(Received for publication, October 16, 1996, and in revised form, February 3, 1997)
From the Department of Medicine, Division of
Cardiology, Pulmonary Diseases, and Angiology, Heinrich Heine
University, Moorenstrasse 5, D-40225 Düsseldorf, Federal Republic
of Germany, and the § Tumor Biology Section, Radiation
Biology Branch, NCI, National Institutes of Health,
Bethesda, Maryland 20892-1002
Nitric oxide (NO) is a widespread signaling
molecule involved in the regulation of an impressive spectrum of
diverse cellular functions. Superoxide anions (O2) not only
contribute to the localization of NO action by rapid inactivation, but
also give rise to the formation of the potentially toxic species
peroxynitrite (ONOO
) and other reactive nitrogen
oxide species. The chemistry and biological effect of
ONOO
depend on the relative rates of formation of NO and
O
2. However, the simultaneous quantification of NO and
O
2 has not been achieved yet due to their high rate of
interaction, which is almost diffusion-controlled. A sensitive
spectrophotometric assay was developed for the simultaneous quantification of NO and O
2 in aqueous solution that is based on the NO-induced oxidation of oxyhemoglobin (oxyHb) to methemoglobin and the O
2-mediated reduction of ferricytochrome
c. Using a photodiode array photometer, spectral changes of
either reaction were analyzed, and appropriate wavelengths were
identified for the simultaneous monitoring of absorbance changes of the
individual reactions. oxyHb oxidation was followed at 541.2 nm
(isosbestic wavelength for the conversion of ferri- to ferrocytochrome
c), and ferricytochrome c reduction was
followed at 465 nm (wavelength at which absorbance changes during oxyHb
to methemoglobin conversion were negligible), using 525 nm as the
isosbestic point for both reactions. At final concentrations of 20 µM ferricytochrome c and 5 µM
oxyHb, the molar extinction coefficients were determined to be
465-525 = 7.3 mM
1
cm
1 and
541.2-525 = 6.6 mM
1 cm
1, respectively. The
rates of formation of either NO or O
2 determined with the
combined assay were virtually identical to those measured with the
classical oxyhemoglobin and cytochrome c assays,
respectively. The assay was successfully adapted to either kinetic or
end point determination in a cuvette or continuous on-line measurement
of both radicals in a flow-through system. Maximal assay sensitivity was ~25 nM for NO and O
2. Cross-reactivity with
ONOO
was controlled for by the presence of
L-methionine. Generation of NO from the NO donor spermine
diazeniumdiolate could be reliably quantified in the presence and
absence of low, equimolar, and high flux rates of O
2.
Likewise, O
2 enzymatically generated from
hypoxanthine/xanthine oxidase could be specifically quantified with no
difference in absolute rates in the presence or absence of concomitant
NO generation at different flux rates. Nonenzymatic decomposition of
3-morpholinosydnonimine hydrochloride (100 µM) in
phosphate buffer, pH 7.4 (37 °C), was found to be associated with almost stoichiometric production of NO and O
2 (1.24 µM NO/min and 1.12 µM O
2/min).
Assay selectivity and applicability to biological systems were
demonstrated in cultured endothelial cells and isolated aortic tissue
using calcium ionophore and NADH for stimulation of NO and O
2
formation, respectively. Based on these data, a computer model was
elaborated that successfully predicts the reaction of NO and
O
2 with hemoprotein and may thus help to further elucidate these reactions. In conclusion, the nitric oxide/superoxide assay allows the specific, sensitive, and simultaneous detection of NO and
O
2. The simulation model developed also allows the reliable prediction of the reaction between NO and O
2 as well as their kinetic interaction with other biomolecules. These new analytical tools
will help to gain further insight into the physiological and
pathophysiological significance of the formation of these radicals in
cell homeostasis.
Nitric oxide (NO) is a widespread intracellular and intercellular
signaling molecule involved in the regulation of diverse physiological
and pathophysiological mechanisms in the cardiovascular system and the
central and peripheral nervous systems and in immunological reactions
(for review, see Refs. 1-3). In contrast to the current view that NO
is extremely unstable in vivo, but in agreement with its
functioning as a paracrine mediator, NO can travel significant distances to reach target cells neighboring the NO-generating cell (4,
5). Along this migration, in particular at higher concentration, NO can
interact with molecular oxygen to form higher nitrogen oxides
(e.g. NO2 and N2O3),
which can either react with other biomolecules such as thiols and
amines or simply hydrolyze to form nitrite
(NO2) and nitrate
(NO3
). Furthermore, NO rapidly reacts
with reduced hemoproteins and oxygen-derived radicals. The extent of
either of these reactions largely depends on the microenvironmental
conditions under which NO is released, most important, the
concentration of other bioreactants (6, 7).
There is increasing evidence that the effective concentration of NO in
a given biological tissue is not solely determined by its rate of
enzymatic formation, but also by its rate of degradation and
scavenging by other biomolecules. For example, the progression of
atherosclerotic lesions has been attributed to an increased oxidative
stress within the vascular wall and, consequently, to an accelerated
breakdown of NO (8, 9). One important oxygen-derived metabolite capable
of inactivating NO is superoxide (O2), which reacts with NO at
an almost diffusion-controlled rate (10) to form peroxynitrite
(ONOO
) (Equation 1).
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
The simultaneous monitoring of NO and O2, although highly
desirable, was believed to be technically unfeasible due to the extremely rapid reaction between both radicals. We describe here an
analytical technique that allows, for the first time, the simultaneous and sensitive quantification of both NO and O
2 and, under
certain conditions, additionally helps to unmask the proportion of
peroxynitrite and other reactive nitrogen oxide species formed. The
results obtained with this new assay could be successfully simulated by a computer model that predicts the reaction of both radicals with hemoproteins and thus may give valuable new insights into the reaction between NO and O
2 in biological systems.
