Oxidation of cerebral cytochrome aa3 by oxygen plus carbon dioxide at hyperbaric pressures.

Oxidation of cerebral cytochrome plus carbon dioxide at hyperbaric

aa, by oxygen pressures

F. G. HEMPEL, F. F. JOBSIS, J. L. LAMANNA, M. R. ROSENTHAL, AND H. A. SALTZMAN F. G. Hall Laboratory Pharmacology, Duke

for Environmental University Medical

Research and Department of Physiology Center, Durham, North Carolina 27710

HEMPEL, F. G., F. F. J~BSIS, J. L. LAMANNA, M. R. ROSENTHAL, AND H. A. SALTZMAN. Oxidation of cerebral cytochrome pressures.

aa

by oxygen

plus

carbon

dioxide

at hyperbaric

J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 43(5): 873-879, 1977. -The reduction-oxidation level of cytochrome aa:, in the intact cerebral cortex of cerveau isole or pentobarbital-anesthetized cats was monitored by means of dual-beam reflectance spectrophotometry. Respiratory gases containing varying fractions of carbon dioxide and oxygen were administered at increased pressures, allowing titration of the cytochrome redox state from maximum reduction during nitrogen ventilation to the completely oxidized state. We show that the fully oxidized state could be reached with about 5% CO,-95% 0, inspired at 4 ATA. Maximum oxidation was achieved only through the relaxation of oxygeninduced vasoconstriction of the cerebral vessels, as our data indicated that large blood volume responses accompanied the increases in inspired carbon dioxide. With our technique, we have established that under resting air-breathing conditions, cytochrome aa:% is about 85% reduced in the cat cerebral cortex, and that the absorption peak for cytochrome aa in situ is located near 602 nm. A free energy change of an additional 8 kcal is calculated to occur with donation of an electron pair from 85% reduced cytochrome aa:, to oxygen as compared to a 1% reduction level. brain;

mitochondria;

respiration;

cat

ORIGINALLY DEVELOPED by Chance (1, 2) to study absorption changes in turbid suspensions and recently used to observe reflection from the living cerebral cortex (11, 23), the dual-wavelength spectrophotometer functions by comparing optical density changes at a wavelength of interest with the optical density at a nearby reference wavelength. Ordinarily the wavelengths of interest are the cypeaks of reduced cytochrome au,, b, and c, in the mitochondria, and the reference wavelengths chosen to be the closest isobestic points between reduced and oxidized states of the cytochrome, and, in living tissue, the closest points at which the spectral extinction difference between oxy- and deoxyhemoglobin equals that difference at the sample (cytochrome) wavelengths. Reflectance spectrophotometry of the cortical cytochromes relies on the fact that as these compounds are reduced, they absorb more of the incident measuring wavelength and hence less reflected light returns from the brain. Since the reduction-oxidation level of the cytochromes depends on oxygen delivery to the electron

and

transport system (3, lo), dual-wavelength reflectance spectrophotometry offers a noninvasive means of following the dynamics of oxygen utilization in the exposed cerebral cortex. Previous investigations with reflectance spectrophotometry have shown that, by increasing inspired oxygen pressure, cytochrome au, in the cat and rabbit cerebral cortex could be oxidized beyond the steady-state reduction level recorded during room air breathing (23). Five percent carbon dioxide potentiated the effect, but there were no indications that a saturation point could be reached at 1 ATA. At the substrate end of the respiratory chain, hyperbaric oxygen combined with the metabolic demand of convulsions led to a 40% increase in oxidation of the pyridine nucleotide, NADH, beyond the normoxic level (16). This amount approximately equaled the increased reduction of NAD found with anoxia, implying that brain NAD was at least halfreduced under usual resting, air-breathing conditions. There are, therefore, indications that the capacity of the cerebral mitochondria for oxygen utilization extends beyond those tensions at which the brain normally operates. To investigate the extreme range of cytochrome oxidation attainable in vivo, we have submitted cats to hyperbaric conditions and monitored the redox state of cytochrome au, in the cerebral cortex with variations in inspired oxygen and carbon dioxide partial pressures. Our work will show that cytochrome aat3, typically almost 100% oxidized in oxygenated mitochondrial suspensions in vitro, is found to be highly reduced in the brain cortex in vivo during resting air-breathing conditions. METHODS

