1 Biophysics Group, Using the
blood-free perfused rat brain, we examined the redox behavior of
cytochrome oxidase of two chromophores, heme
a + a3 and copper.
When perfusate inflow was stopped to induce global ischemia,
the reduction of heme a + a3 was triphasic,
with a rapid phase, a slow phase, and a second rapid phase. In
contrast, the reduction of copper was monophasic after the rapid phase
of heme a + a3. The triphasic
reduction of heme a + a3 was diminished by energy-depleting treatments, such as addition of an uncoupler. The
time course of the reduction of copper was not affected by the energy
depletion. During global ischemia the decrease in creatine phosphate nearly paralleled the reduction of heme
a + a3, whereas ATP
remained at the control level until ~60% of heme
a + a3 was reduced in
the rapid phase. In the slow phase, ATP started to decrease with the
reduction of copper. The redox behavior of copper was similar to the
slow phase of the reduction of heme a + a3 because of
the higher oxygen affinity of copper than of heme
a + a3. Therefore,
the rapid phase of the reduction of heme
a + a3 can be used as
an alarm before a decrease in ATP, whereas the reduction of copper
indicates a decrease in ATP under severe hypoxia. Thus the copper
signal in noninvasive near-infrared spectroscopy is a useful parameter
for the clinical setting.
oxygen supply; mitochondria; uncoupled state; hypoglycemia; near-infrared spectroscopy
WITH ISOLATED MITOCHONDRIA, there have been many
studies on the redox behavior of heme
a + a3 and copper in
cytochrome oxidase related to the mitochondrial energy state and
respiratory rate in vitro (19, 28, 31, 33, 35). It has been established that the redox state of heme a + a3 depends on the
energy state and the oxygen concentration, whereas the redox state of
copper depends only on the oxygen concentration (19, 35). During the
state 3-state 4 transition, heme a + a3 is more
reduced in state 3 than in state 4, but no redox shift is observed with
copper. Thus simultaneous monitoring of redox changes in heme
a + a3 and copper in
cytochrome oxidase offers the possibility of obtaining direct
information on the energy conditions and oxygenation of living tissues.
However, spectrophotometric measurement of the redox behavior of heme
a + a3 in the visible
region has been practically impossible because of the hemoglobin
absorption.
Therefore, we employed a technique for hemoglobin-free perfusion of the
rat brain where blood was completely removed (1, 21). Using this
preparation, we investigated the redox changes in heme
a + a3 and copper in
cerebral tissue, as related to the mitochondrial energy state. The data
obtained with this experiment bridged the gap between isolated
mitochondria and blood-circulating living tissues, in which the redox
behavior of copper has been measured by near-infrared spectroscopy (14,
20, 22, 30, 42).
Materials
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Surgical Preparation
The experiments were performed on male Wistar rats (180-250 g body wt) that were allowed free access to food and water. Anesthesia was induced and maintained with halothane (0.5-2%) during the surgical procedures. Tracheotomy and femoral venous catheterization were performed. After paralysis with pancuronium bromide (0.1 mg/kg iv), rats were ventilated with a positive-pressure rodent respirator. Tidal volume and respiratory rate were adjusted to maintain normal arterial PCO2 (35-40 mmHg). Skin and muscle overlying the calvaria were removed, and the parietal bone was thinned to make light penetration into the brain effective. The method of brain perfusion was based on the procedure of Inagaki and Tamura (21). Both common carotid arteries were exposed, and the external carotid arteries were ligated. After injection of heparin (500 U/kg), bypass catheters were implanted in each of the common carotid arteries without interrupting brain circulation. The rat brain was perfused with FC-43 via the bypass catheters, and the perfusate flowed out from the catheter that was implanted via the inferior vena cava into the right atrium. The flow rate was maintained at 6 ml/min, and the perfusion pressure was monitored. The perfusate temperature was kept at 30°C. Bipolar electroencephalograms (EEG) were recorded from the parietal region of each hemisphere, with electrodes placed in direct contact with the bone. The spontaneous EEG during perfusion was similar to that during the surgical preparation under anesthesia. The spontaneous EEG was maintained for at least 4 h during perfusion.The study was approved by the Hokkaido University Committee on the Ethics of Animal Investigation.