One of the few spectrophotometric techniques that allows the quantification of NO in aerobic aqueous solutions (the oxyhemoglobin assay) is based on the stoichiometric conversion by NO of oxyhemoglobin (oxyHb1; Fe2+) to methemoglobin (metHb; Fe3+) (17) (Equation 5).
![]() |
(Eq. 5) |
One of the most commonly used spectrophotometric assays for
the quantification of O2 is based on the rapid
reduction by O
2 of ferricytochrome c
(Fe3+) to ferrocytochrome c (Fe2+)
(Equation 6),
![]() |
(Eq. 6) |
The photometric
assay for the simultaneous determination of NO and O2
described in this paper is based on a combination of the
above-mentioned detection principles: the NO-mediated oxidation of
oxyHb and the O
2-induced reduction of ferricytochrome
c. For the separate recording of either signal, a
programmable diode array spectrophotometer (DU-7500i, Beckman, Munich,
FRG) was used. In contrast to classical dual-wavelength
spectrophotometers, the diode array technique has the advantage of
allowing parallel monitoring of multiple wavelengths almost at the same
time (e.g. an entire absorption spectrum can be derived
within 0.1 s). As the internal memory of these machines is usually
limited in capacity to the storage of a couple of full spectra or a few
hundred data points only, a series of programs were developed for the
continuous visualization of a large number of data points on the
photometer screen and for the on-line transfer of raw data between
photometer and personal computer to allow unlimited data sampling and
post-processing of photometric recordings. Measurements were performed
as either end point or kinetic determinations using glass or disposable plastic cuvettes or under conditions of continuous flow using a
flow-through cuvette with a total volume of 80 µl (QS 178-010, Hellma, Mühlheim, FRG). If not otherwise indicated, all
measurements were carried out in 100 mM phosphate buffer,
pH 7.4, at 37 ± 0.02 °C using a thermostatted cuvette holder
connected to a D 8-L water bath (Haake, Karlsruhe, FRG).
Consecutive spectral scans between 350 and 600 nm were recorded on oxidation of oxyHb to metHb using either
aqueous solutions of authentic NO or spermine NONOate as NO donor.
Changes in absolute spectra obtained with the diode array technique
were virtually identical to those observed using a conventional
dual-wavelength spectrophotometer (17). Difference spectra were derived
by subtraction of individual spectral scans from the spectrum obtained
with pure oxyHb at time 0 (t0 min). Increasing
concentrations of oxyHb (0.1-7.0 µM) were reacted with
excess NO or SIN-1, and the difference in absorbance between 401 and
411.5 nm (isosbestic point for oxyHb to metHb conversion) was recorded.
Photometric readings were converted to NO concentrations using a molar
extinction coefficient of 401-411.5 = 49.5 mM
1 cm
1 as determined under the
conditions of this study. Reproducibility for end point determinations
(repeated measurements of NO standards) was better than 4%
(coefficient of variance = 3.4; n = 12). The molar
extinction coefficient for the cytochrome c assay
(
550 = 19.5 mM
1
cm
1) was determined by reacting increasing concentrations
(5-30 µM) of ferricytochrome c with a 3-fold
molar excess of ascorbic acid or the O
2-generating
hypoxanthine/xanthine oxidase system (n = 3 each) and
recording changes in absorbance at 550 nm. The extinction coefficients for the NO-induced conversion of oxyHb to metHb
(
525-541.2) and for the O
2-mediated
conversion of ferri- to ferrocytochrome c
(
465-525) in the combined nitric oxide/superoxide assay were determined basically in the same manner, but in the presence of
the respective other hemoprotein. Determinations were performed under
conditions identical to those of the kinetic investigations. In those
experiments where O
2 was enzymatically generated using the
hypoxanthine/xanthine oxidase system, the buffer was additionally supplemented with 25 µM EDTA.
All compounds used in this study
were either analytical grade or otherwise of the highest purity
available. ABTS (2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid,
diammonium salt), catalase (from bovine liver, thymol-free; 20,000 units/mg), 18-crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane), ferricytochrome c (from horse heart, containing 2%
ferrocytochrome c and 2.7% water), hemoglobin (human,
lyophilized), hypoxanthine, manganese dioxide (MnO2;
activated), L-methionine, potassium superoxide (KO2), and superoxide dismutase (from bovine erythrocytes;
4400 units/mg of protein) were purchased from Sigma (Deisenhofen, FRG). Xanthine oxidase (from bovine milk, phosphate-free lyophilisate; 0.18 unit/mg) was obtained from Boehringer (Mannheim, FRG). Sodium nitrite
(NaNO2), hydrogen peroxide (H2O2;
30%), and pyrogallol (1,2,3-trihydroxybenzene) were from Merck
(Darmstadt, FRG). Spermine NONOate (Cayman Chemical Co., Inc., Ann
Arbor, MI) was dissolved in argon-gassed 0.01 M NaOH. SIN-1
(kindly donated by Dr. K. Schönafinger, Hoechst Marion Roussel,
Frankfurt/Main, FRG) was dissolved in distilled water adjusted to pH
5.0. S-Nitrosoglutathione was synthesized according to Hart
(19) and dissolved in de-aerated citrate buffer, pH 2.0. NO donor stock
solutions were kept on ice in the dark for up to 3 h. Aqueous
solutions of authentic NO were prepared essentially as described (20).