Cats of either sex and indeterminate age and breeding were prepared for exposure of the brain by anesthesia with ether, thiopental sodium, or ketamine. After installation of, a tracheal breathing tube, the brain stem was transected at the plane between the superior and inferior colliculi to produce the cerveau isole state. Transection was performed with a commercial electrolytic lesion device through burr holes on the interaural line. Some animals did not receive brain-stem transections, but were anesthetized with pentobarbital sodium, given to effect. Maintenance doses, approximately 10 mg/kg per h, were given to the anesthetized cats during

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874 the course of long experiments The brain was exposed bilaterally by cutting windows approximately 15 x 15 mm from the skull and removal of the dura. The femoral artery and vein were cannulated and a heating pad, controlled by a rectal thermistor probe, was applied to keep the animals at normal body temperature. Cats were paralyzed with intravenous gallemine triethiodide (Flaxedil) and ventilated at a rate and tidal volume which produced blood gas values near 90 Torr Pa,, and 20 Torr Pa,?(,, during air breathing. The optical components of the dual-beam spectrophotometer were installed in a large hyperbaric chamber. Penetrations through the chamber wall led to the spectrophotometer electronic chassis and recording equipment outside. Investigators and technicians accompanied the cat inside the chamber during pressurization, and arterial blood samples were drawn for PO,, Pco2, and pH determinations at pressure, using previously published methods (24), Respiratory gases were administered from premixed cylinders, via the respirator with valve attached to the intake hose, at pressures of 1, 2, 3, or 4 ATA. Approximately 4 h were allowed for di soap on of anesthesia in the cerveau isd~ pre rbatx.ti ons. ta acquisition began immediately aft,er s esrgery kentobarbit,al-anesthetized animalt5 S Instrumentatixn for detection of the cortical cyto2,a :g redraction-oxidation state has been pubIready (23. In brief, fiber optics bundles conght fkom t.he two spectrophotometer monochromators to the brain. Flashes were directed alternately at a frequency of 30 EIz. The sample m.onochromator was set at 605 m, corresponding to the absorption peak for reduce cytochrome a~, while the reference monochromator as fixed at, 590 nm, representing the point at which the spectral extinction difference between oxy- and deoxyhemoglobin equals that difference at 605 nm. Light from the fiber optics bundle was directled into the suprasylvian gyrus at an angle of about 45” from its dorsal surface and perpendicular to the A-P (sagittal) axis. The diffuse glow out of the cortex was collected by a microscope objective focused on a 3.2mm-wide optical field and received on a photomultiplier tube in the microscope body. Care was taken to exclude specular reflections from the observation site. The difference voltage between reflected sample wavelength minus reference was recorded on a pen writer negative-feedback circuit, wh output from the photomulti illumination half of the cycle, isplayed. These variations in high-voltage sup photomultiplier tlube served as indices of the of quenching of monitoring light by changes in vascular volume. Thus vasodilation and vasoconstriction, or relative blood volume, was followed concurrently with the cytochrome redox signal. An electrocorticogram was also recorded, using a silver button electrode on the pial surface just outside the observation area. Calibration of the 590 minus 605 signal was made athing under normal resting conditions. ction-oxidation point, oxidations are sig-

HEMPEL

ET

AL.

naled in a downward direction, representing more reflected sample light and less absorption of it. The opposite occurs during cytochrome reduction in which a positive pen deflection signals less reflected light relative to the reference (590 nm) wavelength. Redox changes are expressed as a percent change in the balance between 59O- and 605-nm reflected light. RESULTS

In Fig. 1, a sample trace is shown to illustrate both the manner in which data are taken and the effects of increasing inspired oxygen and carbon dioxide tensions on the reduction level of cortical cytochrome aa, and blood volume. A cat breathing the equivalent of air (10% 0, at 2 ATA) is given pure oxygen at 2 ATA, with the result that cytchrome au, reduction level shifts slightly toward oxidation. On reaching a steady state with oxygen, a. mixture of 2.5% C02, balance air, at 2 ATA is given, causing a further, larger oxidation in spite of the lower inspired oxygen tension in the latter mixture. There is no visible vascular reaction to breathing pure oxygen in this animal, but the admixture of CO, stimulates a large vasodilatory response and the blood volume trace illustrates the increased quenching of the reference beam by an expanding vascular volume. An increased blood volume is signaled by a positive pen deflection. Titration of the cytochrome with oxygen, a procedure whereby each time a redox plateau is established a subsequent increment in arterial oxygen tension leads to a step change in cytochrome redox state, is illustrated in Fig. 2. The addition of a constant fraction of CO, to oxygen-nitrogen mixtur s the effect of resetting the redox level toward er oxidation and the titrated oxidations are new superimposed on the new steady redox state reached with the elevated arterial CO, partial pressure. The blood volume trace mirrors 605-590