Spectrophotometric Measurement
Two methods of optical measurement were employed to monitor the redox behavior of cytochromes: one to measure spectra of the perfused brain and the other to monitor redox changes in heme a + a3 and copper in cytochrome oxidase simultaneously and continuously by dual-wavelength spectrophotometry.A photodiode scanner (model MCPD2000, Otsuka Electronics, Osaka, Japan) with quartz optical fibers and a 150-W halogen lamp as a light source were used to measure absorption spectra of the rat brain in the visible region. The measuring light from the halogen lamp directly illuminated the parietal region of the rat skull through a light guide. The emergent light from the brain collected by an another light guide was coupled with a monochrometer and detected by photodiodes with resolutions of 2 nm to scan wavelength ranges between 400 and 900 nm. The distance between the two fibers was ~5 mm. Sampling time of each scan was 0.8 s. The spectrum that was obtained with fully oxygenated perfusion (FC-43 equilibrated with 95% O2-5% CO2 and 1% halothane) was used as a reference.
Redox changes in heme a + a3 and copper in cytochrome oxidase were measured simultaneously and continuously with a two-channel dual-wavelength spectrophotometer (Unisoku, Osaka, Japan). The measuring lights from a halogen lamp filtered through four interference filters (605-, 620-, 780-, and 830-nm wavelengths) were illuminated on the center of the parietal region of the rat skull through the light guide. The light transmitted through the cranial bone and cerebral tissue was coupled with the photodetection system through another light guide on the hard palate. The redox behavior of heme a + a3 and copper was monitored using 605- to 620-nm and 830- to 780-nm wavelength pairs, respectively. The redox states of heme a + a3 and copper were expressed as a percentage of the total absorbance change. The full scale of the absorbance change was obtained with perfusion with oxygenated FC-43 (equilibrated with 95% O2-5% CO2 and 1% halothane) and anoxia with the interruption of perfusion. The experimental setup is shown in Fig. 1.
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Biochemical Analyses
At predetermined times, the brain was frozen in situ using the liquid nitrogen funnel procedure as described by Ponten et al. (34). This has been accepted as the preferred method of freezing to minimize metabolic changes. The brain was chiseled out during intermittent irrigation with liquid nitrogen. Brains were stored at ![]() |
RESULTS |
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Difference Spectra of the Rat Brain
Figure 2A shows difference spectra of the rat brain at 400-700 nm during anoxic insult induced by the interruption of perfusion. The spectrum of the fully oxygenated perfusion (FC-43 equilibrated with 95% O2-5% CO2 and 1% halothane) was used as a reference and taken as a flat baseline. After the cessation of perfusion, as the anoxic insult was continued, three absorption peaks at ~605, 550, and 520 nm became prominent. These were due to the reduction of the
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Redox Behavior of Heme a + a3 and Copper in Cerebral Tissue
Normal energy conditions. After aerobic perfusion with the oxygenated FC-43, brain anoxic insult was induced by interrupting perfusion. Figure 4 shows the redox responses of heme a + a3 and copper in cytochrome oxidase to the transient anoxic insult induced by the interruption. The redox states of heme a + a3 and copper were expressed as a percentage of the total absorbance change. In response to the cessation of perfusion, the reduction of heme a + a3 and copper started. The time course of reduction of heme a + a3 was triphasic, as shown in Fig. 3. The reduction of copper was monophasic, similar to that of cytochrome c. However, copper was fully reduced ~2 min after full reduction of heme a + a3. After perfusion was started again, the time course of the oxidation of heme a + a3 was biphasic, and the rapid oxidation phase accounted for 70% of the total absorbance change, which was the same magnitude as the rapid reduction phase. The oxidation of copper was monophasic, similar to the reduction. Copper was restored to the fully oxidized state within 4 min, but restoration of heme a + a3 required >8 min. The spontaneous EEG disappeared within 60 s after the cessation of perfusion, when the reduction of heme a + a3 shifted from the rapid phase to the slow phase. At this stage, >70% of heme a + a3 was reduced, but only ~20% of copper was reduced. When ischemia continued for >5 min, the spontaneous EEG did not recover within 60 min after reperfusion was begun, although heme a + a3 and copper had already been restored to the fully oxidized state.
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Energy-depleted conditions. Figure 5 shows the redox responses of heme a + a3 and copper of cerebral tissue of the uncoupled preparation, where we used the uncoupler of CCCP. After the switch to the FC-43 solution containing CCCP, heme a + a3 was transiently reduced ~70% and then the partial reduction of ~40% was maintained. Copper was kept in the fully oxidized state, then was transiently reduced (~30% reduction). The spontaneous EEG disappeared after heme a + a3 and copper were transiently reduced. In the presence of the uncoupler, the ischemic insult caused rapid and monophasic reduction of heme a + a3 from the 40% partially reduced state. Reperfusion caused the monophasic oxidation of heme a + a3, an overshoot to an 80% reduction, then return to the previous level of the uncoupled state (~30% reduction from normal conditions). Copper recovered to the previous level.