Briefly, argon (quality = 5.0, >99.99% argon; Linde AG,
Unterschleissheim, FRG) was passed through a closed all-glass
system comprising two scrubbing bottles, one containing an alkaline
pyrogallol (5%, w/v) solution for removal of traces of oxygen and the
other containing potassium hydroxide (10%, w/v) to scavenge higher
oxides of nitrogen, connected in series with a three-necked beaker
containing saline (0.9% NaCl) for the dissolution of NO. After
flushing with argon for 30 min, the gas flow was switched to NO
(quality = 3.0, >99.9% NO; AGA GAS GmbH, Hamburg, FRG) and
maintained for a further 45 min. Aliquots were transferred to air-tight
syringes via a septum with the system kept under positive pressure with
NO to avoid changes in NO concentration due to re-equilibration between
the aqueous and gas phases. Concentrations ranged between 1.7 and 2 mM NO, depending on the ambient pressure and temperature.
Dilutions were made in deoxygenated and argon-flushed saline, and
concentrations were determined immediately before application using the
gas-phase chemiluminescence reaction with ozone (21). Stock solutions
of O
2 (0.1-10 mM) were prepared by dissolving
solid KO2 in water-free dimethyl sulfoxide containing 0.5%
crown ether (22). Aqueous stock solutions of ONOO
were
prepared by mixing NaNO2 and H2O2
in a quenched-flow reactor as described (12). Peroxide contamination
after treatment with MnO2 was typically below 0.05% as
assessed using the horseradish peroxidase-catalyzed ABTS cation radical
formation as described (23). Frozen aliquots (final concentration = 250-350 mM) were stored at
80 °C for up to 4 weeks
with only minor decomposition. Dilutions were made in 0.01 M NaOH and used immediately. Decomposed ONOO
was prepared by dilution of the stock solution in 1 M
hydrochloric acid and readjustment with NaOH to pH 13 after 5 min.
Oxyhemoglobin was prepared as described in detail elsewhere (17).
Aliquots of the stock solution (1.9-2.5 mM, expressed as
concentration in heme) were stored at
80 °C for up to 6 months.
Ferricytochrome c was used without further purification.
Endothelial cells
(ECs) were harvested enzymatically from porcine aorta using collagenase
and cultured in M199 medium (Boehringer Mannheim) containing 20%
newborn calf serum. Antibiotics were omitted from the medium from day 5 after isolation. The cells were passaged twice using trypsin/EDTA,
grown to confluence on plastic dishes (Primaria, Falcon, Heidelberg,
FRG), and characterized morphologically by their typical
cobblestone-like growth pattern as well as by uptake of acetylated low
density lipoproteins. Absence of contamination with smooth muscle cells
>2% was verified by counterstaining of cell nuclei with bisbenzimide
and with an antibody against smooth muscle -actin. ECs were
collected from a single Petri dish using gentle trypsinization
beginning 10 ± 1 min before the start of incubation. The
resulting cell suspension was subjected to gentle centrifugation at
200 × g, and cells were washed twice with
Krebs-Henseleit buffer, resuspended in 5 ml of Krebs buffer containing
10% newborn calf serum, and kept at 37 °C in a culture tube. ECs
were added to the incubation cuvette immediately before the start of
the experiment, and the cell suspension was subjected to continuous low
speed stirring using an electromagnetic cuvette-stirring unit.
Anesthetized male Wistar rats (300-350 g) were killed, and the
thoracic aortas were carefully removed, cleaned up of fat and connective tissue, and cut into 4-5-mm rings. The endothelial integrity of these vascular rings was verified by their ability to
relax, in an organ bath, on addition of 1 µM
acetylcholine by 70% of the precontraction level achieved with
phenylephrine. Vascular rings were kept in phosphate-buffered saline at
37 °C for up to 30 min before addition to the incubation cuvette.
Vascular tissue was kept at the bottom of a 3-ml cuvette without
stirring.
Simulations for development of a
mathematical model to explain the observed results were run using
Stella II (Version 1.02; High Performance Systems, Hanover, NH) on a
Power Macintosh 7200/90 (Apple Computers) with numerical regression of
the Runge-Kutta fourth derivative (24) (for the rate equations used,
see "Results"). Calculated metHb and ferrocytochrome c
concentrations were plotted as a function of time and converted into
absorbance units using the molar extinction coefficients for the
NO/oxyHb and O2/ ferricytochrome c reactions
determined for the combined assay (see "Results").
The raw data output of the
photodiode array spectrophotometer was transferred via the serial
interface to a personal computer and stored in individual files for
later processing by a commercial graphics and data analysis software
(Origin 4.0; MicroCal Inc., Northampton, MA). Base-line smoothing was
achieved by applying the method of moving average. With an increasing
number of consecutive data points set in relation to one original data
point, base-line noise decreases, and thus, sensitivity increases
provided the length of the averaging interval does not exceed the
length of the signal to be monitored (for the effect of this procedure
on base-line noise, see Fig. 2). In contrast, assay
sensitivity for fast kinetics is improved, and changes in peak shape
are avoided by selecting rather short intervals. Usually, an averaging
interval of 5-10 s was used for rapid kinetics and 30-240 s for
slower reactions. This flexibility in choice of the most appropriate averaging interval adds to the general advantage of the diode array
technique over conventional spectrophotometry, i.e. the possibility of adjusting the data processing with respect to the signal
without loss of original data points after the actual measurement has
taken place.
Results are presented as means ± S.D. The rates of formation in
either assay system are reported as net values corrected for the blank
determined under the same conditions, but in the absence of an NO- or
O2-generating system. As pilot tests revealed that the
presence of oxyHb causes slow, superoxide dismutase-independent reduction of ferricytochrome c over time, all measurements
for direct comparison of the "classical" cytochrome c
assay with the combined NO/O
2 assay were performed in the
presence of 5 µM oxyHb. Intra- and interassay comparisons
were made applying the Mann-Whitney U test for unpaired
variables, and a p value of <0.05 was accepted to denote
statistical significance.
