(Received for publication, July 12, 1995; and in revised form, October 23, 1995)
From the
The rapid and spontaneous interaction between superoxide
(O) and nitric oxide (NO) to yield the
potent oxidants peroxynitrite (ONOO
) and
peroxynitrous acid (ONOOH), has been suggested to represent an
important pathway by which tissue may be injured during inflammation.
Although several groups of investigators have demonstrated substantial
oxidizing and cytotoxic activities of chemically synthesized
ONOO
, there has been little information available
quantifying the interaction between O
and NO in the absence or the presence of redox-active iron. Using
the hypoxanthine (HX)/xanthine oxidase system to generate various
fluxes of O
and H
O
and the spontaneous decomposition of the spermine/NO adduct to
produce various fluxes of NO, we found that in the absence of
redox-active iron, the simultaneous production of equimolar fluxes of
O
and NO increased the oxidation of
dihydrorhodamine (DHR) from normally undetectable levels to
approximately 15 µM, suggesting the formation of a potent
oxidant. Superoxide dismutase, but not catalase, inhibited this
oxidative reaction, suggesting that O
and not hydrogen peroxide (H
O
) interacts
with NO to generate a potent oxidizing agent. Excess production of
either radical virtually eliminated the oxidation of DHR. In the
presence of 5 µM Fe
-EDTA to insure
optimum O
-driven Fenton chemistry, NO
enhanced modestly HX/xanthine oxidase-induced oxidation of DHR. As
expected, both superoxide dismutase and catalase inhibited this
Fe-catalyzed oxidation reaction. Excess NO production with respect to
O
flux produced only modest inhibition
(33%) of DHR oxidation. In a separate series of studies, we found that
equimolar fluxes of O
and NO in the
absence of iron only modestly enhanced hydroxylation of benzoic acid
from undetectable levels to 0.6 µM 2-hydroxybenzoate. In
the presence of 5 µM Fe
-EDTA,
HX/xanthine oxidase-mediated hydroxylation of benzoic acid increased
dramatically from undetectable levels to 4.5 µM of the
hydroxylated product. Superoxide dismutase and catalase were both
effective at inhibiting this classic
O
-driven Fenton reaction.
Interestingly, NO inhibited this iron-catalyzed hydroxylation reaction
in a concentration-dependent manner such that fluxes of NO
approximating those of O
and
H
O
virtually abolished the hydroxylation of
benzoic acid. We conclude that in the absence of iron, equimolar fluxes
of NO and O
interact to yield potent
oxidants such as ONOO
/ONOOH, which oxidize organic
compounds. Excess production of either radical remarkably inhibits
these oxidative reactions. In the presence of low molecular weight
redox-active iron complexes, NO may enhance or inhibit
O
-dependent oxidation and hydroxylation
reactions depending upon their relative fluxes.
It is becoming increasingly apparent that certain types of
inflammatory tissue injury are mediated by reactive metabolites of
oxygen and nitrogen. For example, it has been demonstrated that
administration of superoxide dismutase is effective at attenuating the
tissue injury observed in experimental models of arthritis, chronic gut
inflammation, and immune complex-induced pulmonary
injury(1, 2, 3) . Furthermore, models of
joint, bowel, and lung inflammation have been shown to be associated
with enhanced production of nitrogen oxides derived from the free
radical nitric oxide (NO)(4, 5, 6) . Indeed,
recent studies have demonstrated that inhibition of NO synthase also
provides substantial protection against the inflammatory tissue injury
observed in these models of acute and chronic
inflammation(4, 5, 6) . These data suggest
that both superoxide (O) and NO are
important mediators of inflammation-induced tissue injury and
dysfunction. The mechanisms by which O
and NO may either separately or in tandem mediate tissue injury
during inflammation remain the subject of active debate.
Recent
chemical studies have demonstrated that O and NO rapidly interact via a radical-radical reaction at a
diffusion-limited rate (k = 6.7
10
M
s
) to generate the
potent oxidant peroxynitrite (ONOO
)(7) .
