(Received for publication, August 14, 1996, and in revised form, December 9, 1996)
From the Department of Biological and Chemical
Sciences, Central Campus, University of Essex, Colchester, Essex CO4
3SQ, United Kingdom and the § Chimbio Department, Institute
of Chemical Physics, Russian Academy of Sciences,
Moscow 117977, Russia
Normal human venous blood was studied by electron
paramagnetic resonance (EPR) spectroscopy at 196 °C. The EPR
signal of free radicals in frozen blood is shown to have the same
radiospectroscopic parameters and properties as the signal of the
globin based free radical, ·Hb(Fe(IV)=O), formed in the reaction
of purified methemoglobin (metHb) with H2O2 and
therefore has been assigned as such. The globin-based radicals and
metHb exhibited significant variation (fluctuations) in different
frozen samples taken from the same liquid blood sample. In any given
sample a high concentration of free radicals was associated with a low
concentration of metHb and vice versa, i.e. the
fluctuations were always of opposite sense. No such fluctuations were
observed in the concentration of two other paramagnetic components of
blood, transferrin and ceruloplasmin. The time course of free radical
formation and decay upon the addition of H2O2
to purified metHb was studied at three different molar ratios
H2O2/metHb. This kinetic study together with
the results of an annealing experiment allow us to propose a mechanism
for the formation and decay of the globin-based radical in blood.
Within this mechanism, the source of H2O2 in
blood is considered to be dismutation of O
2 radicals produced
via autoxidation of Hb. We postulate that the dismutation is
intensified on the phase separation surfaces during cooling and
freezing of a blood sample. The fluctuations are explained within this
hypothesis.
The reaction between heme proteins and peroxides is known to proceed via formation of two intermediates, a ferryl heme iron, Fe(IV)=O, detected by optical spectroscopy, and a globin-associated free radical ·Hb(Fe(IV)=O), detected by EPR1 spectroscopy (1-21). These transient species have been implicated in the mechanism of oxidative stress associated with human diseases. In fact, formation of a ferryl form of the heme protein was demonstrated for either ferrous or ferric heme-mediated oxidation of a wide range of biologically important substrates in the presence of lipid hydroperoxides or H2O2. These substrates include liposomes (22, 23), low density lipoprotein (24, 25), cholesterol (26), salicylate (26), styrene (27), olefins (28), unsaturated fatty acids (16, 29), and the drug chlorpromazine (13, 30). It is not clear whether the ferryl iron or the free radical plays the critical role in the oxidation. Ferryl species have been demonstrated in rat heart after a period of induced ischemia (31) and in intact red blood cells (10). However, no globin-centered free radicals have been found so far in living systems. It should be noted, however, that a kind of free radical EPR signal has been detected in whole blood (32, 33). This appears unexceptional, since most biological tissues are characterized by a free radical EPR signal (32-38). The problem with interpretation of the signal in blood was in the fact that radicals in most tissues are generally thought to be associated with the respiratory chain in mitochondria (32, 33, 35, 38). There are, however, very few mitochondria in blood, and the nature of the free radicals observed in this tissue is a matter of controversy (32, 33).
Although pro-oxidant activity of the ferryl form of heme and/or of the globin radicals has been discussed for a long time, the actual relevance of the heme protein reaction with peroxides in vivo is not established. The aim of this study was to apply the method of EPR spectroscopy to whole human blood in order to enquire whether those free radicals observed in blood (32, 33) are the radicals formed through the reaction of hemoglobin with hydrogen peroxide. We show in this study that the free radical EPR signal in purified methemoglobin (metHb) oxidized by H2O2 and that in frozen human blood are identical.
Furthermore, we show that the intensity of the free radical EPR signal
in blood measured at 196 °C varies by a factor of 5 for a series
of samples made by freezing from the same blood sample kept in
vitro at room temperature. This variation of free radical
concentration is clearly not an instrumental error, since the
concentration of the Cu(II) ions of ceruloplasmin, measured from
another signal within the same EPR spectrum, is bound by only a 5%
variation. The effect of "fluctuation" of the concentration of free
radicals in frozen blood was found to be associated with reverse
fluctuations in the concentration of metHb in the same frozen samples.
These opposing fluctuations in metHb and free radical concentrations in
frozen blood can be explained in terms of the interaction of metHb with
H2O2.
