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Hyperthermia stimulates nitric oxide formation: Electron paramagnetic resonance detection of ·NO-hemoglobin in blood
Session 4: Nitric Oxide and Peroxynitrite 4:21
PHENOXYL RADICAL FORMATION ADDITION WITH NITRIC OXIDE BUTYLPHENOLS Allan L. Wilcox and Edward G. Janzen,
AND REVERSIBLE AND 2,6-DI-m-
National Biomedical Center for Spin Trapping and Free Radicals, Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104 USA The chemistry of nitric oxide is relatively unexplored. We have asked the question, does ‘NO react with phenols to give the phenoxyl radical, and if so does the phenoxyl radical react with nitric oxide? Is the reaction reversible and what is the structure of the ‘NO adduct of the phenoxyl radical? Nitrites and Cnitroso compounds are possibilities. By EPR spectroscopy it can be shown that phenoxyl radicals are formed slowly from the reaction of ‘NO and 2,6-di-E-butylphenol derivatives or tocopherol. Further reactions of ‘NO with the former can be demonstrated but not for the latter. The EPR signal of the tocopheroxyl radical disappears at the same rate in the presence or absence of ‘NO. For the 2,6-di-m-butyl phenols such as BHT the nitric oxide adducts are C-nitroso compounds with addition to the 2- or 4-positions as established by NMR spectroscopy. Nitric oxide adduct formation is reversible is some cases. The rate of addition of ‘NO to phenoxyl radicals is surprisingly slow as evidenced by the fact that the EPR signal is broadcncd by excess ‘NO in a similar fashion to the broadening observed with dioxygen. These observations will be described in homogenous as well as micellar solutions,
4:~s
~~RTRERhm ST~TFX~ mTmc oxm~ FORMATION: ELECTRON PARAMA GNETIC RESONANCE DETECTION OF *NO-HEMOGLGBIN IN BIGOD Carry R. Buettner. David M. Hall, Ronald D. Matthes & Carl V. Gisoltl. ESR Facility Science,
The University
Prolonged dilation oxide
hyperthermia
results
of the splanchnic (*NO)
has
vasodilator,
been
vascular
circulatory
events
bed,
be
a
hyperthermia
oxide within the
samples.
These
the classic
results
an enhanced
the
catastrophic
Using electron
rats.
we
observed
complex.
nitrogen
triplet
suggest
the
(*NO-Hb) hyperfine
structure, aN = 17.5 G. centered at g = 2.012. species was not seen in corresponding arterial stimulates
nitric potent
spectroscopy to scan whole blood uiuo from the portal vein of
hyperthermic displays
Because
severe
of nitric
USA
life-threatening to
precipitating
of a nitric oxide-hemoglobin
This signal
a
that lead to heatstoke.
paramagnetic resonance sampies collected in unrestrained
that
of Exercise
, IA 52242
bed.
demonstrated
the local release
splanchnic
in
vascular
we hypothesized
would trigger
formation
and Department
of Iowa, Iowa City
This blood
that severe hyperthennia
local release
of
*NO within
the
splanchnic circulation, which may explain the sudden loss of compensatory splanchnic vasoconstriction that immediately preceeds death from heatstroke.
499
NITRIC OXIDE REQUIRES SUPEROXIDE BACTERICIDAL ACTIVITY Luca Brunelli and Joseph S. Beckman, Department of Anesthesiology, The University Birmingham,
Birmingham,
Alabama,
TO
EXERT
of Alabama
4:22
at
35233 USA
It is still unclear whether nitric oxide (NO) is directly toxic because, both in uivo and in vitro, secondary reactions with the ubiquitous superoxide (02.) cannot be easily ruled out. NO and 02. will react at near diffusion limit (Huie and Padmaja, Free Rad Res Comm 1993, in press) to form the potent oxidant peroxynitrite (ONOO). E.coli exposed to 1 mM NO for up to one hour in both aerobic and anaerobic conditions did not show decreased viability. However, ONOOkdling was proportional to its concentration with an LDs0 of 0.25 mM after ten minutes of exposure. The latter finding is in agreement with previous reports (Zhu et al, Arch Biochem Biophys 298, 452; 1992). The decomposition of the sydnonimine SIN-l also produces ONOO- through the release of NO and 02.. SIN-l killing was proportional to its concentration with an LDso of 0.5 mM after one hour exposure. The bactericidal activity of SIN-I was enhanced by 02-, since exposure to 0.5 mM SIN-l plus pterin/xanthine oxidase (X0) resulted in 0.1% survival. Interestingly, the addition of FeEDTA to the same amounts of SIN-l and pterin/XO, resulted in almost complete protection. Pterin/XO alone or together with FeEDTA showed only a slight decrease in viability. Our results prove that NO alone is not toxic to E.&i. The presence of 02. is required for NO to exert bactericidal activity through the production of ONOO-. The release of 02. by SIN-1 is likely to be the limiting factor in ONOO- production. Furthermore, ONOO- exerts a much greater toxicity to E.coli compared to 02., hydrogen peroxide (H202) and hydroxyl radical (OH’). The protectlon exerted by FeEDTA can be due to decreased 02.because of its funneling to OH’. Alternatively, Fe could react directly with NO, thus reducing the other fundamental chemical species m ONOO- production.