nm

-\--ACy tot hrome

OXIDATION

lO%O; 90%4

07

.-

--

h 25 % CO;? , ,975 % Air

2 ata

Blood

Volume

Increase

1

FIG. 1. Reflection changes signaling cortical cytochrome a+ oxidation (top) and cerebral blood volume increase (bottom) in responses to gases inspired at 2 ATA. Arterial blood is drawn where indicated by asterisks.


CYTOCIIROME

auS OXIDATION

WITII

HYPERBARIC

generally common to 9 of 13 animals on which techni. tally acceptable studies were completed. As the Pa,,,‘, increased above ase line in two steps by raising the inspired CO:!, redox state was likewise displaced toward oxidation. For each of the essentiallv isocapnie blood conditions, the titration effect by oxygen could be shown at each arterial CO, tension, up to arterial PO, values of about 900 Torr. Beyond the 900 Torr Pa,,,, increasing oxygen to more than 2,400 Torr did not produce further cytoehrome oxidation. Contrasting data representative of four animals are shown in Fig. 3B. There was virtually no oxygen dependence of the reduction-oxidation level of cytochrome aa3 at each of the three arterial CO, tensions in this animal group, but there was a large oxidation each time the arterial CO, was raised. Cytochrome reduction with nitrogen ventilation indicated that an oxygen-labile response could occur nevertheless In those animals represented by Fig. 3& cerebral blood volume reactions to CO, did not differ qualitatively from the group in Fig. 3A e The foregoing observations make it clear that added t on the cytochrome signal in the CO, has a potent Further work was required to direction of oxid examine the maximum oxidation possible with hypercapnic hyperoxia. Figure 4 illustrates one study demonstrating the extreme oxidation to which cytochrome aa may be extended in vivo. An animal breathing the equivalent of 5% O,-95% N, at 4 ATA) was given producing a small but discernible oxygen at 4 oxidation and a slight decrease in blood volume. The inspired CO, fraction was then raised successively to 1.5%, XI%, 5.4%, and 1596, balance oxygen, at 4 ATA. Oxidations and blood volume increases accompanied each change. With the 5.4% inspired CO, fraction, equivalent to breathing about 22% CO, at sea level, the

the cytochrome oxidation trace, increasing pari passu with cytochrome oxidation. Data from the study chosen for Fig. 3A show features Cytochrome OXiDATlON

aa __ 8

----/

minutes

51000

VOLUME

increuse

8

titration of cytochrome CZCZ,~redox level with oxygen near 20 (base line for this animal) and 66-80 Torr (produced by inspiring 2.5% CO, at 3 ATA). An oxidation on increasing Pao, from 960 to 1,920 may be due to incomplete equilibration time. Bottom: blood volume changes. Asterisks denote blood sampling times, preceding each new gas mixture. A slow drift toward oxidation of the cytochrome and a gradual blood volume increase occurred in this experiment and occasionally in others. FIG.

875

OXYGENATION

2. Top:

at. Pact,, values

OXIDATION of Cyt. 8a3

42 u

16

I

I8

17 0

4

* PaC02

v

585

59

60

+

17

I

”

I

2

3

4

5

6

7

8

9

IO

Ii

I2

13

14

I

I5

1

16

.

17

.

18

.

19

.

20

I

1

.

.

21

22

23

24

FIG. 3. Titration of cytochrome CZCL~ with oxygen Each curve joins points representing different isocapnic blood conditions: a) preparation representative of the group reactive to increased Pao,; and b> preparation showing little reaction to increasing Pa,:!. Redox states are plotted relative to anoxic, reduced condition.

1

25

PeQ~ x 100 mmHg Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1977 American Physiological Society. All rights reserved.

876

HEMPEL CYTW-ROME

aa

OXIDATION

ET

AL.