Therefore, we conclude that the reaction of metHb with endogenous H2O2 takes place in whole blood. We consider this an important issue, since the intermediates of the reaction, namely the globin-based free radical and/or the ferryl form of hemoglobin are shown to be potentially harmful pro-oxidants (13, 16, 22-30).
The methodological conclusion drawn from our results is that freezing biological tissues in liquid nitrogen for EPR measurements is not always as free of drawbacks as generally thought. The method of freezing has gradually become conventional in the EPR studies of biological systems (32, 33, 37, 39-44), since it has replaced the method of sample lyophilization extensively used before. It was shown that lyophilization, i.e. drying biological tissue in vacuum, results in poorly reproducible EPR spectra (35, 36, 45). Freezing samples has never been reported to have such a drawback. To our knowledge, this study gives the first example of freezing resulting in low reproducibility of the concentration of paramagnetic centers fixed in biological tissue.
Venous blood was donated by consenting adults and collected under air in glass tubes containing a small amount of sodium citrate used as an anticoagulant. Blood without any anticoagulant was also tested over a limited time scale (1 min) following donation and showed no difference with respect to sodium citrate-treated blood. Dilution by anticoagulant was taken into account in concentration measurements. In some experiments, red blood cells were separated from plasma by centrifugation, 2000 rpm, 20 min.
Human metHb was prepared, and its concentration was determined (in
terms of heme iron) as described elsewhere (21). The level of catalase
contamination in the preparations (21) was found to be less than
1011 M. The concentration of
H2O2 (Sigma) was calculated by
the addition of catalase (4000 units/ml) to solutions in an oxygen
electrode and measuring the amount of O2 evolved, assuming
a stoichiometry [H2O2]added:[O2]produced = 2:1 (46). The reaction of metHb with H2O2 was
conducted in 50 mM phosphate buffer, pH 7.4, at
37 °C.
EPR samples of blood, metHb/H2O2 mixtures, and blank samples (distilled water or phosphate buffer) were made singly or in duplicate using syringes and plastic tubes as described elsewhere (21). Either fast (~10-s) or slow (~60-s) freezing of the samples in liquid nitrogen was used and resulted in no difference in the effects observed. (The rate of freezing was varied by the rate of immersion of the plastic tubes with samples into the liquid nitrogen.)
EPR Spectra MeasurementThe EPR spectra of frozen blood
were measured on a Bruker ER 220D spectrometer equipped with an Aspect
2000 data system. An ER 4102 ST Universal X-Band Resonator
(TE102 mode) was used. The g-factors of EPR signals were
determined by reference to the external magnetic field value measured
by a Bruker ER 035 Gaussmeter and of the microwave frequency measured
by a Systron Donner 6235A frequency counter. Kinetic measurements in
the metHb/H2O2 system were performed using a
Varian E109 X-band spectrometer equipped with an Archimedes 440 computer. All EPR measurements were performed at 196 °C using a
quartz finger Dewar containing a frozen sample immersed in liquid
nitrogen. A sample was fixed inside the finger with a glass rod in
order to hold it down and avoid sample dancing due to liquid nitrogen
boiling. The sample was long enough that the bottom of the rod was well
above the upper edge of the working zone of the cavity. The boiling of
the liquid nitrogen was a necessary condition for preventing
overheating, but it was controlled by making the bubbles small and
frequent with proper fixing of the sample with the rod. The microwave
power was kept at a 200-milliwatt instrumental setting unless stated
otherwise. Modulation frequency was 100 kHz. The time constant was
0.1 s. The EPR spectra of the blanks were subtracted from the
spectra of blood and the metHb/H2O2 mixture.
Such a subtraction excluded all background signals, such as any signals
of the quartz Dewar or the cavity or the broad signal caused by oxygen
dissolved in liquid nitrogen.
The EPR signal intensities in blood were measured against fixed
standard signals using the D subroutine (dual consideration) of the EP routine (EPR program of Aspect 2000).
