Fig. 4). It was found that the reaction reached almost steady
state after 60 min. After dioxygen in the reaction mixture is
consumed, the supply of dioxygen to the reaction solution
220ANALYTICAL SCIENCES FEBRUARY 2007, VOL. 23
aqueous solution of DMPO after heating at 70˚C for 30 min (a). Four
strong signals in the mid-section are of the DMPO-OH radical (AH
1.50 mT, AN 1.50 mT), and two side signals are of Mn used as an
external reference. The ESR spectrum of DMPO aqueous solution
heated under an argon atmosphere (b); under aerobic conditions in
ultra-pure water (c); under an argon atmosphere in ultra-pure water (d).
The ESR spectrum of DMPO-OH radical observed in an
(external reference), which is the highest at around pH 8.5.
Effect of pH on the relative intensity of DMPO-OH vs. Mn
should be the rate-determining step.
DMPO-OH formation in an aqueous solution of DMPO was
also observed at 37˚C after 24 h, indicating that radical trapping
experiments in an intact animal using DMPO over 20 h may
provide misleading results (Fig. 5). However, in the standard
experimental procedure, spin-trapping experiments should be
carried out at room temperature or lower, and over a short time,
DMPO-OH formation could be neglected.
The mechanism of DMPO-OH radical formation remains
uncertain; however, we report here the observed evidence that
DMPO should react with dioxygen in water to generate DMPO-
OH radical under aerobic conditions at 70˚C.
In conclusion, DMPO-OH radical was detected after heating a
DMPO aqueous solution at 70˚C for 30 min. In the presence of
dioxygen, DMPO-OH production was enhanced. The catalytic
participation of metal ions was also suspected, because
decreased formation of DMPO-OH was observed when regular
purified water was replaced with ultra-pure water.
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7. B. Tuccio, R. Lauricella, J. C. Bouteiller, and P. Tordo, J.
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221ANALYTICAL SCIENCES FEBRUARY 2007, VOL. 23
reference) with time. The ESR signal of DMPO-OH radical in a
2.5% DMPO solution adjusted at pH 7.4 using a phosphate buffer,
was recorded just 1 min after taking out the reaction tube which was
heated at 70˚C and then cooled at 0˚C for 15 s in a water-bath.
The relative intensity of DMPO-OH vs. Mn (external
solution of DMPO (dissolved in milliQ water) under aerobic conditions.
ESR signal of DMPO-OH radical observed in an aqueous