#

5.4

%co2

4 ata

FIG. 4. Upper trace: oxidation of aa, with increasing percent CO, in oxygen at 4 ATA. MiddZe: blood volume reaction. Bottom: electrocorticogram. Animal is breathing 5% O,-95% N, at 4 ATA when the traces begin.

t5% co* 85%0 19

minutes

I

02 mV

11

II ’

ECOG

most extreme oxidation plateau was reached, because inspiration with 15% CO, introduced a shift toward reduction and a decrease in blood volume. On resumption of oxygen breathing, the redox trace and blood volume trace returned to their previous levels. Arterial blood gases were not analyzed for the series represented by Fig. 4. The electrocorticogram was markedly depressed when 5.4% CO, was breathed. Complete electrical silence ensued when 15% CO, was administered, but this isoelectric condition is not easily visualized when displayed on the slow time scale of Fig. 4. Figure 5 pools data from animals, three of which were ventilated at 4 ATA with 5% O,-95% Nz, pure 02, and 5.4% CO,-94.6% OZ, while the remaining four were exposed to all increasing CO, fractions up to 15%. The four data points in Fig. 5 at each inspired CO, fraction represent the latter group. The zero oxidation level for these cats was established with nitrogen ventilation, and the apparent 100% oxidation state was reached with 5.4% CO,-94.6% 0, at 4 ATA. On this scale, the average percent oxidation level for cytochrome aa found while breathing the equivalent of air (5% O,-95% N, at 4 ATA) was 14.5% (12.0-18.8%). Pure oxygen at 4 ATA produced an additional 2.3% average oxidation (range O-6.4%) beyond the 14.5% “air-breathing” level, and subsequent increases in CO, each produced greater oxidations until the apparent 100% oxidized state was reached with 5.4% CO,-94.6% 0, at 4 ATA. Thus the entire span of cytochrome redox states from 100% reduction to probable 100% oxidation of the cytochrome pool has been established with anoxic and with hyperoxic, hypercapnic atmospheres. Accordingly, about 85.5% of the oxidizable cytochrome seems to exist in a reduced state in the cerebral cortex of these cat preparations under resting air-breathing conditions. While more animals were used as cerveau isole preparations and fewer animals were anesthetized with pentobarbital in our samples, the responses of the latter group to increased gas tensions did not differ quantitatively from the former group. Data from both sets were pooled in the determination of cytochrome oxidation ranges.

01

2

3

4

5

6

7

8

9

10

11

12

13

14

15

INSPIRED CO2 (%) FIG. 5. Percent oxidation of cortical cytochrome of inspired CO, fraction in oxygen at 4 ATA. curves bracket data points from 4 animals.

aa3 as a function Solid and dashed

It was necessary to verify the contribution by cytochrome aa, to these 590-605 nm signals, since other oxygen- or CO,-dependent optical reactions of the tissue may have influenced our measurements. To do this, reflection (absorption) spectra were generated by means of a scanning dual-wavelength technique whereby the reference wavelength was held at 590 nm and the sample monochromator was driven through the wavelengths from 590 to 630 nm while the animals were breathing oxygen at 4 ATA, 2.1% CO,-97.9% 0, at 4 ATA, and 5.4% CO,-94.6% 0, at 4 ATA. On subtraction of the base-line oxygen spectrum from the 2.1% CO, spectrum, and subtraction of the oxygen spectrum from the 5.4% CO, spectrum, the difference reflection spectra shown in Fig. 6, A and B, respectively, are derived. The absorption of the cerebral cortex in this spectral region, deduced from its increased reflection of measuring light as cytochrome oxidation occurs, is broad but has a peak located near 602 nm. Secondary peaks fall at 593-594 nm, 611-613 nm, and 602-622 nm, the latter band being most prominent. These subsidiary peaks were identifiable in several animal trials. Figure 6C results from subtraction of the 2.1% CO, spectrum from the 5.4% CO, spectrum. Since hemoglobin should


CYTOCHROME

cza3 OXIDATION

WITH

1

590

,

HYPERBARIC

1

596

I

1

602

1

I

608

877

OXYGENATION

1

1

614

1

I

I

620

1

1

626

590

1

1

596

1

I

602

1

I

608

I

1

614

I

I

620

I

I

626

WAVELENGTH 6. Reflection spectra indicating cortical cytochrome aa absorption, derived by inspiring gases at 4 ATA. A: 2.1% CO,-97.9% 0, minus oxygen; B: 5.4% CO,-94.6% 0, minus oxygen; C: Curve B minus curue A; D: reduced minus oxidized spectrum of brain mitoFIG.