Beckman and co-workers (8) have suggested that the interaction
between these two free radicals to yield ONOO
and its
conjugate acid, peroxynitrous acid (ONOOH), enhances dramatically the
toxicity of either O
or NO alone.
Indeed, it has been demonstrated in vitro using preformed or
chemically synthesized ONOO
/ONOOH that these oxidants
are capable of directly oxidizing carbohydrates(8) ,
sulfhydryls(9) , lipids(10, 11) , and DNA
bases (12) as well as mediating bacteriocidal and endothelial
cell toxicity(13, 14) . It has also been demonstrated
that the simultaneous production of NO and O
by macrophages may result in the formation of
ONOO
/ONOOH(15) . However, a series of recent
reports demonstrate that NO may actually inhibit
O
-dependent, iron (or
hemoprotein)-catalyzed lipid peroxidation in
vitro(16, 17, 18, 19, 20) .
This apparent ``antioxidant'' activity of NO has prompted
some investigators to suggest that the interaction between
O and NO is an important detoxification
pathway of potentially injurious
O
-derived reactive oxygen metabolites
and thus may actually represent an endogenous anti-inflammatory
pathway. Indeed there is increasing evidence to suggest that NO may
protect cells and tissue against reactive oxygen metabolite-mediated
oxidative
damage(16, 17, 18, 19, 20) .
Wink et al.(17) have shown that exogenous NO protects
Chinese hamster lung fibroblasts, rat H4 hepatoma cells, and rat
mesencephalic dopaminergic cells against reactive oxygen
metabolite-induced cell injury. Assreuy et al.(16) recently demonstrated that although generation of NO
by activated macrophages is cytotoxic to Leischmania major,
simultaneous generation of NO and O
or
addition of authentic ONOO
failed to induce any
microbicidal activity. Finally, several investigators have demonstrated
that exogenous administration of NO inhibits ischemia-induced
microvascular dysfunction produced by
O
-dependent adherence and emigration of
neutrophils in the post capillary venules in
vivo(21, 22, 23) .
The reasons for
these apparent discrepant results are not clear. However, recent
evidence by Rubbo et al.(10) suggests that the
relative fluxes of the two free radicals may be an important
determinant as to whether NO enhances or inhibits
O-dependent, iron-catalyzed lipid
peroxidation (10) . In these studies the effects of NO were
assessed only in the presence of ferric iron. A recent preliminary
study from our laboratory suggests that the effects of NO on
O
-dependent oxidative reactions may be
quite different depending upon whether redox-active transition metals
are present or absent(24) . Therefore, the objectives of the
present study were to: (a) systematically quantify the
oxidizing and hydroxylating activity of NO and/or
O
in the absence or the presence of
redox-active iron and (b) characterize these reactions using
different fluxes of each radical. The physiological significance of our
findings is discussed.