To
select the optimal wavelengths that would allow the simultaneous and
independent monitoring of the redox conversion of either hemoprotein,
spectral changes of the individual reactions were compared. The
classical wavelengths of either assay (i.e. 401 and 411.5 nm
for the oxyHb assay and 550 nm for the cytochrome c assay)
could not be used for a combined assay as they overlap with opposite
spectral changes of the respective other reaction (see difference
spectra depicted in Fig. 1, A and B). However, an
isosbestic point was found at 525 nm, a wavelength at which absorbance
changes of either reaction were negligible. 541.2 nm, which represents
an isosbestic point for the ferri- to ferrocytochrome c
conversion, was chosen for recording of the changes in oxyHb concentration, and 465 nm, a point at which absorbance changes for the
conversion of oxyHb to metHb are negligible, was chosen for monitoring
the ferri- to ferrocytochrome c conversion (Fig. 3,
A-C).
H2O2, which can be formed by the spontaneous or
enzymatic (superoxide dismutase-mediated) dismutation of O2,
has the potential to interfere with either hemoprotein (25, 26), in
particular at higher rates of O
2 generation. Therefore,
catalase was added, and a final concentration of 100 units/ml was found
to be sufficient to completely prevent erroneous absorbance changes
(false positive oxyHb oxidation and partial reversal of ferricytochrome
c reduction) caused by this oxidant. Although a hemoprotein
as well, catalase did not affect the absorbance spectrum of the
ferricytochrome c/oxyHb mixture at this low concentration.
Likewise, the measured rates of NO formation generated by 20 µM spermine NONOate were virtually identical in the
absence and presence of this concentration of catalase using either the
combined assay or the classical oxyHb technique (n = 2 each) (data not shown).
Initial experiments were carried out with equimolar concentrations of
ferricytochrome c and oxyHb (5 µM each), which
required slightly different detection wavelengths and molar extinction coefficients (data not shown). By systematic variation of the concentration ratio of either hemoprotein (5:5, 5:10, 5:15, and 5:20
and 10:5, 15:5, and 20:5 for ferricytochrome c and oxyHb, respectively; n = 2-3 for each concentration ratio)
and the use of the hypoxanthine/xanthine oxidase system for generation
of O2 and spermine NONOate for generation of NO, it was found
that the trapping efficacy O
2 approached an optimum at a
ferricytochrome c/oxyHb molar ratio of 4:1. The recovery of
NO at a ferricytochrome c/oxyHb ratio of 4:1 was not
significantly different from that at a ratio of 1:1 as evidenced by
comparison of the rates of NO generation from 20 µM
spermine NONOate under both conditions (586.0 ± 12.5 versus 594.6 ± 38.1 nM NO/min at 4:1 and
1:1 ferricytochrome c/oxyHb, respectively, at 37 °C
and pH 7.4; n = 5). However, O
2 recovery
increased by almost 50% at higher ferricytochrome c
concentrations, both in the absence and presence of simultaneous
NO generation (901.2 ± 136.6 versus 604.3 ± 93.7 nM O
2/min, respectively, with 25 µM hypoxanthine and 0.75 milliunit of xanthine oxidase at
37 °C and pH 7.4; p < 0.05, n = 5).
There was no significant difference in measured O
2 rates
between 15 and 20 µM ferricytochrome c. Concentrations higher than 20 µM ferricytochrome
c could not be tested in the presence of 5 µM
oxyHb due to technical limitations (too high an absorbance for
auto-zeroing of the diode array photometer). Final concentrations of 20 µM ferricytochrome c and 5 µM
oxyHb were thus chosen for all further experiments. The molar
extinction coefficients under these conditions were determined to be
465-525 = 7.3 mM
1
cm
1 for the conversion of ferri- to ferrocytochrome
c using ascorbate as reductant and
541.2/525 = 6.6 mM
1 cm
1 for the oxyHb to
metHb conversion using spermine NONOate as NO donor. Assay
reproducibility was better than 4% (coefficient of variance = 3.7; n = 12).
Selectivity of specific wavelengths for the
measurement of NO and O2 was assessed as follows. Repeated
addition of aliquots of an aqueous solution of authentic NO (final
concentration = 0.2-1 µM) to a mixture of oxyHb and
ferricytochrome c in phosphate buffer, pH 7.4, produced
stepwise increases in the absorbance difference between 541.2 and 525 nm, indicative of metHb formation, without inducing any changes in the
absorbance difference between 465 and 525 nm, demonstrating that the
cytochrome c signal was unaffected (n = 5)
(data not shown). Likewise, selective gradual increases in metHb
formation were seen when NO was continuously generated by the NO donor
compound spermine NONOate (Fig. 4, upper panel). Measured rates of NO formation by spermine NONOate in the
combined assay were identical, within experimental error, to those
obtained with oxyHb alone (Table I), demonstrating that NO detection is not influenced by the presence of cytochrome
c. Likewise, addition to the ferricytochrome
c/oxyHb mixture of aliquots of a stock solution of
KO2 in dimethyl sulfoxide resulted in a stepwise reduction
of ferricytochrome c without changes in the hemoglobin
signal (n = 3) (data not shown). In the presence of the
O
2-generating hypoxanthine/xanthine oxidase system, a
selective increase in the absorbance difference between 465 and 525 nm
was observed (Fig. 4, middle panel). In the presence of the
same concentration of both spermine NONOate and hypoxanthine/xanthine
oxidase, i.e. under conditions of simultaneous generation of
NO and O
2, measured individual rates of radical formation were
identical to the separately determined rates (Fig. 4, lower
panel). This indicates that the concentrations of oxyHb and
ferricytochrome c present in the combined assay are
sufficient to effectively trap all of the NO and O
2 generated.