An EPR spectrum of a blood sample covering both g = 6 and g = 4.3 signals (H0 = 1350 G; H = 1000 G) was accumulated at NS = 20. This spectrum was considered
as the standard for the metHb and Tf signal determination in a series
of blood samples. The EPR spectra of every sample in the series
(experimental spectra) were measured at the same conditions but at a
lower NS (NS = 2), such a restriction resulting from a large
number of samples in a series. A difference spectrum, (experimental
spectrum)
k × (standard spectrum), was constructed for every sample using the D subroutine. The higher NS in
the standard spectrum minimizes the signal:noise ratio in the resultant difference spectrum. The factor k was then varied. The value
of k, at which the g = 4.3 signal completely
disappeared from the difference spectrum, leaving the g = 6 line
only, was taken as a measure of the g = 4.3 signal intensity in
the sample. Similarly, the value of k, when the g = 6 signal disappeared, leaving only the g = 4.3 signal, was taken as
the g = 6 signal intensity. (If an experimental spectrum had by
chance the same partial contribution of g = 6 and g = 4.3 signals as the standard spectrum, then both signals disappeared, of
course, at the same value of k, so that the difference
spectrum was transformed to a flat line.) An identical procedure was
used in determination of the ceruloplasmin (Cp) and free radical signal
intensities, with the difference that the spectra were measured at
H0 = 3300 G and
H = 200 G in
order to cover the g = 2.05 and g = 2.005 signals.
In the purified metHb experiments the EPR signals of metHb and free radicals were not contaminated with any other EPR lines (after subtraction of the constant base line). Therefore, the D subroutine was not necessary, and the simple peak-to-trough measurement of the signal intensities was appropriate.
Finally, the intensities of the blood EPR signals (g = 6 and g = 2) expressed in units of standard signals (the values of k when a blood sample was used as a standard) were related to the intensities of the signals in the purified metHb/H2O2 system expressed in the peak-to-trough units (mm). This was done by using a sample from the metHb/H2O2 series as a standard (with signal intensities already measured in mm) when applying the D subroutine to a blood sample (with already known k values). Therefore, a factor transforming k values in the blood series to the mm units used for signal intensities in the metHb/H2O2 series was found. This allowed us to determine the concentration of the high spin metHb (in terms of heme iron) and of the free radicals in the frozen samples of blood on the basis of the quantitation procedure developed for the metHb/H2O2 system (20). The legitimacy of the procedure for blood was verified by the fact that the line shapes and saturation behavior of the signals were the same in blood and purified metHb.
AnnealingThe frozen samples were annealed in a unit
comprising a transfer Dewar, a nitrogen evaporator, and a temperature
controller. After annealing, the samples were recooled to 196 °C.
The EPR spectra were always measured at this temperature.
A typical EPR spectrum
of normal human blood measured at 196 °C is shown in Fig.
1. This spectrum consists of the signals of at least
four different paramagnetic centers. The signal with g = 6 is
caused by heme Fe(III) ions in the high spin state (2, 32, 33, 47, 48)
and is assigned to metHb. The EPR signal with g = 4.3 is assigned
to transferrin Fe(III) ions complexed by
HCO3
ions (32, 33, 48, 49). The signal
with g = 2.05 is the perpendicular component of the anisotropic
EPR signal of Cu(II) ions (50). This signal is due to Cp, the major
copper-containing protein found in blood (48, 51). A slightly
asymmetric singlet with a g-factor g = 2.005 and the
peak-to-trough width of 17 G is observed in the high field wing of the
ceruloplasmin signal. This signal is attributed to the presence of free
radicals in blood (32, 33). These free radicals were found to be
present in the cell fraction of blood. Plasma does not exhibit any free radical EPR signal (data not shown).