Nitric oxide was generated
using the spontaneous decomposition of the Sp/NO. Sp/NO spontaneously
decomposes at 37 °C and pH 7.4 at a known and constant rate (t = 39 min; (29) ) to yield 2
mol of NO/mol of adduct. Sp/NO solutions were prepared fresh each day
as a 10 mM stock solution in ice-cold 10 mM NaOH and
stored on ice until used. Various concentrations of Sp/NO (0-200
µM) were incubated at 37 °C in a total reaction volume
of 500 µl containing 20 mM potassium phosphate buffer (pH
7.4) and 0.15 M NaCl. The initial rate of NO generation via
Sp/NO decomposition at 37 °C for 50 and 100 µM Sp/NO
was determined electrochemically using a NO-specific electrode (World
Precision Instruments, Sarasota, FL) and was found to be 0.87 ±
0.13 and 2.36 ± 0.12 nmol NO/min, respectively. These values
agreed well with those calculated for 50 and 100 µM Sp/NO
(1.1 and 2.2 nmol NO/min, respectively) based upon the published t
= 39 min at 37 °C and pH 7.4 (29
Hydroxylating activity of the various systems described
above was quantified by measuring the hydroxylation of BA to HB. At pH
7.4, the principal products of BA hydroxylation are the
monohydroxylated derivatives, 2-, 3-, and 4-hydroxybenzoate, with
2-hydroxybenzoic acid representing approximately 60% of total
product(32) . Fluorescence emission of the HB derivative
represents greater than 95% of total emission detected at 410 nm (i.e. for the three purified derivatives at equimolar
concentrations and physiological pH(32) ). 500-µl reaction
volumes containing 20 mM potassium phosphate buffer (pH 7.4)
and 0.15 M NaCl. 1.0 mM benzoic acid, 0.5 mM HX, and various concentrations of xanthine oxidase or Sp/NO were
incubated for 60 min at 37 °C. For some experiments, catalase (15
µg/ml) or superoxide dismutase (100 µg/ml) were included in the
reaction volumes, whereas in other experiments catalase and superoxide
dismutase were omitted and 5 µM Fe-EDTA
was included. Following the 60-min incubation period, reactions were
terminated by dilution with 0.5 ml of cold phosphate-buffered saline
(pH 7.4). Production of HB was quantified by measuring the fluorescence
obtained with excitation and emission wavelengths of 290 and 410 nm,
respectively. The concentration of HB was determined using HB
standards. All fluorescence emission measurements and spectra were
obtained using an Aminco/Bowman Series 2 luminescence spectrometer (SLM
Instruments, Inc., Rochester, NY).
Figure 1:
Oxidation of dihydrorhodamine to
rhodamine in the presence of constant superoxide production and various
fluxes of nitric oxide. All samples contained 15 µg/ml catalase and
were analyzed as described under ``Materials and Methods.''
Superoxide was generated by the HX/xanthine oxidase system at 1.0
nmol/min in 20 mM potassium phosphate buffer (pH 7.4) at 37
°C. Various fluxes of NO were achieved by increasing the
concentration of Sp/NO.
Figure 2: Interaction between NO and peroxynitrite. NO was continuously produced by 50 µM Sp/NO in 50 mM potassium phosphate buffer (pH 7.4) at 37 °C and was quantified using electrochemical detection. Peroxynitrite was dissolved in ice-cold 0.1 N NaOH containing 0.1 mM diethylenetriaminepentaacetic acid. The arrows indicate the time points at which a 1.00-µl addition of the base only or of the alkaline peroxynitrite solution (containing 11 nmol) were made.
Figure 3: Oxidation of dihydrorhodamine to rhodamine in the presence of constant nitric oxide production and various fluxes of superoxide. All samples contained 15 µg/ml catalase and were analyzed as described under ``Materials and Methods.'' NO was produced at a flux of 1.0 nmol/min by Sp/NO in 20 mM potassium phosphate buffer (pH 7.4) at 37 °C. Superoxide was generated by the HX/xanthine oxidase system.
Figure 4:
Effect of superoxide on
peroxynitrite-mediated oxidation of dihydrorhodamine. Superoxide was
generated by dissolving 50 mg of KO in ice-cold 0.1 N NaOH containing 0.1 mM diethylenetriaminepentaacetic
acid. Aliquots of this solution were then mixed with 35 nmol of
alkaline ONOO
prepared as described in the legend to Fig. 2. Aliquots of ONOO
or ONOO
and O
were added to 50 µM of dihydrorhodamine in a 1.0-ml reaction volume containing 50
mM potassium phosphate buffer (pH 7.4) at 37 °C and
incubated for 30 min. RH production was quantified as described under
``Materials and Methods.''