As for NO, the measured rates of O
2 formation using the
combined assay were not significantly different from those determined
with the classical cytochrome c assay (Table I). The slightly (<10%) smaller rates of O
2 formation using the
combined NO/O
2 assay compared with the classical cytochrome
c assay (Table I) may be explained by reaction of
O
2 with oxyHb, which, although comparatively slow (27), may
partially compete with the trapping by ferricytochrome c
under these conditions. Absorbance changes for the ferri- to
ferrocytochrome c conversion elicited by high concentrations
of hypoxanthine/xanthine oxidase (producing flux rates of ~1
µM O
2/min) were completely (>96%) abolished in
the presence of 100 units/ml superoxide dismutase, confirming the specificity of the signal for O
2. In a separate set of
experiments, the recovery of NO and O
2 at low (0.1 µM/min) and high (1 µM/min) flux rates was
additionally tested in a crossover design under conditions of
simultaneous formation of both radicals at either equimolar generation
rates or at a 10-fold excess of one radical over the other (Table
II). Under all conditions, i.e. at equimolar flux rates as well as at 10-fold higher generation rates of NO over
O
2 and vice versa, individual rates of formation significantly differed neither from each other nor from the rates measured in the
absence of the respective other radical (compare Tables I and II).
|
|
Maximal assay sensitivity suitable for monitoring relatively slow
changes in cellular metabolic activity was calculated to approach 25 nM for NO and O2 production, respectively, using 240 s as the time interval for data post-processing, applying the
method of moving average (see "Experimental Procedures" for details). Shorter averaging intervals (10-30 s), which are recommended for routine monitoring of rapid concentration changes, decrease the
detection limit to ~100-60 nM for either radical
(calculated for a signal-to-noise ratio of 3:1).
Because NO and O2
react at an almost diffusion-controlled rate to form ONOO
and because the oxyHb assay has been reported to cross-react with
ONOO
(28), we sought to investigate what absorbance
changes occur on addition of peroxynitrite to the combined
NO/O
2 assay. Consecutive addition to the ferricytochrome
c/oxyHb mixture of authentic ONOO
resulted in
stepwise contrary changes in the absorbance difference for each signal,
indicating oxidation of both oxyHb and ferrocytochrome c
(Fig. 5). Interestingly, the extent of oxidation was
dependent on the concentration of ONOO
as well as on the
concentration of oxyHb and ferrocytochrome c, with
decreasing efficacy at lower hemoprotein concentrations. The quantum
yield of this reaction as estimated from the sum of oxidized
ferrocytochrome c and oxyHb in relation to the concentration of ONOO
applied was in the range of 3-10% at 100 and 10 µM ONOO
, respectively (n = 3). Controls with solutions of decomposed ONOO
at
identical pH were found to have no effect (n = 2),
confirming that the observed changes in the absorbance difference were
not the result of an artifactual change in pH. Moreover, the difference spectrum of the reaction of ONOO
with oxyHb was
indistinguishable from that obtained with oxyHb and authentic NO,
whereas that of ferrocytochrome c and ONOO
was
the mirror image of that obtained with ferricytochrome c and
O
2. Thus, whereas ONOO
appears to mimic NO in
its reaction with oxyHb, it produces spectral changes opposite to those
elicited by O
2. Methionine was found to inhibit the oxidation
by ONOO
of ferrocytochrome c and oxyHb in a
concentration-dependent manner (tested at 1-50
mM; maximal effects at 20 mM), presumably via oxidation of its thioether group to form the corresponding sulfoxide. Other known ONOO
scavengers such as ascorbate, cysteine,
ebselen, and glutathione (all tested at 0.01-1 mM) could
not be used in this assay because of redox interferences with one or
the other hemoprotein.
In Vitro Applications
In addition to the validation
experiments described before, a separate set of investigations was
performed using SIN-1 as model compound for the simultaneous generation
of NO and O2 (29). Addition of SIN-1 to the assay mixture
resulted in a time-dependent increase in absorbance for
both the NO and O
2 signals (Fig. 6). Increases
in ferrocytochrome c formation by SIN-1 were completely inhibited in the presence of 5 units/ml superoxide dismutase
(n = 2). The release of NO and O
2 from 100 µM SIN-1 at pH 7.40 and 37 °C was determined to be
1.24 ± 0.07 µM NO/min and 1.12 ± 0.04 µM O
2/min (n = 3), which is in
good agreement with the proposed mechanism of oxidative breakdown of
sydnonimines (30, 31). Interestingly, the increase in the cytochrome
c-related signal turned into a decrease of about the same
rate after all of the oxyHb was consumed by reaction with NO. This is
explained by a reoxidation of the formed ferrocytochrome c
by ONOO
, which is produced when NO is no longer scavenged
by oxyHb and was found to continue until all of the ferrocytochrome
c was completely converted to ferricytochrome c.
In agreement with this assumption, addition of fresh oxyHb to such
incubations resulted in a reversal of this phenomenon, with further
increases in both the NO and O
2 signals until all of the oxyHb
was consumed again (n = 2) (data not shown). Addition
of 20 mM L-methionine completely prevented ferrocytochrome c reoxidation without affecting the initial
rates of NO and O
2 formation (Fig. 6, inset). These
data suggest that, using this assay, measurements made in the presence
and absence of methionine provide a straightforward means to
additionally test for the presence of already formed
ONOO
. Identical results were obtained when NO and
O
2 were generated using spermine NONOate and
hypoxanthine/xanthine oxidase (data not shown).