chondria illustrating X-X was generated to spectrum B.

cytochrome au3 region. Spectrum outlined by in a second animal under conditions identical

remain saturated in transit through the brain with this degree of hypercapnia and at this oxygen pressure, a possible hemoglobin artifact in the spectrum of Fig. 6C is avoided, and the 590-605 nm signal giving rise to the broad spectrum should be due to cytochrome aa reduction or oxidation and probably not to substances producing the subsidiary spectral peaks. Only a single peak around 593 nm drops out during the subtraction giving rise to Fig. 6C, suggesting that this peak may be due to hemoglobin saturation changes. Figure 6D illustrates the reduced minus oxidized absorption spectrum of cytochrome aa, in brain mitochondria. The fit of our reflection spectra to it is within limits, especially since oxidized cytochrome aa, absorbs near 598 nm (30) and would be expected to shift in vivo determinations toward the lower wavelengths. Since cytochrome a reportedly contributes from 85 to 100% to the 603-605 nm band, and cytochrome a:, contributes from about 0 to 15% (30, 31), cytochrome a redox changes should account for most of the reflectance data we recorded.

tion of 5% CO, as reported previously (23). Under hyperbaric conditions, however, the combination of carbon dioxide with oxygen, as illustrated by Figs. 4 and 5, can be used to establish a maximally oxidized state of cytochrome aa in the cat cerebral cortex, assuming that the saturation of the oxidation effect we observed would indicate that the oxidation limit had been reache”d. Between the 100% reduced and 100% oxidized states, there exist ordered steps in cytochrome acx3 redox levels with increasing oxygen and carbon dioxide. While the blood remains isocapnic, the titration effect of oxygen could be demonstrated (in about 70% of the animals) up to arterial tensions near 900 Torr, or breathing 0, at 1.5 ATA. Extension to higher oxygen pressures did not oxidize more cytochrome, apparently because cerebrovascular controls regulated the steadystate delivery of oxygen to the cortex. For this reason, pure oxygen could not be used to obtain maximum oxidation, even at 4 ATA. Little oxidation of cytochrome 2.3% beyond the air breathing redox level, occurred aa3, because of the vasoconstrictor effect of pure oxygen on the cerebral vessels. Lambertsen (14) has shown that DISCUSSION there is only a slight elevation in oxygen tension of Ordinarily, the only condition in intact brain tissue cerebral venous blood when oxygen is breathed at 3.5 for which the oxidation-reduction levels of the members ATA, compared to air-breathing levels. of the mitochondrial respiratory chain can be deterWhat criteria do we use to assume that complete mined on an absolute scale is the anoxic condition. cytochrome oxidation has been achieved with the hyperHere, the electron transport components are in a 100% capnic, hyperoxic conditions employed in this study? reduced state. To find the physiologically significant The first factor to be considered is that of oxygen redox level associated with normal brain function, and delivery. Carbon dioxide has a profound effect on oxybreathing air, the 100% oxidized cytochrome state must gen transport to the brain and on the cytochrome be determined as well. At ambient atmospheric pres- reduction-oxidation state. The vascular volume expands sure, oxygen cannot apparently be delivered in suffi- under the influence of COZ, and since blood volume in ciently high concentrations to the brain cortex to estab- the cat brain is directly proportional to blood flow with lish the completely oxidized state, even with the addi- a correlation coefficient of 0.96 (Zl), blood flow is known Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 29, 2018. Copyright © 1977 American Physiological Society. All rights reserved.