Fig. 5shows that neither O nor NO generation alone at fluxes of
1.0 nmol/min in the
absence of exogenous Fe
-EDTA and the presence of
catalase was capable of producing more than 3.0 µM RH. In
contrast, approximately 12 µM RH was produced with the
simultaneous production of equimolar fluxes of
O
and NO. Under conditions where both
NO and O
were present, the addition of
superoxide dismutase (0.1 mg/ml) inhibited RH production by more than
75%. The initial rate of RH production in the presence of 1.0 nmol/min
NO and 1.0 nmol O
/min was almost
10-fold higher when compared with the rate achieved with a
O
flux of 10 nmol/min (data not shown).
Figure 5:
Effect of superoxide dismutase on the
oxidation of dihydrorhodamine to rhodamine in the presence of equimolar
fluxes of NO and O. Assays were
performed in the presence of 15 µg/ml of catalase as described
under ``Materials and Methods.'' The fluxes of both NO and
O
were constant at
1.0 nmol/min.
Superoxide dismutase (SOD; 0.1 mg/ml) was added as described
under ``Materials and Methods.''
Figure 6:
A, the effect of increasing NO flux on
O/H
O
-dependent
production of rhodamine in the presence of 5 µM
Fe
-EDTA. Rhodamine was quantified as described under
``Materials and Methods.'' A constant flux of
1.0
nmol/min was used for both O
and
H
O
in all assays. B, the effect of
increasing O
/H
O
flux on rhodamine production in the presence of 5 µM
Fe
-EDTA. Rhodamine was quantified as described under
``Materials and Methods.'' NO flux was constant at
1.0
nmol/min in all assays.
The addition of catalase or
superoxide dismutase to solutions containing Fe-EDTA
and HX/xanthine oxidase attenuated RH production by >90% (Fig. 7). The generation of NO in Fe
-EDTA
solutions in the absence of O
and
H
O
resulted in production of very small amounts
RH (
2.0 µM), whereas the simultaneous production of
O
/H
O
and NO in
the presence of Fe
-EDTA oxidized approximately 13
µM DHR.
Figure 7:
Rhodamine production in the presence of
equimolar fluxes of NO and O and in the
presence of 5 µM Fe
-EDTA. Rhodamine was
quantified as described under ``Materials and Methods.'' NO,
O
, and H
O
were
all generated at
1.0 nmol/min each. Catalase (CAT; 15
µg/ml) or superoxide dismutase (SOD; 100 µg/ml) were
included in some reaction volumes.
Figure 8:
A, hydroxylation of benzoic acid in the
presence of constant O production and
various fluxes of NO. Assays were performed as described under
``Materials and Methods.'' The data were obtained at a
constant O
flux of
1.0 nmol/min.
All solutions contained 15 µg/ml catalase in 20 mM potassium phosphate buffer (pH 7.4) at 37 °C. B,
hydroxylation of benzoic acid in the presence of constant NO production
and various fluxes of O
. Assays were
performed as described under ``Materials and Methods.'' A
constant NO flux of
1.0 nmol/min was used. All solutions contained
15 µg/ml catalase in 20 mM potassium phosphate buffer (pH
7.4) at 37 °C.
Fig. 9demonstrates that neither O nor NO alone were capable of mediating significant hydroxylation
in the absence of Fe
-EDTA. Equimolar fluxes (
1.0
nmol/min) of each radical produced more than 510 nM HB, which
was inhibited by 60% by the addition of superoxide dismutase (0.1
mg/ml), suggesting that O
and NO
interact to yield an oxidant with only modest hydroxylating activity.
Figure 9:
Production of 2-hydroxybenzoate in the
presence of equimolar fluxes of NO and
O. The fluxes of NO and
O
were constant at
1.0 nmol/min
each in all assays. Catalase (15 µg/ml) was also present in all
assays. Superoxide dismutase (SOD) was added as described
under ``Materials and Methods.''
Figure 10:
A, the effect of increasing NO flux on
O/H
O
-dependent
hydroxylation of benzoate in the presence of iron. Assays were
performed as described under ``Materials and Methods.'' All
samples contained 5 µM Fe
-EDTA, and
O
/H
O
fluxes
were held constant at
1.0 nmol/min in 20 mM potassium
phosphate buffer (pH 7.4) at 37 °C. B, the effect of
increasing O
and H
O
fluxes on O
/H
O
and NO-dependent hydroxylation of benzoate in the presence of
iron. Assays were performed as described under ``Materials and
Methods.'' All samples contained 5 µM
Fe
-EDTA, and the NO flux was constant at
1.0
nmol/min.