The on-line measurement of NO and O2 under conditions of
continuous flow was demonstrated using a perfusion system with either phosphate buffer or Krebs-Henseleit buffer supplemented with 5 µM oxyHb and 20 µM ferricytochrome
c, which was passed through a flow-through cell placed in
the cuvette holder of the diode array spectrophotometer, and continuous
recording of absorbance changes at 465, 525, and 541.2 nm. The flow
rate of the system was adjusted to values of 1.0-10.0 ml/min by means
of a low pulse peristaltic pump (Minipuls 3, Gilson Medical Electronics
Inc., Middleton, WI), and calibration for NO and O
2 was
performed by coinfusion of small volumes (10-40 µl/min) of NO and
KO2 standards, respectively, using a high precision
infusion pump (Precidor, Infors AG, Bottmingen, Switzerland). Provided
dilution by the coinfusion of the total ferricytochrome
c/oxyHb concentration in the buffer was kept below 0.5%,
blanks with coinfused saline instead of NO or KO2 did not
produce any artifactual changes in absorbance. Linearity of the
photometric response was demonstrated for NO concentrations in the
range of 0.05-3 µM (r = 0.97;
n = 15). Reproducibility was comparable to that for end
point determinations (coefficient of variance = 5.6%;
n = 24). Using this system, the flux rates of NO and
O
2 formation by three different classes of NO donor compounds
(spermine NONOate, S-nitrosoglutathione, and SIN-1) were
investigated (Fig. 7). Whereas the spontaneous decomposition in phosphate buffer of any one of the model compounds was
found to be associated with the release of small amounts of NO, only in
the case of SIN-1 was NO release accompanied by a stoichiometric
generation of O
2.
Biological Applications
Having demonstrated the validity of
the method for measuring NO and O2 in relatively simple
in vitro systems, it was of interest to see whether or not
it would be applicable to more complex biological systems without
further modification. Two different biological sources were used to
demonstrate, in a first pilot investigation, the suitability of the
combined NO/O
2 assay for application to intact cells and
tissues: cultured aortic ECs and isolated vascular tissue. ECs in
suspension were found to continuously release NO and O
2 at
low, yet detectable rates (n = 3) (Fig.
8A). NO was enhanced severalfold over basal
release rates in the presence of the calcium ionophore A23187, whereas
the signal for O
2 was markedly suppressed (Fig.
8B). In contrast, addition of NADPH (data not shown) or NADH
to ECs in suspension selectively increased the signal for O
2,
leaving the NO signal virtually unaffected (Fig. 8C). In
freshly isolated resting aortic tissue, the basal release of NO was
below the quantifiable limit. Addition of NADH markedly increased the
formation of O
2 (Fig. 8D), and this increase was
fully inhibitable by superoxide dismutase (n = 5) (Fig.
8E). These data clearly demonstrate that this method can
also be used for reliable kinetic measurement of NO and O
2 in
biological samples.
Simulation Model
In our experiments, NO and O2 were
formed simultaneously at specified rates. Under these conditions,
either radical can undergo various reactions. To better understand the
NO/O
2 interaction, a theoretical model that allows the
prediction of experimental results via a mathematical simulation was
elaborated, which is based on the following assumptions and
experimental evidence.
Superoxide can react with ferricytochrome c (Equation 6;
k6 = 2 × 106
M1 s
1) (32), with NO to form
ONOO
(Equation 1; k1 = 6 × 109 M
1 s
1) (10), or
dismutate spontaneously to give hydrogen peroxide (Equation 7;
k7 = 5 × 105
M
1 s
1) (33).
![]() |
(Eq. 7) |
![]() |
(Eq. 8) |
The simulation was thus performed using the following rate equations:
d[metHb]/dt = k5[oxyHb][NO] (where
k5 = 3 × 107
M1 s
1), d[ferrocytochrome
c]/dt = k6[ferricytochrome
c][O
2]
k8[ONOO
][ferrocytochrome
c] (where k8= 1.6 × 105 M
1 s
1),
d[NO]/dt = kNO
k5[oxyHb][NO]
k1[O
2][NO] (where
kNO = rate of NO generation),
d[O
2]/dt = ksup
k6[ferricytochrome
c][O
2]
k1[O
2][NO]
k7[O
2]2 (where
ksup = rate of O
2 generation),
d[ONOO
]/dt = k1[O
2][NO]
k8[ONOO
][ferricytochrome
c]
k2[ONOO
],
d[NO3
] = k2[ONOO
], and
d[H2O2]/dt = k7[O
2]2.
As depicted in Fig. 9, the simulated data are in good agreement with
the data obtained experimentally. The most striking result from these
simulations is the finding that ONOO is not formed to any
appreciable extent until >90% of the oxyHb is consumed. In addition
to the high efficiency of oxyHb in trapping NO, ferricytochrome
c plays a significant role in maintaining low O
2
levels. When the steady-state concentrations of NO and O
2 were
calculated at flux rates of ~1 µM/min for either
radical in the presence of 5 µM oxyHb and 20 µM ferricytochrome c, these concentrations
were maintained at <1 nM for NO and <10 nM
for O
2. These data suggest that there is very little
ONOO
formation in the presence of micromolar amounts of
both hemoproteins. However, when oxyHb was exhausted, there was
significant ONOO
formation as the levels of NO rose to
~80 nM, which is sufficiently high for O
2 to be
scavenged by NO instead of ferricytochrome c.