PHENYL N-~~~~~~~~~~~~~~ (PBN) As A ~R~cmsoR OF NITROGEN OXIDES RESULTING FROM IRON (III)CATALYZED HYDROLYSIS AND HYDROXYL RADICAL ADDUCT FORMATION Walee Chamulitrat, Carol E. Parker, Kenneth B. Tomer, and Ronald P. Mason Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 USA. PBN is a spin trap commonly employed to detect free radicals in vivo which has been shown to have both adverse and beneficial effects on various biological functions. We report here evidence that PBN is decomposed by two pathways leading to the generation of nitric oxide and nitrite. The first pathway is by the hydrolysis of PBN which is catalyzed by ferric iron. The second pathway is by hydroxyl radical adduct formation of PBN. Nitric oxide was trapped by cysteine and ferrous iron to form a [(cys)2Fe(N0)2J-3
complex which was measured by use
of electron paramagnetic resonance CEPR) spectroscopy. Benzaldehyde was determined from both reaction mixtures. Mechanistically, we propose that PBN is hydrolyzed by ferric iron or attacked by hydroxyl radical leading to a transient species, tert-buiyl hydronitroxide which is further oxidized to a nitric oxide source. Our in vitro data imply that PBN may decompose to nitric oxide when used in biological systems which are under high oxidative stress conditions.
4:24
PHENOXYL RADICAL FORMATION ADDITION WITH NITRIC OXIDE BUTYLPHENOLS Allan L. Wilcox and Edward G. Janzen,
AND REVERSIBLE AND 2,6-DI-m-
National Biomedical Center for Spin Trapping and Free Radicals, Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104 USA The chemistry of nitric oxide is relatively unexplored. We have asked the question, does ‘NO react with phenols to give the phenoxyl radical, and if so does the phenoxyl radical react with nitric oxide? Is the reaction reversible and what is the structure of the ‘NO adduct of the phenoxyl radical? Nitrites and Cnitroso compounds are possibilities. By EPR spectroscopy it can be shown that phenoxyl radicals are formed slowly from the reaction of ‘NO and 2,6-di-E-butylphenol derivatives or tocopherol. Further reactions of ‘NO with the former can be demonstrated but not for the latter. The EPR signal of the tocopheroxyl radical disappears at the same rate in the presence or absence of ‘NO. For the 2,6-di-m-butyl phenols such as BHT the nitric oxide adducts are C-nitroso compounds with addition to the 2- or 4-positions as established by NMR spectroscopy. Nitric oxide adduct formation is reversible is some cases. The rate of addition of ‘NO to phenoxyl radicals is surprisingly slow as evidenced by the fact that the EPR signal is broadcncd by excess ‘NO in a similar fashion to the broadening observed with dioxygen. These observations will be described in homogenous as well as micellar solutions,
4:~s
~~RTRERhm ST~TFX~ mTmc oxm~ FORMATION: ELECTRON PARAMA GNETIC RESONANCE DETECTION OF *NO-HEMOGLGBIN IN BIGOD Carry R. Buettner. David M. Hall, Ronald D. Matthes & Carl V. Gisoltl. ESR Facility Science,
The University
Prolonged dilation oxide
hyperthermia
results
of the splanchnic (*NO)
has
vasodilator,
been
vascular
circulatory
events
bed,
be
a
hyperthermia
oxide within the
samples.