Much of the vascular and tissue injury observed in certain
models of inflammation have been shown to be inhibited by either
superoxide dismutase or NO synthase inhibitors, suggesting that both
O and NO are important mediators of
tissue injury and
dysfunction(1, 2, 3, 4, 5, 6) .
Because neither O
nor NO are
particularly potent oxidants or cytotoxins, it has been suggested that
O
and NO may combine to produce the
potent cytotoxic oxidants ONOO
and ONOOH(8) .
Indeed, this hypothesis has generated tremendous interest because it
has provided a biochemical rationale to account for the remarkable but
perplexing protective effects of intravenous administration of L-arginine analogs (NO synthase inhibitors) or superoxide
dismutase in these pathophysiologic models of tissue injury and
inflammation(1, 2, 3, 4, 5, 6) .
Numerous studies have been published describing the physicochemical and
cytotoxic properties of chemically synthesized
ONOO
(8, 9, 10, 11, 12, 13, 14) .
However, there is a paucity of information quantitatively
characterizing the interaction between O
and NO under physiologic conditions. Thus, we have attempted to
systematically quantify the interaction between NO and
O
in the absence or the presence of
redox-active iron.
Data obtained in the present study demonstrate
that in the absence of iron-catalyzed reactions, simultaneous
generation of equimolar fluxes of O and
NO synergize to yield an oxidant or oxidants capable of oxidizing DHR
to RH ( Fig. 1and Fig. 3). Because catalase was present
throughout these experiments and because superoxide dismutase decreased
RH production by 90%, we propose that O
but not H
O
nor OH
interacts with NO to yield the oxidant or oxidants (Fig. 5). These data also confirm a previous report (31) that found that neither O
,
H
O
, nor NO per se is capable of
oxidizing substantial amounts of DHR in the absence of redox active
metals such as iron or hemoproteins. Only oxidants such as those
derived from Fenton-type reactions, ferryl hemoproteins, or
ONOO
/ONOOH are potent enough oxidizing agents to
oxidize DHR. Indeed, decomposition of peroxynitrous acid to nitrate has
been suggested to proceed via a rate-limiting isomerization reaction
that yields a potent oxidizing agent capable of hydroxylating organic
substrates(8) . Thus, we also assessed the ability of
O
and NO to interact (in the absence of
iron and H
O
) to hydroxylate BA. We found a
similar pattern of hydroxylation of BA as observed for DHR oxidation in
that equimolar fluxes (1.0 nmol/min) of O
and NO appeared to synergize to hydroxylate BA to HB (Fig. 8, A and B), although the magnitude of
this hydroxylation reaction was rather small (<15% that with iron
and H
O
present). Although we have not
definitely identified ONOO
/ONOOH as the oxidants
produced in this system, we expect that this would be the likely
reaction pathway because of the rapid interaction between
O
and NO and because of the lack of
alternative explanations for the production of equally potent oxidants.
We speculate that the decreased production of RH or HB in the
absence of iron and HO
but in the presence of
either excess NO or O
may be accounted
for on the basis of either secondary chemical interactions occurring
directly between NO or O
and ONOOH. It
may also be due to the interaction between NO or
O
with free radical intermediates of
DHR or BA to yield adducts with diminished fluorescence. The latter
possibility does not appear to be a major pathway because we did not
observe dramatic inhibition of DHR oxidation by excess NO in the
iron-containing system (Fig. 6A) nor did nitrosation of
HB by NO-derived nitrosating agents attenuate its fluorescence (data
not shown). The former hypothesis appears to be the more viable
explanation.