Fluctuation of metHb and Free Radical Concentration in Frozen Blood
In a series of samples taken from blood exposed to air
in vitro, we have found that the EPR signal intensity of the
free radicals and of metHb varies from sample to sample around some
average values. Fig. 2A shows the data for
the g = 6 and g = 4.3 signals (see frame A in Fig. 1)
measured for a number of EPR samples consecutively taken from the same
liquid blood sample and frozen at various time following donation. For
the same set of samples, the intensities of the g = 2.005 and
g = 2.05 signals were also measured (Fig. 2B). A
significantly greater variation in the EPR signal of metHb (g = 6)
is seen compared with that of the Tf signal (g = 4.3). Similarly,
the variation of the free radical EPR signal (g = 2.005) is much
greater than that of the Cp signal (g = 2.05) measured from the
same spectrum and with the same absolute error (see Fig. 1B). We used a formalism described in details elsewhere (20) to express the intensities of the g = 6 and g = 2.005 signals in terms of heme concentration of high spin metHb and of the free radicals, respectively (Fig. 2C). The average concentration
of metHb is approximately 1 order of magnitude greater than the average concentration of the free radicals. When the data for free radicals are
magnified by a factor of 8, it is clearly seen that the variations in
concentration of these two centers, metHb and radicals, are not
independent; they are inversely related, i.e. in a sample with a greater metHb concentration than its mean value, the
concentration of free radicals is found to be lower than its mean value
(Fig. 2C). Similar variations in metHb and free radical
concentrations were found for duplicate samples taken from blood and
frozen at the same moment. In fact, any two samples taken randomly from a set of either consecutively or simultaneously frozen samples, which
had significantly different metHb concentrations, displayed a
difference in the concentrations of free radicals, the higher concentration of metHb being in the sample with the lower concentration of free radicals and vice versa. Moreover, we found that in
some cases the concentration of metHb (and of the free radicals) is significantly different in the opposite ends of the same sample, with a
higher metHb concentration associated with a lower concentration of the
free radicals and vice versa (results not shown).
Thus, these variations of concentration of metHb and free radicals are not time-dependent oscillations in liquid blood but are statistical fluctuations around mean values observed in frozen samples. We therefore conclude that the fluctuations in metHb and free radical concentration are linked and are caused by sample freezing. The effect of fluctuations was observed independently of whether the blood samples were frozen in liquid nitrogen quickly (10 s) or slowly (1 min). The amplitude of the fluctuations in metHb and free radical concentration was found to decrease with time of blood exposure to air. In a 5-h experiment the fluctuations were detectable during the first 3 h after donation, their amplitude becoming indistinguishable from the instrumental error after 4 h (results not shown). After the fluctuations stopped, the radical concentration became approximately equal to the lower limit of the range within which the concentration previously fluctuated. It should be noted that dark venous blood turned bright pink by 4 h of exposure to air, indicating a significant increase in oxygenation.
Effect of Annealing a Blood SampleFreezing a sample of blood
at 196 °C stops the reactions in which metHb and free radicals
were involved when the blood was liquid. Such reactions, or some of
them, may resume upon heating of the sample at successively increasing
temperatures. This procedure is known as stepwise annealing and is
widely used in radiation chemistry (39, 41, 52, 53). The result of the
annealing experiment is shown in Fig. 3. A frozen sample
of blood was annealed, first, at
60 °C for 30 min. During this
period, the sample was repeatedly (at 5, 10, and 20 min) taken from the
annealing unit for EPR measurement at
196 °C and then placed back
into the unit for proceeding with the annealing at
60 °C. The last
EPR measurement was made after 30 min of annealing. Then the
temperature in the annealing unit was increased to
50 °C, and a
new step of annealing was performed in the same manner. The temperature
was successively increased in such a way by 10 °C centigrade steps
up to
20 °C. Then the sample was recooled in a similar way at
40 °C (the second incubation at
40 °C, Fig. 3),
and the second annealing at
20 °C followed. The recooling at
40 °C was performed twice more (the third and the fourth
incubation at
40 °C).
The annealing experiment shows that concentration of metHb in a frozen
sample of blood increases with time if the sample is stored at a
temperature above 40 °C. This increase is due to hemoglobin
autoxidation. The rate of this process is strongly dependent on the
temperature being highest at
10 °C, when only 5-min storage was
sufficient for a 30-fold increase in metHb concentration compared to
the initial value before annealing (Fig. 3). The free radical
concentration rises to a maximum and then decreases as the annealing
temperature is increased, the maximum concentration being reached at
40 °C. Repeated recooling of the sample to
40 °C after
annealing at
20 °C showed significant increase in the free radical
concentration with a concomitant decrease in the upward trend of the
metHb concentration. For example, when the concentration of metHb is
decreased by 3.9 µM during e.g. second
incubation at
40 °C the increase in the free radical concentration
is 0.3 µM, i.e. ~ 8% (Fig. 3).
The following conclusion can be drawn from the annealing experiment.
Two processes are initiated in a frozen sample of blood in the
temperature range between 60 °C and
10 °C. The first is Hb
autoxidation, which leads to the increase of metHb concentration. The
second is a process in which the free radicals are formed while metHb
concentration decreases, i.e. the concentration of metHb and
radical are inversely related. The time courses depicted in Fig. 3 are
therefore complex because of a superposition of these two processes
with different temperature dependences.