Two other conditions that are important for understanding the chemical
reactions presented here, i.e. the simultaneous generation of NO and O2 in the absence of either oxyHb or ferricytochrome c, were simulated using our computer model. As seen from the
experimental data, ferrocytochrome c is rapidly oxidized
after oxyHb is consumed. Presumably, it is ONOO
that is
responsible for this reaction as ferrocytochrome c is not
readily oxidized by either NO or O
2 under these conditions. When no oxyHb is present, ferrocytochrome c concentrations
never rise above 10 nM as determined by simulation
(assuming equimolar flux rates of 1 µM/min for NO and
O
2, respectively). This is consistent with the observation
that after oxyHb was exhausted, oxidation of ferrocytochrome
c continued until it was completely converted into
ferricytochrome c. How can ONOO
formation be
so dominant under these conditions (no oxyHb), while in the absence of
ferricytochrome c, oxyHb is readily oxidized with little
contribution from ONOO
? In the absence of ferricytochrome
c, the O
2 concentration rises to ~40
nM and slowly decays, which appears to be due to the
dismutation kinetics (Equation 7) and reaction with NO (Equation 1) as
the oxyHb concentration decreases. Under these conditions, NO is
limiting; and hence, the factors that determine the kinetics are
k5[oxyHb] and
k1[O
2]. Since O
2 never rises
much above 45 nM (note: in the absence of either
ferricytochrome c or oxyHb, the steady-state level is ~30
nM), the NO reaction with oxyHb will dominate. Conversely, in the sole presence of ferricytochrome c (0 µM oxyHb), NO continues to rise to levels of ~80-100
nM, where the NO/O
2 reaction becomes significant
(note: under these conditions, O
2 concentrations do not rise
above 1 nM). Since ferricytochrome c consumes
O
2, NO levels would be expected to rise gradually until levels
are reached where NO can compete for O
2. Since the
concentration of ferrocytochrome c in the assay is only 20 µM, with a second-order rate constant for O
2 of
2 × 106 M
1
s
1, it is not unexpected that significant
ONOO
formation will occur at NO concentrations above 10 nM. These two different conditions clearly demonstrate the
importance of oxyHb in preventing the formation of the deleterious
molecule, ONOO
. As the reaction between NO and
O
2 is near the diffusion-controlled limit, the importance of
the role of hemoglobin in controlling the formation of
ONOO
is often overlooked.
The most important finding of this study is that it is technically
feasible to simultaneously monitor NO and O2 formation. The
new nitric oxide/superoxide assay, for the first time, allows parallel,
specific, and sensitive quantification of NO and O
2 under
conditions of simultaneous generation, although the chemical reaction
between these radicals proceeds at an almost diffusion-controlled rate.
Experimental data obtained with this assay were successfully simulated
by a mathematical model that may provide a better understanding of the
reaction of either radical with biomolecules such as hemoproteins. Moreover, the results obtained with cultured endothelial cells and
isolated vascular tissue unambiguously demonstrate that this assay
methodology is applicable to biological systems, too. Taken together,
these new analytical tools may help to gain new insights into the
physiological and pathophysiological significance of co-generated NO
and O
2 for maintenance and disturbance of cellular homeostasis.
The most striking feature of this new assay is that the chosen
concentrations of hemoproteins are sufficient to almost completely trap
all of the NO and O2 generated at any flux rate tested, thus
allowing reliable quantification of either radical. Furthermore, this
assay not only allows the quantification of the formation of NO or
O
2, but also the study of their interaction. When one of the
two hemoproteins is consumed entirely, e.g. when either all
of the oxyHb is oxidized to metHb or all of the ferricytochrome c is reduced to ferrocytochrome c, NO and
O
2 react at an almost diffusion-controlled rate to form
ONOO
. More rapid than the isomerization of
ONOO
to NO3
is its
reaction with oxyHb or ferrocytochrome c (see Figs. 6 and
9). This, in addition, offers the possibility of using this assay for
competitive kinetic studies. Addition of methionine, for example, an
effective ONOO
scavenger, to the assay mixture of oxyHb
and ferricytochrome c can unmask the presence of already
formed ONOO
. Thus, differences in the measured rates of
formation of metHb or ferrocytochrome c in the absence and
presence of methionine can be taken as indirect evidence for the
involvement of ONOO
.
Recently, it has been claimed that readings obtained with the classical
oxyhemoglobin assay may not exactly reflect the amount of NO formed due
to cross-reactivity of the assay with ONOO (28). This
interpretation is potentially misleading and requires re-evaluation in
light of the findings of the present study. It is true that
ONOO
can oxidize oxyHb (see Fig. 5), and the spectral
changes observed are indistinguishable from those elicited by NO.
Nevertheless, the absolute amount of NO formed (i.e. before
its conversion to ONOO
,
NO2
, or
NO3
, the ratio of which depends on the
incubation conditions (7)) is correctly reflected even by the classical
oxyHb assay as, contrary to other NO assay techniques, the photometric
signal always represents the sum of both NO and ONOO
. The
nitric oxide/superoxide assay broadens analytical perspectives as it
not only detects NO and simultaneously quantifies O
2, which probably accounts for most of its inactivation, but also allows the
differentiation between conditions under which NO may or may not have
already reacted with O
2 to form ONOO
. In
contrast, in biological systems, Clarke-type electrodes (28), gas-phase
chemiluminescence (21), and porphyrinic microsensors (39) allow only
the quantification of the portion of NO that escapes reaction with
O
2, molecular oxygen, and hemoproteins. Moreover, in contrast
to the classical cytochrome c assay (40) and probably other
assay systems for O
2 as well, the nitric oxide/superoxide assay allows the correct quantification of the rate of O
2
formation even in the presence of concomitantly generated NO. In
summary, compared with classical analytical methods for separate
measurement of either NO or O
2, this new assay offers
specific, reliable, and simultaneous quantification of both
radicals.