Although the direct reaction of ONOOH with either NO or
O has not been definitively
demonstrated, it has been suggested to be thermodynamically
possible(33, 34) . Koppenol et al.(34) have calculated Gibbs free energies (
G)
of -36 and -27 at neutral pH and 25 °C.
Although reaction rates are not forthcoming from calculated
thermodynamic values, the possibility of the interaction of ONOOH with
NO or O is at least indicated.
Therefore, competing reactions involving excess NO or
O
with ONOOH could be a possible
mechanism for the decreased DHR oxidation and BA hydroxylation in the
absence of iron-catalyzed reactions. Albeit, our data indicate that
excess NO or O
may be acting (at least
in our system) as modulators of the pro-oxidant characteristics of
ONOO
/ONOOH, and by extension, we suggest that under
similar conditions in vivo, excess NO or
O
may act as an endogenous modulator of
ONOOH-mediated tissue damage. On the other hand, depending on the ratio
of fluxes of O
to NO, oxidation and
hydroxylation reactions may be either enhanced or inhibited in the
absence of iron (Fig. 1, Fig. 3, and Fig. 10).
Under conditions of limiting O flux,
excess NO will instead be auto-oxidized in the presence of molecular
oxygen-producing nitrogen oxides (e.g. NO
,
N
O
, or NO
) that are not potent
oxidizing or hydroxylating agents but are potent N-nitrosating
agents (35) . We have recently demonstrated that
O
will effectively inhibit NO-mediated N-nitrosation reactions(36) , and contrary to a recent
report(37) , we detected no significant change in xanthine
oxidase activity (measured via urate production) in the presence of 200
µM Sp/NO, which produces a flux of
4.0 nmol NO/min
(data not shown). Furthermore, the production of hydrogen peroxide from
xanthine oxidase was not inhibited by the presence of 200 µM SP/NO or 1 mM DEA/NO (data not shown). On the other hand,
increased production of O
at higher
xanthine oxidase concentrations is concomitant with increased urate
production (a potent free radical scavenger). Whereas urate-mediated
inhibition cannot be totally discounted, it apparently was not a
significant factor at O
fluxes below
1.0 nmol/min, as indicated by the similarity in the shape of curves
shown in Fig. 1and Fig. 3. Moreover, total elimination
of possible urate interference was achieved with the use of pterin (500
µM) as substrate in place of HX, yet the data were
virtually identical to those shown in Fig. 3(data not shown).
When the same oxidation and hydroxylation experiments were performed
in the presence of O,
H
O
, and 5 µM Fe
-EDTA, qualitatively different results were
obtained. Generation of O
and
H
O
in the presence of Fe
-EDTA
but not NO stimulated oxidation of DHR producing approximately 15
µM RH compared with the formation of 20 µM RH
in the presence of NO (1.0 nmol/min; Fig. 6A). These
data suggest that as the ratio of NO/O
increased from 0 to 2, oxidation of DHR increased by
approximately 30% (Fig. 6A). As the ratio was increased
further to 4.5, RH production was reduced by 40%. These data are
reminiscent of those reported by Rubbo et al.(10) using xanthine oxidase-dependent iron-catalyzed lipid
peroxidation. As expected, generation of O
and H
O
(1.0 nmol/min each) in the
presence of iron increased production of HB tremendously (i.e.
4000 nM) (Fig. 10A). Remarkably, the
addition of NO to this iron-catalyzed hydroxylation system dramatically
inhibited hydroxylation of BA such that equimolar fluxes of NO
inhibited hydroxylation by 80% (Fig. 10A). Kanner et al.(38) recently suggested that NO may modulate
iron-mediated oxidative reactions by forming nitrosyl complexes with
ferrous iron or by the direct interaction of NO with
H
O
. The sequence of reactions involving NO and
iron may proceed as follows:
The efficiency of such interactions could explain the results in Fig. 10A. Indeed it is well known that NO binds under
physiological conditions with ferrous heme containing compounds (e.g. hemoglobin and myoglobin), and moreover these reactions
are the chemical basis of current methodology used for NO
detection(39) . An alternative and more likely explanation for
this dramatic inhibitory effect of NO may be that NO shunts
O away from iron-catalyzed
OH
formation by the Fenton reaction and toward the
formation of an oxidant (e.g. ONOO
/ONOOH)
with only weak hydroxylating activity.