The radiospectroscopic
properties of the free radical signal in blood (the g-factor, signal
width, and saturation curve) are similar to those reported for the free
radicals observed at 196 °C in the model reaction of purified
metHb with hydrogen peroxide (2, 3, 5, 6, 20, 21). To check the
suggestion that the free radicals in the two systems are the same, we
have compared the EPR spectra, measured at the same instrumental
conditions, of the blood free radicals and of the radicals formed
during H2O2-mediated oxidation of metHb. The
result of this comparison is shown in Fig. 4.
The fact that the free radical signal increases upon annealing the
blood sample at 40 °C (Fig. 3) was used to obtain an intense signal of free radicals in blood. Two samples of blood, one giving a
strong and the other a weak free radical signal were chosen. The sample
with the strong signal was then annealed at
40 °C to make its
intensity even stronger (Fig. 4A). The subtraction of the
two spectra (the factor of subtraction was chosen 0.76) eliminated the
background signal of ceruloplasmin (g = 2.05), leaving a pure
singlet of the free radicals in blood (Fig. 4C). Free
radicals in the purified metHb system were obtained by the addition of
H2O2 to metHb (Fig. 4D). The line
g = 2.03 is due to the parallel component of the EPR signal of
peroxyl radicals formed in the reaction (20, 21). The EPR signals of
blood free radicals and metHb free radicals are superimposable (Fig. 4E). The saturation curves of the signals in the two systems
are also similar (Fig. 5). We suggest therefore that the
free radicals formed in blood and in purified metHb after the
H2O2 addition are the same.
Kinetic Behavior of metHb and Free Radical in the Model Reaction
The concentrations of high spin metHb and free radicals
were monitored during the course of the reaction between metHb and H2O2 (Fig. 6). Three different
initial molar ratios metHb/H2O2 (1:1, 1:3, and
1:10) were used. It can be seen in Fig. 6 that there are no
fluctuations in the free radical and metHb concentrations in the model
reaction, since values for duplicate samples were always very close
(<5%). Within 15 s after mixing, the concentration of metHb
decreased by ~75% due to oxidation to the ferryl species (20, 21).
Simultaneous to this change the radicals were formed (Fig. 6). We shall
refer to this fast process as stage 1. There follows a period of
relatively slow change in the concentration of both species (stage 2),
which can turn into virtual steady state in the case of a significant
initial excess of H2O2 over metHb (20, 21). The
duration of this period is proportional to the initial
H2O2 concentration and corresponds to the time taken for full H2O2 decomposition (Fig. 6). The
concentration of H2O2 was monitored in parallel
experiments by measurement of O2 evolution after the
addition of catalase to the reaction mixture at different times. The
time taken for complete consumption of H2O2
measured in this way corresponded to the duration of stage 2 measured
by EPR spectroscopy (results not shown). Upon complete exhaustion of
H2O2, the radical began to decay, and the metHb concentration began to increase (stage 3). This decay of the free radicals is a first order reaction (20) and is believed to be an
autoreduction process involving modification of the heme protein (2).
It should be emphasized that the observed concentration of free radicals is low (<10%) compared with the amount of metHb transformed to the ferryl form (this is considered to be a characteristic feature of the reaction of metHb (or metMb) with H2O2) (2-4, 17). Also, the mean concentration of the radicals in stage 2, i.e. during the steady state or the period of slow change, decreases with increasing initial H2O2 concentration (Fig. 6).
The technique of freezing biological samples in liquid nitrogen
for EPR measurements has become conventional. An advantage of the
technique, as compared with the alternative method of sample lyophilization, is good reproducibility of results. This study, however, shows that when aliquots from the same blood sample are frozen
(196 °C), significant variations in EPR signal intensities of
metHb and free radicals are found, while signals for Tf and Cp are
reproducible (Fig. 2). It follows, therefore, that freezing blood in
liquid nitrogen does not fix free radicals and metHb in the
concentrations present in liquid blood prior to freezing but instead
results in fluctuating concentrations of these centers. The fact that
the concentrations of metHb and free radicals in blood are inversely
related (Figs. 2 and 3) suggests that these two species are involved
either in a single redox reaction or in a system of interrelated redox
reactions.