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Relationship Between the Redox State of Heme a + a3, Copper, and Changes in Cerebral High-Energy Phosphate Levels
To interpret the redox behavior of heme a + a3, we simultaneously performed biochemical determinations of the cerebral energy states as references for optical measurement. As shown in Fig. 7A, cerebral high-energy phosphate concentrations were measured at the following points: in the full-oxidation phase (control), post-rapid phase, post-slow phase, and full-reduction phase. Ischemia-induced changes in cerebral ATP, ADP, AMP, and PCr are presented in Table 1. The control levels of ATP, ADP, and AMP in the perfused brain were similar to those obtained in other experiments, but PCr was lower (39). The percent changes in PCr and ATP are shown in Fig. 7B, where changes in heme a + a3 and copper are also shown. After perfusion was stopped, PCr decreased rapidly to 40% of the control level at the post-rapid phase and became nearly zero at the post-slow phase. ATP remained at the control level at the post-rapid phase and decreased to 70% of the control level at the post-slow phase. After the end of the slow phase, ATP decreased sharply in proportion as heme a + a3 was almost fully reduced. Here, it must be noted that the reduction of heme a + a3 paralleled the decrease in PCr, not ATP, whereas the reduction of copper paralleled the decrease in ATP. These changes in adenylate nucleotides were reflected by the energy charge of the adenylate pool.
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Figure 8 shows the effects on the redox states and EEG of reoxygenation after transient ischemia. When the perfusion was stopped, the EEG became flat (Fig. 8, b), concomitantly with the reduction of heme a + a3 and copper. At the slow reduction phase of heme a + a3 before the second rapid phase, the perfusion was started. Heme a + a3 and copper were reoxidized immediately. At 90 s after reperfusion, EEG recovered transiently. After a few minutes, a stable EEG was recorded.
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DISCUSSION |
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Hemoglobin-Free Brain Preparations
For precise examination of the energy dependence of the redox state of cerebral cytochrome oxidase in situ, we prepared hemoglobin-free perfused rat heads, from which trace amounts of blood were completely removed. Because of the artificial perfusion system, the mitochondrial energy state could be manipulated without circulatory effects by addition of an uncoupler and hypoglycemia. Our preparation of perfused rat brain was kept under aerobic conditions, where ATP content was the same as in the normal aerobic rat brain (39). It was found that, to maintain aerobic conditions in the brain tissue, a flow rate >6 ml/min and perfusion pressure >160 mmHg, higher than the mean arterial pressure of normal rats, were required. To clearly characterize the relationship between the redox states, ATP, and PCr levels, we used complete ischemia in this study. The redox behavior in graded hypoxia, the control experiment, is reported elsewhere (32).Cytochrome Oxidase in Perfused Brain
Many reports have been published demonstrating the effect of energy state on the respiratory carriers of isolated mitochondria. The oxygen concentrations for the half-maximal reduction (p50) of NADH are 0.08 µM in state 3 and 0.09 µM in state 4 (37). In cytochrome c, there is a linear increase of the p50 with increasing respiratory rate from 0.03 to 0.27 µM (37). The p50 of heme a + a3 in cytochrome oxidase is 0.16 µM in state 3 and 0.08 µM in state 4. That of copper A is 0.08 µM and is independent of the energy state (19).The energy dependence of the oxidase was explained by the midpoint potential (Em) and spectral contributions of these components to each wavelength. The Em of copper A is +240 mV, which is not affected by ATP (13). In state 3 the Em of heme a3 is +375 mV, which is much higher than that of copper A, whereas at +220 mV the Em of heme a is lower than that of copper A. In contrast, in state 4 and the uncoupled state the Em of +155 mV of heme a3 becomes lower than that of copper A, whereas the Em of heme a is +275 mV (28). In the spectral contribution, copper A accounts for >85% of the total absorption at 830 nm and is independent of the energy state (4). On the other hand, heme a in the presence of ATP accounts for 72-89% of total absorption at 605 nm compared with the almost equal contributions of the two components in the absence of ATP (28). In addition to the above Em data, kinetic measurements have revealed that heme a is the initial acceptor of electrons from cytochrome c and that copper A might accept electrons from heme a (7).
Recently, amino acid sequences and structural prediction methods suggest that copper A is close to the docking site for cytochrome c (38). However, photoactivated ruthenium-labeled cytochrome c donates electrons to the copper A dimer next to heme a, showing the reaction with the copper first and with the heme a second, the rate constants being 6:1 in favor of the copper A-to-heme a reaction being faster than the cytochrome c-to-copper reaction (44). Our results are explained by these kinetic data. In the perfused brain, cytochrome c, heme a (+a3), and copper were reduced in that order, and the rapid reduction phase of heme a (+a3) was similar to that of cytochrome c. This is in good agreement with in vitro observations; that is, the oxygen affinity of copper is highest and that of heme a + a3 is second in state 3, and heme a and cytochrome c show similar oxygen affinities (19, 33).