878 to be increased. At a given arterial oxygen tension, cytochrome aa, is oxidized with carbon dioxide increments until saturation is reached with 5.4% CO, in oxygen at 4 ATA, equivalent to breathing 22% CO, at 1 ATA (Fig. 4). The increases in blood volume which accompany increases in inspired CO, at 4 ATA is further evidence that augmented oxygen transport to the spectrophotometer observation site is taking place. Thus, the vasodilatation and enhanced blood flow effects that carbon dioxide in oxygen bring about are important for maximal oxidation of the cytochrome. The driving force toward oxidation by hyperbaric oxygen on NADH has been well documented by Chance and his colleagues (3, 4) in living and isolated systems. In yeast cells, titratable oxidations occurred beyond 1 ATA oxygen pressure, but a long plateau existed in NADH oxidation state between lOA and 1 ATA. Although observations on pyridine nucleotide in yeast cells may not be immediately applied to cytochrome redox states in living brain, it is clear that high-pressure oxygen can override the usual electron flow rates in the respiratory chain It may explain why 30% of the animals showed no cytochrome titrations with oxygen, but responded to carbon dioxide. In these animals, the respiratory chain functions along a redox plateau similar to that in yeast cells, remaining in a relatively steady state until the vasoconstrictive regulation is modified by carbon dioxide. Perhaps then, the 70% population responded more passively to oxygen increments, implying poorer autoregulation of oxygen delivery in the latter group. The second mechanism by which carbon dioxide inhalation may lead to total in vivo cytochrome oxidation involves alterations of energy metabolism in the brain, with modifications in respiratory chain substrate supply and oxidatiye phosphorylation. Inhibition of the glycolytic pathwa:; at the phosphofructokinase step has been demonstrat,ed i :a extreme hypercapnia in cats (Pa,Y,,, > 200 Torr) with Gwered concentrations of metabolites beyond the enzvin:.ati c lock (25). Normoxia with moderate hypercapnia = 61 Torr) reduced the rate of phosphorylation of TP in rat brain, and at the in high-energy phosphate same time led to a utilization (13). Th ct was an increased ATPI ADP ratio. Severe ercapnia had a similar effect in cat brain (25). In isolate itochondria, succinate oxidation declines to 8% ntrol rates when the incubation mixture is e rated with 15% CO, (12). Other respiratory chain rates declined about 60%. The pH was held at 7.3. amieson and Chance (9) have ria that azide-reduced shown in suspended mitoc cytochrome aa3l is oxidized to a greater degree by 5 ATA 0, when the pH is lowered from 7.4 to 6.7. Hence, while the addition of CO, may 1ea.d to cytochrome oxidation through a mechanism involving a lowered intracellular pH, it is also likely that hypercapnia deprives the respiratory chain of re ucing equivalents available via glycolysis and the citri.c acid cycle. Third, oxygen alone may slow the rate of substrate utilization. Hyperbaric oxygen inhibits the glycolytic pathway (8), and also affects the oxidation of interme-

HEMPEL

ET

AL.

diates in the citric acid cycle in the brain in vitro (29). There seems to be little hyperoxic inhibition of the respiratory chain-linked dehydrogenases in mitochondria, however (7), and Teng and Harris concluded that sulfhydryl group oxidation, often given as the biochemical lesion of hyperbaric oxygen, does not occur, and SH groups on cellular dehydrogenases are not affected (28). Cerebral ATP and creatine phosphate levels remain unchanged during hyperbaric oxygenation, even after seizures begin (19), even though a possible stimulation of ATPase activity may occur (6). We conclude that the conditions for maximal cytochrome aa oxidation in the cerebral cortex have been met when oxygen and carbon dioxide are inspired at the high partial pressures employed during this study (Figs. 4 and 5). The supply of reducing equivalents from substrates becomes limiting and oxygen is superabundant. In such a case, the mitochondrial respiratory state, according to the scheme of Chance and Williams (5) approaches the completely oxidized condition of state 2, or “starved” mitochondria. Relative to this maximally oxidized state of cytochrome aa,, about 85% of the cytochrome aa 3 pool is reduced in the cat cerebral cortex under resting air breathing conditions. Accordingly, the brain seems to function within a 15% threshold of its anoxic limit. It is intriguing that cytochrome aa is about 85% reduced under normal conditions. If oxygen values reported to produce cytochrome oxidation in vitro, lop7 M (20) and 0.06 Torr (27), apply to the in situ cortex then the mitochondria should be highly oxidized in the light of the oxygen tensions usually found in cerebral tissue. Local PO, measurements in the dog cerebral cortex by Lubbers et al. (15) show that a majority of brain sites have oxygen tensions between 11 and 30 Torr, with a range of l-90 Torr. Higher PO, values, ranging from 18.8 to 60.4 Torr, with a mean of 38.7 Torr have been found in the cat cortex (18). A still higher cerebral Po2, averaging 43 Torr, was reported by Mitnick and Jobsis, using the intracellular oxygen indicator pyrenebutyric acid (17). That the cytochrome pool is indeed reduced in the functioning brain suggests that the affinity of cytochrome aa for oxygen is considerably higher in isolated mitochondria than it is in the undisturbed in vivo state. A highly reduced cytochrome aa, in normoxia could possibly explain the initiation of synthesis or increase in activity of this enzyme with increases in the oxygen needs of the organ or tissue involved. Cytochrome aa activity has been correlated with the rate of oxygen consumption in organs and whole organisms (22, 26). Presumably, an inefficient or partially blocked electron transfer from the cytochrome to oxygen is compensated for by increases in the amount of aa, available, slowly adjusting to meet oxygen demand. Finally, we may compute the free energy advantage at the final step of 0, reduction gained by the existence of cytochrome aa in an 85% reduced state rather than the oxidized state observed in vitro. If AF = -2.303 RT log (oxidized species)/(reduced species), then at equilibrium in isolated mitochondria