It was shown as early as 1956 that free radicals are formed when metHb reacts with hydrogen peroxide (1). The reaction is fast (within seconds) and results in metHb oxidation at two sites; heme is oxidized to the ferryl form Fe(IV)=O, and a free radical is formed on the globin ·Hb-(Fe(IV)=O). The nature of the protein-based radical is still under discussion. Formation of free radicals was demonstrated for H2O2 oxidation of both metHb and metMb using fast freezing techniques (2-4, 6, 16, 17, 20, 21) as well as in the liquid phase either directly (5, 7, 9, 12, 15) or by spin trapping (11, 14-16, 18). Many investigators agree that at least two different kinds of radicals are formed on the protein (12, 16-18, 20). Formation of peroxyl radicals has been proven for both metHb and metMb. However, while direct measurement showed peroxyl radicals to be the dominant type in frozen solutions of metMb treated with H2O2 (16), this kind of radical constituted only a fraction of the total concentration of radicals in the metHb system (20). The peroxyl radical in metMb was recently shown to be derived from oxygen addition to the Trp-14 radical (18, 19), although there is also evidence that tyrosine radicals are formed with metMb (7-9, 12) and metHb (15).
Our study provides strong evidence that the free radicals observed in frozen blood samples and the free radicals resulting from oxidation of purified metHb by hydrogen peroxide are the same. The parameters of the EPR signals of blood free radicals and the globin-based radicals coincide (Fig. 4). Both EPR signals have similar saturation curves (Fig. 5). The fluctuations in metHb and the free radical concentrations in frozen blood samples are inverse (Fig. 2C), implying that an excess of free radicals is formed when the metHb drops, as is the case in the reaction of purified metHb with H2O2 (Fig. 6).
The globin-centered free radicals decay rapidly in model systems (1-5, 15). On the other hand, it is possible to observe similar radicals in blood at a virtually constant (within the fluctuations) level during several hours. We propose that the reactions shown in Scheme 1 are responsible for the observed concentrations of metHb and the globin-centered free radical in blood. (For clarity of presentation, the reactions in the scheme are not balanced and indicate only the pathways of the species interaction and transformation.)
[View Larger Version of this Image (17K GIF file)]
Scheme 1.
Generally, the balance between Hb and metHb can be described in terms
of two reactions, Hb autoxidation to metHb (reaction 1) and metHb
reduction by enzyme methemoglobin reductase (reaction 2). Autoxidation
of Hb is associated with formation of the superoxide anion radical
O2 (reaction 1), which either dissociates from heme in
oxyhemoglobin after intramolecular electron transfer from Fe(II) to
oxygen (54) or can be formed, as has been shown for myoglobin (55), in
a bimolecular reaction between molecular oxygen and a weakly bound
complex between water and deoxyhemoglobin. It has been shown recently
that superoxide production correlates well with the known bell-shaped
dependence of Hb autoxidation on oxygen partial pressure (56).
Superoxide radicals dismutate, producing hydrogen peroxide (reaction
3). The steady state concentration of H2O2 in
bovine red blood cell under physiological conditions, i.e.
in the presence of H2O2 decomposing enzymes
catalase and glutathione peroxidase, has been reported to be 2 × 10
10 M (57). Kinetic comparison of
H2O2 production and oxyhemoglobin autoxidation
indicated that reactions 1 and 3 probably represent the main route of
H2O2 generation in red blood cells (57).
Once formed, we suggest, hydrogen peroxide in blood reacts with different forms of hemoglobin in the ways known from biochemical model studies. Our data on the kinetic behavior of metHb and free radicals in the model reactions (Fig. 6) show that the free radical concentration in stage 2, i.e. in the stage of slow changing/steady state concentration, is greater when the molar excess of H2O2 is lower. This can only be understood if both the rate of production and the rate of decay of the radical are H2O2 concentration-dependent (reactions 4 and 5, respectively, of Scheme 1) and the decay process is favored at a high excess of H2O2. We have attempted to simulate the time courses in Fig. 6, employing a mechanism similar to that shown in Scheme 1 except that it incorporates the reactions between metHb and H2O2 only, and the ferrous state was omitted. This simulation does not perfectly fit the experimental data, especially in the initial stage of the reaction (first 2 min). However, it does reproduce several essential features, namely 1) relatively low radical yield compared with initial protein concentration (often reported in the literature (2-4, 17)); 2) the existence of the steady state/slow changing stage followed by free radical decay and ferryl reduction to the metHb; 3) longer steady state at higher initial H2O2 concentration; 4) the lower concentration of the free radical in the steady state at higher initial [H2O2]. We were only able to simulate these important characteristics seen in Fig. 6 by assuming a [H2O2]-dependent decay pathway for the radicals. No steady state could be achieved if only two reactions, a very fast radical formation (stage 1 is less than 15 s; see Fig. 6) and relatively slow radical decay (t1/2 = 8-10 min in stage 3; Fig. 6) were used.