In the present study the copper was difficult to reduce and remained oxidized until significant heme a + a3 reduction. In contrast to our results, copper reduction occurs very early in the hypoxic process in exchange-transfused cats (36). On the other hand, in the purified enzyme, Cooper and Springett (10) reported that copper reduction occurs late in the hypoxic process and simultaneously with heme a reduction. The differences may result from spectral overlap of residual hemoglobin in vivo (32). Alteration in the spectral and thermodynamic properties of some of the cytochromes can occur in the purified process in vitro (23).
Copper in Cytochrome Oxidase
Although many metalloproteins have charge-transfer bands in the near-infrared region, cytochrome oxidase is unique, in that the extinction coefficient is very high in the oxidized form (>3 mMIn contrast to heme a + a3, the redox behavior of copper was found to be monophasic with the various energy states. This agreed with the redox behavior of copper calibrated in vitro; that is, the time course of the reduction of copper was monophasic in state 3, in state 4, and in the uncoupled state (19, 35), since the oxygen affinity of copper is independent of the energy state and the degree of the oxidation-reduction state of copper depends only on the tissue oxygen concentration (19). As shown in Fig. 7, ATP content was almost the same as that in control conditions, although 15% of copper was reduced at the post-rapid phase of heme a + a3. This shows that the reduction of copper is a critical condition in hypoxia. This is confirmed by the fact that, as shown in Fig. 8, the EEG recovered after the readministration of oxygen at this stage.
Characterization of Energy-Dependent Redox Behavior
When these mitochondrial observations are considered, the observed triphase of the reduction of heme a + a3 in normal cerebral tissue can account for this complex behavior of cytochrome oxidase. The first rapid phase of the reduction, ~70% of the total absorbance change, can be mainly attributed to the reduction of heme a. This was confirmed by the result showing that cerebral ATP remained at the control level during this rapid phase; that is, the rapid phase was independent of the tissue energy conditions. Thus the slow phase could be attributed to heme a3, which depends on the energy state. The slow phase was diminished under the energy-depleted conditions, such as uncoupled and hypoglycemic conditions. With the in vitro calibration of isolated mitochondria, the reduction of heme a3 does not reach full reduction in the presence of ATP (5, 40). However, in in vivo living tissue, heme a3 can be fully reduced because of the depletion of ATP, which is consumed during ischemia. Thus, in the second rapid phase, heme a3 started to be rapidly reduced because of the loss of cerebral ATP (Fig. 7), so the slow phase and the subsequent rapid phase were due to the reduction of heme a3 accompanied by a decrease in the energy charge of the adenylate pool.The energy-dependent redox behavior can be summarized as follows. In the perfused brain, a decrease in oxygen supply due to the perfusion interruption caused reduction of heme a. PCr compensated for the decrease in ATP concomitant with the reduction. When PCr disappeared, reduction of heme a3 started with a decrease in ATP and the energy charge. After reduction of heme a, copper A was reduced. An excellent correlation of the redox state of copper A with cerebral ATP was obtained in the perfused brain; nevertheless, the redox state of copper A in vitro was independent of the energy state. The redox behavior of copper A was similar to the slow reduction of heme a + a3, which is attributed to heme a3 under these conditions. This assumption was confirmed by the changes in energy charge shown in Fig. 7.
The present data emphasize the critical importance of the measurement of the redox behavior of heme a + a3 in addition to that of copper for assessment of the tissue energy state. The latter has been measured by near-infrared oxygen monitoring in the clinical field (3, 6, 12, 15, 26, 27, 43). Thus, despite the difficulty of heme a + a3 measurement in the visible region under normal blood supply conditions, an effort must be made to introduce bedside energy monitoring as a replacement for magnetic resonance spectroscopy.