These
the classic
results
an enhanced
the
catastrophic
Using electron
rats.
we
observed
complex.
nitrogen
triplet
suggest
the
(*NO-Hb) hyperfine
structure, aN = 17.5 G. centered at g = 2.012. species was not seen in corresponding arterial stimulates
nitric potent
spectroscopy to scan whole blood uiuo from the portal vein of
hyperthermic displays
Because
severe
of nitric
USA
life-threatening to
precipitating
of a nitric oxide-hemoglobin
This signal
a
that lead to heatstoke.
paramagnetic resonance sampies collected in unrestrained
that
of Exercise
, IA 52242
bed.
demonstrated
the local release
splanchnic
in
vascular
we hypothesized
would trigger
formation
and Department
of Iowa, Iowa City
This blood
that severe hyperthennia
local release
of
*NO within
the
splanchnic circulation, which may explain the sudden loss of compensatory splanchnic vasoconstriction that immediately preceeds death from heatstroke.
499
NITRIC OXIDE REQUIRES SUPEROXIDE BACTERICIDAL ACTIVITY Luca Brunelli and Joseph S. Beckman, Department of Anesthesiology, The University Birmingham,
Birmingham,
Alabama,
TO
EXERT
of Alabama
4:22
at
35233 USA
It is still unclear whether nitric oxide (NO) is directly toxic because, both in uivo and in vitro, secondary reactions with the ubiquitous superoxide (02.) cannot be easily ruled out. NO and 02. will react at near diffusion limit (Huie and Padmaja, Free Rad Res Comm 1993, in press) to form the potent oxidant peroxynitrite (ONOO). E.coli exposed to 1 mM NO for up to one hour in both aerobic and anaerobic conditions did not show decreased viability. However, ONOOkdling was proportional to its concentration with an LDs0 of 0.25 mM after ten minutes of exposure. The latter finding is in agreement with previous reports (Zhu et al, Arch Biochem Biophys 298, 452; 1992). The decomposition of the sydnonimine SIN-l also produces ONOO- through the release of NO and 02.. SIN-l killing was proportional to its concentration with an LDso of 0.5 mM after one hour exposure. The bactericidal activity of SIN-I was enhanced by 02-, since exposure to 0.5 mM SIN-l plus pterin/xanthine oxidase (X0) resulted in 0.1% survival. Interestingly, the addition of FeEDTA to the same amounts of SIN-l and pterin/XO, resulted in almost complete protection. Pterin/XO alone or together with FeEDTA showed only a slight decrease in viability. Our results prove that NO alone is not toxic to E.&i. The presence of 02. is required for NO to exert bactericidal activity through the production of ONOO-. The release of 02. by SIN-1 is likely to be the limiting factor in ONOO- production. Furthermore, ONOO- exerts a much greater toxicity to E.coli compared to 02., hydrogen peroxide (H202) and hydroxyl radical (OH’). The protectlon exerted by FeEDTA can be due to decreased 02.because of its funneling to OH’. Alternatively, Fe could react directly with NO, thus reducing the other fundamental chemical species m ONOO- production.
PHENYL N-~~~~~~~~~~~~~~ (PBN) As A ~R~cmsoR OF NITROGEN OXIDES RESULTING FROM IRON (III)CATALYZED HYDROLYSIS AND HYDROXYL RADICAL ADDUCT FORMATION Walee Chamulitrat, Carol E. Parker, Kenneth B. Tomer, and Ronald P. Mason Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 USA. PBN is a spin trap commonly employed to detect free radicals in vivo which has been shown to have both adverse and beneficial effects on various biological functions. We report here evidence that PBN is decomposed by two pathways leading to the generation of nitric oxide and nitrite. The first pathway is by the hydrolysis of PBN which is catalyzed by ferric iron. The second pathway is by hydroxyl radical adduct formation of PBN. Nitric oxide was trapped by cysteine and ferrous iron to form a [(cys)2Fe(N0)2J-3
complex which was measured by use
of electron paramagnetic resonance CEPR) spectroscopy. Benzaldehyde was determined from both reaction mixtures. Mechanistically, we propose that PBN is hydrolyzed by ferric iron or attacked by hydroxyl radical leading to a transient species, tert-buiyl hydronitroxide which is further oxidized to a nitric oxide source. Our in vitro data imply that PBN may decompose to nitric oxide when used in biological systems which are under high oxidative stress conditions.
4:24