Our data confirm and extend
the results recently reported by Rubbo et al.(10) ,
who demonstrated that increasing fluxes of NO with respect to
O and H
O
modestly stimulated iron-catalyzed lipid peroxidation followed by
inhibition when fluxes of NO exceed those of
O
. Furthermore, these same
investigators demonstrated that NO could partially inhibit
ONOO
-induced lipid peroxidation(10) .
Interestingly, in our studies in the presence of iron, conditions under
which total inhibition of hydroxylation occurred resulted in only a
modest 33% inhibition of DHR oxidation ( Fig. 6versus 10), suggesting that NO is shunting O
away from iron-catalyzed OH
formation and toward
the formation of a potent oxidant with weak hydroxylating activity.
Two major physiological implications arise from our present study. (a) The role of ONOO at sites where both
O
and NO are produced (i.e. inflammatory foci) is dependent upon the relative fluxes of NO and
O
in the extracellular space. Our data
suggest that excess production of one radical over the other may act as
an endogenous modulator of ONOO
formation such that
the steady state levels of this potent cytotoxic oxidant never
accumulates above a certain amount. Indeed, the spontaneous
acid-catalyzed decomposition of another potent oxidant, hypothiocyanous
acid, is an example of autocatalytic regulation of oxidant
formation(40) . (b) NO may enhance or inhibit
oxidation and hydroxylation reactions depending upon the absence or the
presence of low molecular weight, redox-active metals such as iron.
Normally, there is little low molecular weight iron (e.g. amino acid, carbohydrate, or nucleotide chelates of iron) present
in most cells and tissue, with the vast majority of this metal
sequestered in its redox-inactive, protein-bound forms such as
ferritin- or transferrin-bound iron. However, it is known that certain
reductants (e.g. ascorbate and
O
) are very effective in releasing iron
from ferritin by reducing ferritin-bound Fe
to
Fe
, which is no longer capable of being bound by the
protein(41, 42) . In addition to ferritin, there is
also a small but significant pool of low molecular weight iron chelate (e.g. non-protein-bound iron) located within cells. Studies by
Deighton and Hider (43) have identified this low molecular
weight iron chelate as a glutamate-iron complex (molecular weight of
1000-1500) that can easily exchange its iron with other more
potent chelators.
Important consequences of the
oxidant/hydroxylation-dependent flux of NO and
O are temporal and spatial
considerations. To achieve the maximum oxidant resulting from NO/
O
, the site orientation and timing of
the formation of these two species is crucial. The timing of the
superoxide production relative to the NO can be distinctly different in vivo and have a limited overlap under some immunological
and pathophysiological conditions. For instance, superoxide formation
of neutrophils reaches a flux 10 times higher than that of NO within
the first few minutes after treatment with phorbol ester(36) .
However, the flux of superoxide formed quickly subsides within an hour,
whereas the NO production continues for several hours. The time overlap
in which the flux of these two radicals is one to one is for a very
limited time; therefore the amount of peroxynitrite formed is small.
Conversely, cytokine-stimulated alveolar macrophage are thought to
generate both NO and O
at the same
sustained rate for long period of time, implying that the oxidant
formed may be intentionally held high in this specific cell
line(15) . Yet, RAW macrophages appear to generate solely
nitrosating agents via the NO/O
reaction without the
presence of superoxide (35) . This switching between oxidation,
hydroxylation, and nitrosation appears to be well orchestrated in the
immune response to pathogens and appears to be critical in host
defense. Although NO and superoxide can be generated from the same cell
type and cytokine influence, kinetic considerations must be carefully
examined to determine the reactive intermediates involved.