CYTOCHROME

aa

OXIDATION

WITH

HYPERBARIC

OXYGENATION

in state 4 (approx 1% reduction of aaJ AF = -(2.303) (199 cal*mol-l*oC-l)

(310°C) log 99/l

and AF = -2836 cal, i.e., 2.836 kcal less is available during the reaction with 0, than if aa, were in the standard 50% reduced condition used for thermodynamic calculations. On reduction to the 85% level AF=

-1,418 log 15/85 = 1,103 cal

Therefore the free energy available at the aa level on donation of an electron to atomic oxygen is 3,939 cal

879

per electron or approximately 8 kcal per electron pair. Hence, the 85% reduction level of cytochrome aa in vivo provides an additional step of 8 kcal over that available in the in vitro situation. A free energy change of 8 kcal thermodynamically required for phosphorylation of ADP to ATP can thus be met at or near the final step of oxygen reduction. This may represent an immediate response to meet cellular demands for energy. The authors are grateful to Mr. Albert Boso and Mr. Richard Steele for their excellent technical assistance. This work was supported by Research Grant HL-07896 from the National Institutes of Health. Received

for publication

24 January

1977.

REFERENCES B. Rapid and sensitive spectrophotometry. III. A douapparatus. Rev. Sci. Instr. 22: 634-638, 1951. CHANCE, B. Spectrophotometry of intracellular respiratory pigments. Science 120: 767-775, 1954. CHANCE, B., D. JAMIESON, AND H. COLES. Energy-linked pyridine nucleotide reduction: Inhibitory effects of hyperbaric oxygen in vitro and in vivo. Nature 206: 257-263, 1965. CHANCE, B., D. JAMIESON, AND J. R. WILLIAMSON. Control of the oxidation-reduction state of reduced pyridine nucleotide in vivo and in vitro by hyperbaric oxygen. In: Proceedings of the Third International Conference on Hyperbaric Medicine, edited by I. W. Brown and B. G. Cox. Washington, D.C.: NAS-NRC Publ. 1404, 1966, p. 15-41. CHANCE, B., AND G. R. WILLIAMS. Respiratory enzymes in oxidative phosphorylation. III. The steady state. J. Biol. Chem. 217: 409-427, 1955. GOTTLIEB, S. F., AND S. K. HEMRICK. Oxygen-induced activation or inhibition of Na-K-Mg activated ATPase of beef brain (Abstracts). Federation Proc. 34: 716, 1975. HASHIMOTO, S., S. KOMATSU, E. LA BROSSE, AND R. COWLEY. Effect of hyperbaric oxygenation in vitro on mammalian respiratory chain-linked dehydrogenases. In: Proceedings of the Third International Conference on Hyperbaric Medicine, edited by I. W. Brown and B. G. Cox. Washington, D.C.: NAS-NRC Publ. 1404, 1966, p. 52-60. HAUGAARD, N. Cellular mechanisms of oxygen toxicity. Physiol. Rev. 48: 311-373, 1968. JAMIESON, D., AND B. CHANCE. Effect of high-pressure oxygen on the steady state of cytochromes in rat liver mitochondria. Biochem. J. 100: 254-262, 1966. J~BSIS, F. F. Basic processes in cellular respiration. In: Handbook of Physiology. Respiration. Washington, D.C.: Physiol. Sot., 1964, sect. 3, vol. I, chapt. 2, p. 63-124. J~BSIS, F. F., J. KEIZER, J. LAMANNA AND M. ROSENTHAL. Reflectance spectrophotometry and cytochrome aa in vivo. J.