The use of the photodiode array technique for analysis of spectral
changes associated with the redox conversion of hemoproteins offers
several advantages over conventional photometric techniques. 1) A
number of discrete wavelengths can be continuously monitored in
parallel; 2) the post-processing of photometric signals permits reanalysis of spectral changes without the need for repetition of
experiments originally run at suboptimal speed or sensitivity; 3) the
assay is easy to handle, and the technical equipment required is much
cheaper than that needed for conventional dual-wavelength double-beam
spectrophotometry; and 4) the ability to perform full spectral scans at
any time during the measurement provides a powerful tool for on-line
control of the specificity of the observed absorbance changes.
Post-processing of raw data using the method of moving average resulted
in a remarkably high sensitivity for NO and O2. The detection
limit for both radicals is below the physiologically relevant
concentration range that we (41-43) and others (44, 45) previously
determined in, for example, cultured endothelial cells and intact
vascular tissue. Furthermore, the ratio of formation of either radical
may vary considerably within one cell type depending on its activation
status, as, for example, in macrophages immunologically challenged with
cytokines (46). The nature of a given physiological, pharmacological,
or immunological stimulus as well as the time between cellular exposure
and analytical investigation will influence the ratio of NO,
O
2, and ONOO
formed by these cells and may
explain, at least in part, some of the discrepancy that can be found in
the literature regarding experimental results. Under all these
conditions, the nitric oxide/superoxide assay may help to resolve
controversial issues. Besides future biological applications, this
assay is also suitable for investigating NO and O
2 formation
by experimental drugs such as sydnonimines (see Fig. 6), for clarifying
the mode of bioactivation of other NO donors in different tissues and
cells, and for investigating the biochemical basis of certain
endogenous counter-regulatory mechanisms, which are, for example,
associated with nitrate tolerance (47, 48).
The flexibility in choice of the most appropriate averaging interval
constitutes an important advantage of this assay over conventional
photometric techniques. Data can be easily reanalyzed with extended or
shorter averaging intervals to achieve either greater sensitivity or
better resolution for reactions with unexpectedly fast kinetics without
loss of original data points. This will be of particular interest for
analysis of radical formation in biological systems. Furthermore, the
assay can be used at comparable sensitivity for end point
determinations and continuous on-line measurement. Continuous
quantification of NO and O2 represents an attractive
analytical approach for investigation of reactive oxygen and nitrogen
oxide formation in isolated organs under physiological and
pathophysiological conditions.
The computer model developed from know rate constants allows the
simulation of the reaction of both radicals with oxyHb and ferricytochrome c. From these results, insight is gained
into the important role of redox-active proteins. These hemoproteins are abundantly present within and in close proximity to cells of the
vascular wall and blood cells. Thus, because of the rapid reactions of
NO and O2 with either hemoprotein, it is intriguing to
speculate that hemoglobin and cytochrome c as well as other redox compounds may critically control both the reaction products and
the degradation of NO and O
2 in vivo and, by this,
their biological effects. Since the reaction between NO and O
2
is nearly diffusion-controlled, the importance of these hemoproteins
can hardly be over-stressed, as the presence of micromolar
concentrations already completely prevents formation of the potentially
harmful species, ONOO
. As revealed by computer
simulation, equimolar flux rates of NO and O
2, when
co-generated in the presence of ferricytochrome c and oxyHb,
prevented formation of significant levels of ONOO
(and
should thus prevent formation of other reactive nitrogen oxide
species), as the steady-state concentration of NO is maintained at a
low level by oxyHb. Besides thiols and tyrosine residues in proteins,
the vicinity of oxyHb may be an additional limiting factor for
ONOO
action in biological systems. Which factor(s) may,
however, govern the extent of formation of ONOO
in
vivo awaits further elucidation. Although data from our computer simulation suggest that, in the presence of certain hemoproteins, O
2 may be the limiting factor, this may not be the case under other conditions. To shed light on these questions, one would need to
know what the prevailing local concentrations of O
2 are inside
a cell in the vicinity of NO formation. Such data are not available
yet.
The ability to simultaneously measure NO and O2 at
physiologically relevant levels may be of particular interest for
atherosclerosis research. Indirect evidence from recent studies using
animal and human tissues suggest that the antiatherogenic and
vasodilatory properties of NO are counteracted by an increased
oxidative stress within the vascular wall (for review, see Ref. 8).
Endothelial and vascular smooth muscle cells as well as monocytes,
macrophages, and neutrophils are capable of producing both NO and
O
2 (for review, see Refs. 1, 3, 49, and 50). An imbalance in the formation of these radicals may have deleterious effects on vascular homeostasis. So far, this hypothesis is, however, supported by
indirect evidence only (for review, see Refs. 50-52). In arterial hypertension, it has been speculated that endogenous NO production is
compensatorily enhanced to counteract an increased oxidative stress
within the vascular wall (9, 53, 54). Similarly, an imbalance in these
radicals may be involved in the vascular dysfunction observed in
diabetes mellitus and hyperlipoproteinemia (44, 55) and in the
manifestation of functional disturbances following reperfusion of
ischemic tissue and in myocardial ischemia and reperfusion injury,
which has been shown to be associated with an increased formation of
both oxygen free radicals (56) and NO (57) within the first minutes of
reperfusion. The new assay, with its ability to simultaneously quantify
both radicals in biological tissue, provides us with a powerful tool to
experimentally challenge this yet hypothetical concept and to
investigate the functional relevance of a shift in NO/O
2
balance, providing a basis for the development of new therapeutic
strategies for vascular protection.
We thank G. Alt, K. Knüttel, and R. Spahr for skillful technical assistance.