Hydrogen peroxide can also react directly with deoxyhemoglobin (10, 54), resulting in the ferryl form (reaction 6), which either comproportionates with oxyhemoglobin (10) (reaction 7) or undergoes reduction to metHb via a first order reaction (reaction 8) (21, 58). The autoreduction of the free radicals, which causes radical decay after H2O2 is exhausted (Fig. 6, stage 3) is depicted in Scheme 1 as a first order reaction (reaction 9).
For blood in vitro, reactions 1-9 lead to a steady state
concentration of every species shown in Scheme 1, or rather a
quasi-steady state, because the concentrations change due to slow
oxygenation of blood (during hours). Reaction R1 slows upon increasing
oxygen concentration in blood (54-56, 58), while reaction 2 changes little (presumably). Thus, oxygenation results in a gradual decrease in
the mean metHb concentration (Fig. 2). Nevertheless, one may consider
that within a short time interval (~10 min) reactions 1-9 generate a
steady state concentration of every species. We propose that freezing
blood perturbs this steady state (see below) and that the extent of
this perturbation is different for every sample frozen, causing
fluctuations in the concentration of metHb and free radicals observed
at 196 °C.
In contrast to blood, we found no fluctuations in metHb and free
radical concentrations in the frozen samples of reaction mixture
containing purified metHb and H2O2. To account
for this we propose that it is the concentration of
H2O2 that fluctuates in blood as a result of
freezing in liquid nitrogen. We suggest that the process of freezing
blood creates nonequilibrium high concentrations of
H2O2 as a result of intensified dismutation of
superoxide radicals due to phase transitions. A very similar effect of
intensified disproportionation of ascorbic acid radicals upon freezing
solutions of ascorbic acid in water is documented (39). When ascorbic
acid is autoxidized in water, ascorbic acid free radicals may easily be
observed by EPR spectroscopy. If, however, such a solution is frozen in
liquid nitrogen (~15 s), ascorbic acid radicals disproportionate, and
no EPR signal can be detected in the frozen sample. Alternatively, if
autoxidation proceeds in a water-glycerol (1:1) solution, ascorbic acid
free radicals can be detected at 196 °C as well as at room
temperature because cooling of such a solution down to
196 °C does
not result in phase separation, and the radicals do not vanish due to
intensified disproportionation (39).
Therefore, we suggest that O2 behaves similarly, being
effectively concentrated at the phase separation surfaces as blood freezes. High local concentration causes enhanced disproportionation and formation of a relatively high concentration of
H2O2. The macroscopic distribution of phase
separation surfaces in the sample volume, and their propagation within
the time taken for complete freezing can never be the same in
different samples. Therefore, the concentration of
H2O2 fixed eventually in a blood sample will fluctuate. The higher the O
2 concentration before freezing,
the wider will be the range of possible H2O2
concentrations and, consequently, the wider the range of the metHb and
free radical fluctuation observed.
This mechanism also allows us to explain why the fluctuations decrease
with time. The hemoglobin/myoglobin autoxidation rate decreases upon
increasing O2 pressure, indicating that the deoxy heme
fraction of the protein facilitates the process of oxidation (54-56).
Blood oxygenation, therefore, results in a decrease in the steady state
concentration of metHb (reaction 1 slows compared with reaction 2; see
Scheme 1), which can be seen as gradual decrease of mean intensity of
the g = 6 signal (Fig. 2A). Since one molecule of
superoxide radical is formed per one ferrous heme oxidized to the
ferric form (55), superoxide production during Hb autoxidation in blood
(reaction 1) also decreases at a higher blood oxygenation level (56).