Effects of the Uncoupler and Hypoglycemia on the Redox State of Heme a + a3 and Copper
The uncoupler CCCP changes not only the energy state of mitochondria but also the respiratory rate. On switching from normal FC-43 to uncoupled FC-43, heme a + a3 and copper were transiently reduced. The reduction of heme a + a3 results from the reduction of heme a3, which is caused by an increase in respiratory rate and energy depletion (28, 33, 41). The oxygen supply to cerebral tissue by FC-43 became inadequate with the increase in cerebral oxygen consumption caused by the increasing respiratory rate, and, as a result, copper was also reduced. This is consistent with the previous observation that the seizure induced by pentylenetetrazol administration causes a transient reduction of copper in the rat brain, because the oxygen supply does not compensate for the increasing oxygen demand produced by the seizure (20). After the transient reduction, a decrease in cerebral oxygen consumption owing to the disappearance of the spontaneous EEG may lead the shift of heme a + a3 and copper toward oxidation.Blocking of the glucose metabolism induced by 2-DG decreases the electron flow rate through the respiratory chain and changes the cerebral energy state. Copper, on switching to hypoglycemic FC-43, was slightly hyperoxidized because of the decrease in electron flow, whereas heme a + a3 fluctuated in the redox state. Furthermore, the redox state of heme a + a3 was unstable after reperfusion. The reasons for these fluctuations in heme a + a3 were not clear. However, Inagaki and Tamura (21) reported that heme a + a3 and copper become hyperoxidized in the perfused rat brain with a glucose-free perfusate. This difference in the redox behavior of heme a + a3 may result from insufficient blocking of the glucose metabolism by 2-DG.
According to Kariman et al. (24), addition of 2,4-dinitrophenol in the isolated and perfused rat brain resulted in almost complete disappearance of PCr and ATP peaks after 7 min while the entire 31P nuclear magnetic resonance spectrum is dominated by the Pi peak resulting from a decrease in the PCr-to-Pi ratio of <0.1 from 3.5 before the administration. In the present study, uncoupled FC-43 was perfused for 20 min. The perfused brain without oxidative phosphorylation would have no ATP. Dirks et al. (11) reported that ATP content decreased to 0.58 µmol/g wet wt from 2.30 when the isolated and perfused rat brain was subjected to aglycemia for 25 min. Also, in our preparation under hypoglycemia, ATP would decrease remarkably, but there were a few residual ATP molecules. This was supported by the little shoulder of heme a + a3 in Fig. 6. In both cases, the monophasic reductions of copper were independent of the energy-depleted treatments, whereas the redox behavior of heme a + a3 changed. Although the energy state did not affect copper, it declined because of the decrease in oxygen concentration concomitantly with the reduction of heme a and the decrease in ATP after the disappearance of PCr.
Because the transport of oxidizable substrates into mitochondria is dependent on the proton motive force, the supply of reducing equivalents to NADH affects the cellular respiration. However, the complex behavior of heme a + a3 reduction presented in Fig. 4 could be explained by absorption changes in isolated mitochondria, in which the reducing equivalents were sufficiently supplied. Thus it appeared unlikely that change in the rate of reduction of heme a + a3 reflected the rate of transport of reducing equivalents into mitochondria. However, the supply of reducing equivalents is important. The correlation of the supply with the kinetics of the oxidase, ATP, and PCr levels is a subject for further experiments.
Several investigators have reported respiratory suppression in hepatocytes (8) and myocytes (25) under hypoxic conditions. This mechanism, however, may not apply to the brain, because it has different properties. For example, the oxygen consumption rate is higher in cerebral tissue than in liver, and there is no myoglobin, which is an oxygen buffer. Hepatocytes exhibit a reversible suppression of respiration during prolonged hypoxia (PO2 = 20 mmHg for 3-5 h). Such adaptation to hypoxia may not be physiologically significant for cerebral tissue. However, isolated heart cytochrome oxidase and submitochondrial particles undergo reversible decreases in apparent maximal reaction velocity when incubated under similar conditions (8). This implies that cerebral cytochrome oxidase may adapt to hypoxia unless it has organ specificity.
Conclusion
Previous reports on isolated mitochondria in vitro were confirmed in the perfused brain; that is, the redox state of heme a + a3 depends on the energy state, respiratory rate, and oxygen concentration, whereas the redox state of copper depends only on the oxygen concentration. However, the redox behavior of copper was similar to the slow phase reduction of heme a3, because the apparent affinity for oxygen was higher for heme a3 than for heme a. Therefore, rapid phase reduction of heme a can be used as an alarm before a decrease in ATP, whereas the copper signal reduction under severe hypoxia indicates the occurrence of the decrease. This knowledge will bring us closer to the goal of noninvasive monitoring of changes in tissue oxygenation and the energy state at the bedside. ![]() |
ACKNOWLEDGEMENTS |
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This study was supported in part by Grant-in Aid for Scientific Research 09558108 from the Ministry of Education, Science, and Culture of Japan.
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FOOTNOTES |
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Preliminary results have been presented and published elsewhere (29).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: Y. Nomura, Biophysics Group, Research Institute for Electronic Science, Hokkaido University, Sapporo 060, Japan.
Received 29 April 1998; accepted in final form 17 June 1998.
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