Res. 76: 481-491, 1974. 17. MITNICK, M., AND F. F. J~BSIS. Pyrenbutyric acid as an optical oxygen probe in the intact cerebral cort,ex. J. AppZ. Physiol. 41: 593-596, 1976. 18. NAIR, P., W. J. WHALEN, AND D. BURKE, PO, of cat cerebral cortex: response to breathing N, and 10% 0,. Microvascular Res. 9: 158-165, 1975. 19. NOLAN, R.. J., AND M. D. FAIMAN. Brain energetics in oxygeninduced convulsions. J. Neurochem. 22: 645-650, 1974. 20. OSHINO, N., T. SUGANO, R. OSHINO, AND B. CHANCE. Mitochondrial function under hypoxic conditions: The steady states of cytochrome a + a3 and their relation to mitochondrial energy states. Biochim: Biophys. Acta 368: 298-310, 1974. 21. RISBERG, J., D. ANCRI, AND D. H. INGVAR. Correlation between cerebral blood volume and cerebral blood flow in the cat. Exptl. Brain Res. 8: 321-326, 1969. 22. ROBIN, E. D., AND L. M. SIMON. How to weigh an elephant: Cytochrome oxidase as a rate-governing step in mitochondrial oxygen consumption. Trans. Assoc. Am. Physicians 83: 288-300, 1970. 23. ROSENTHAL, M., J. C. LAMANNA, F. F. Jobsis, J. E. LEVASSEUR, H. A. KONTOS, AND J. L. PATTERSON. Effects of respiratory gases on cytochrome a in intact cerebral cortex: is there a critical PO,? Brain Res. 108: 143-154, 1976. 24. SALTZMAN, H. A., J. V. SALZANO, G. D. BLENKARN, AND J. .A. KYLSTRA. Effects of pressure on ventilation and gas exchange in man. J. Appl. PhysioZ. 30: 443-449, 1971. 25. SCHINDLER, U., E. GARTNER, AND E. BETZ. Energy-rich metabolites and EEG in hypoxia and in hypercapnia. In: Oxygen

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ble beam

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Alveolar partial pressures of carbon dioxide and oxygen measured by a helium washout technique.

Alveolar partial pressures of carbon dioxide and oxygen measured by a helium washout technique.

Effects of elevated oxygen and carbon dioxide partial pressures on respiratory function and cognitive performance.

Hyperbaric oxygen treatment for carbon tetrachloride poisoning.

Inhibition of pathogenic enteric bacteria by hyperbaric oxygen: enhanced antibacterial activity in the absence of carbon dioxide.

Solubility of Carbon Dioxide in Secondary Butyl Alcohol at High Pressures: Experimental and Modeling with CPA.

The Effect of Carbon Dioxide Insufflation Applied at Different Pressures and Periods on Thrombotic Factors.

Water-carbon dioxide solid phase equilibria at pressures above 4 GPa.

Neuromuscular function at hyperbaric pressures: pressure-anesthetic interactions.

Reflectance spectrophotometry of cytochrome aa3 in vivo.

Comparison of cerebral NADH and cytochrome aa3 redox shifts during anoxia or hemorrhagic hypotension.

Oxygen tensions measured in cat cerebral cortex under hyperbaric conditions.

Acute carbon monoxide intoxication and hyperbaric oxygen in pregnancy.

Antagonism of carbon monoxide-mediated brain lipid peroxidation by hyperbaric oxygen.

Carbon dioxide governs the oxygen affinity of crocodile blood.

Partial pressures of oxygen and carbon dioxide in bone and their correlation with bone-blood flow: effect of decreased arterial supply and venous congestion on intraosseous oxygen and carbon dioxide in an animal model.

Resonance Raman study of cytochrome aa3 from Sulfolobus acidocaldarius.

The influence of hydroxyl volatile organic compounds on the oxidation of aqueous sulfur dioxide by oxygen.

Angiogenesis contributes to the neuroprotection induced by hyperbaric oxygen preconditioning against focal cerebral ischemia in rats.

Cardiovascular System Response to Carbon Dioxide and Exercise in Oxygen-Enriched Environment at 3800 m.

Carbon dioxide elimination and oxygen consumption in mechanically ventilated children.

A single-unit carbon dioxide-oxygen sensing microelectrode system.

Oxidation of one-carbon compounds to formate and carbon dioxide in rat liver mitochondria.

Oxygen, Isoflurane and Pentobarbital Killing Methods in Adult Female Sprague-Dawley Rats.