Four hours of blood oxygenation in our experiment resulted therefore in
such low O2 concentrations in liquid blood that freezing did
not cause significant additional H2O2
formation. Under such conditions, the H2O2
concentration does not fluctuate significantly in frozen samples,
therefore causing no detectable fluctuations in metHb and free radical
concentration.
The result of the annealing experiment (Fig. 3) can also be understood within the mechanism shown in Scheme 1.
We postulate that under our experimental conditions a small fraction of
the H2O2 is left unreacted in blood by the end
of the freezing time. This low concentration of
H2O2 reacts with metHb (reaction 4) when a
frozen blood sample is incubated at an elevated temperature. It is
known that small molecules, like water and molecular oxygen, gain
mobility at 90 °C and become free to interact with macroradicals
in frozen water matrices during stepwise annealing of irradiated
samples (39, 52, 53). It is reasonable, therefore, to suppose that the
peroxide molecules are mobile and can react with metHb, creating the
radicals when frozen matrix is kept at
60 °C (Fig. 3). However,
due to low concentration, as we suggest, H2O2
does not induce the radical decay, since it is only in the presence of
an excess of H2O2 over metHb that
H2O2 interacts with the radicals (reaction 5),
resulting in their decay, as was shown in the experiment with purified
metHb (Fig. 6). Under such conditions the concentration of the radicals formed in a blood sample at
60 °C will be nearly stoichiometric with H2O2 and with the concentration of metHb
oxidized to the ferryl form. The growth of the radical upon annealing
at
60 °C is ~1 µM (Fig. 3). We expect therefore
that concomitant loss of metHb in the course of reaction 4 will be not
greater than 1 µM, which is just below our detection
limit (~2 µM), and that is why we do not observe it
(Fig. 3).
Elevating the annealing temperature above 50 °C causes hemoglobin
autoxidation in frozen blood sample (reaction 1); the higher the
temperature, the steeper the increase of metHb concentration (Fig. 3).
The reverse process of metHb enzymatic reduction (reaction 2) is
probably blocked at such temperatures, since it needs two high
molecular weight species to meet each other through diffusion. As a
result of Hb autoxidation, the H2O2 formed
(reactions 1 and 3) causes both radical formation and decay (reactions
4 and 5). The balance between reaction 4 on one hand and reactions 5 and 9 on the other depends on temperature. As temperature increases from
50 °C to
20 °C, the rate of radical formation (reaction 4) increases more slowly than the rate of radical decay (reaction 5 and
9), so that observed concentration of free radicals has a maximum at a
temperature of
40 °C (Fig. 3). Interestingly, a very similar
incubation temperature of
38 °C has recently been used to
demonstrate superoxide radical production in partially oxygenated human
Hb samples (56). Superoxide radicals were directly observed in Hb
samples at liquid helium temperatures, and their signal intensity
depended on the oxygenation status of Hb in the same fashion as did the
rate of autoxidation (56).
When the sample is cooled to 196 °C after annealing at
20 °C,
the concentration of H2O2 produced by
autoxidation is high enough to cause a measurable loss of metHb on a
following incubation at
40 °C via reaction 4. However, the
increase of the free radical concentration at this second incubation at
40 °C is not as big as at
60 °C (Fig. 3) because it is
determined now by both
H2O2-dependent formation (reaction
4) and H2O2-dependent decay
(reaction 5) of the radicals.
Finally, there are no fluctuations in metHb and free radical concentration in the annealing experiment (the curves in Fig. 3 are smooth within each temperature section) because the sample has been kept solid and has been not subjected to a phase transition.
We conclude, therefore, that in spite of the presence of catalase and glutathione peroxidase, there is a steady state concentration of H2O2 in whole human blood, which leads to metHb oxidation to the ferryl form and concomitant production of globin-centered free radical. The concentration of the radicals in whole blood is ~1 µM and depends on the oxygenation status of the blood. As stressed above, the pro-oxidant activity of the ferryl form of heme and/or of the globin radical has been extensively studied in model systems. This allowed many to speculate as to whether these species have relevance to the processes in vivo. Although ferryl hemoglobin formation has been demonstrated in intact red blood cells (10), the present study is the first, to the best of our knowledge, to show globin-based radical formation in whole blood. Our results imply that hemoglobin interaction with endogenously formed hydrogen peroxide does take place in normal human blood.