Brain Research, 360 (1985) 203-213 Elsevier

BRE 11267

203

Non-invasive In Vivo Spectrophotometric Monitoring of Brain Cytochrome

aa 3 Revisited

KHALIL KARIMAN and D. SCOTT BURKHART

Department of Medicine, Duke University Medical Center, Durham, NC27710 (U.S.A.)

(Accepted April 2nd, 1985)

Key words: non-invasive monitoring - - cytochrome c oxidase - - spectrophotometry - - oxygen delivery - - rat brain

Cytochrome aa 3 (cyt aa3) is the main catalyst of cellular oxygen consumption. The properties of cyt aa 3 will define the tissue oxygen requirements and provide an insight into energy supply and demand. Currently dual-wavelength (605-590 or 605-620 nm) reflec- tance spectrophotometry is used to monitor cyt aa 3 redox state in vivo. We have experimentally demonstrated that the compensation for blood contamination in the surveyed tissue by these wavelength pairs is less than optimal. An alternative approach, similar to spec- trophotometric analysis of multicomponent systems used in vitro, is presented in the triple wavelength equation as follows:

Acyt aa 3 = 1.000 (AA605) -0.662 (AAs86.1) + 0.316 (AAs80) Based on a series of experiments performed in cuvette in vitro, isolated perfused rat head in situ, and living rat head in vivo, we have demonstrated that the cyt aa 3 equation fully compensates for changes in cerebral blood volume and saturation. This non-invasive method of in vivo monitoring of cyt aa 3 can provide the means to reliably and accurately determine tissue oxygen delivery under physi- ological conditions.

INTRODUCTION

Cyt aa3 is the oxygen-activating enzyme of cellular

respiration and is probably responsible for more than 90% of oxygen consumption by living organisms9,16.

The properties of cyt aa 3 will not only define the tis-

sue oxygen requirements 3 but also will provide an in- sight into energy supply and demand 2° since it is af-

fected by the energy state of the cell 5. The dual-wavelength spectrophotometric prin-

ciple introduced by Chance 4 has remained the basis

for monitoring the intramitochondrial cytochromes in perfused organs in situ. The in vivo monitoring of

cytochromes, however, has been hampered in the

past by the presence of blood in the surveyed tissue.

The main absorbing components at 605 nm are cyt

aa3 and hemoglobin. For in vivo monitoring of cyt aa 3, investigators have normally selected a reference wavelength which would reflect an equal absorption

change iin hemoglobin while unaffected by cyt aa 3

changes. Two approaches are generally used. In the

first, the 590 nm wavelength is used as reference (i.e.

605-590 nm) because the change in light absorption

related to the oxygenated state of hemoglobin at 590 nm equals 605 nm ('equibestic') 8,1°,12A3,19,23. In the

second, 620 nm is used as reference for cyt aa 3 (605-620 nm)2,ts.

Because of some uncertainties associated with sig-

nals derived from either 605-590 or 605-620 nm, we

have explored the means to improve the reliability

and accuracy of in vivo monitoring of brain cyt aa 3. In

this communication we have demonstrated that these

wavelength pairs used for monitoring this cyto-

chrome do not fully compensate for blood contami- nation in the surveyed tissue. In an effort to circum-

vent this problem, we present an alternative triple-

wavelength equation based on a series of experi- ments performed in cuvette, isolated peffused rat

head and finally live animals.

MATERIALS AND METHODS

Theoretical background The maximum absorption change for cyt aa 3 in the

Correspondence: K. Kariman, Box 3848, Duke University Medical Center, Durham, NC 27710, U.S.A.

0006-8993/85/$03.30 t~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)

204

wavelength region of interest occurs at 605 nm. At this wavelength the principlal interfering component

contributing to the overall absorption change is he-

moglobin. In the brain, marked changes in blood vol-

ume and saturation can occur and must be compen-

sated for if cyt aa 3 is to be reliably monitored. In de-

veloping an expression for cyt aa 3 we will first assume

a condition where only changes in absorbance are a

function of regulations in blood volume and satu-

ration. An isobestic point for hemoglobin (unaf-

fected by saturation but sensitive to volume) occurs

at 586.1 nm and the optical density change at 605 nm

from hemoglobin saturation is oppositely related to

that at 580 nm. The relationship of 586.1 nm to 605

and 580 nm is dependent upon the saturation and vol-

ume state. With this basic information one can devel-

op a linear expression for cyt aa 3 which incorporates

information on hemoglobin and equals zero net re-

sponse to absorption changes from blood volume or

saturation.

According to the B e e r - L a m b e r t - B o u g u e r Law for a system of n components, the absorbance Ai, at

wavelength 2 i can be expressed by

Ai = ~ aijbcj (1) j=l

where aij represents the absorptivity of component j

at wavelength 2 i, b is the path length and cj is the con-

centration of component j. We will assume for the

moment that the path length for one particular prep-

aration is a constant and since the primary interest is

to monitor changes in concentration one can express:

AA i = ~ aijAc j (2) j=l

where AA 1 and ACj indicate a change in absorption

and concentration, respectively. This can now be ap-

plied to describe the absorbance change related to re- duced (Hb) or oxygenated (HbO2) hemoglobin con-

centrations at the three wavelengths used in our sys-

tem:

AA605 = a605 (ACHb) q- a605(ACHbo2) (3)

AA586. ~ = a586.1 (ACHB) + a586.1(AeHbO2) (4)

AAsso = a580 (ACI-Ib) + ass0(ACH~o2) (5)

To solve these equations, one needs to know the

absorptivity constant (a) of each component at the 3

wavelengths. Because of scattering differences be-

tween in vitro and in situ conditions, the absorptivity

constants of Van Assendelft 24 cannot be used and

new ones must be determined. The values will be dif-

ferent for whole blood in a cuvette versus the in situ

condition. Since the determination of the final ex-

pression was based on these in situ experiments we

will focus only on the development of cyt aa 3 equa-

tion in rat brain. Representatives of cuvette experi-

ments, however, will be discussed in the Results. The

isobestic point 586.1 nm represents the wavelength at

which the absorptivity constants of oxidized and re-

duced hemoglobin are equal. In a series of experi-

ments (see below), the relative changes at 605,586.1 and 580 nm wavelengths were determined after addi-

t ion of either fully reduced or oxygenated human

blood. The values related to 605 and 580 nm were

then divided by that of 586.1 to provide a reference

constant for each wavelength. The absorbance

change at each wavelength can now be written as:

AA605 = 0.295(ACHb ) + 0.163(ACubo2 ) (6)

AA586.1 = 1.000(ACHb ) + 1.000(ACHbo2 ) (7)

AAs80 = t.158(ACHb ) q'- 1.575(ACHbo2 ) (8)

Since all the data are normalized to 586.1 nm, the co-

efficients at this wavelength equal one. An approach was used similar to the in vitro spec-

trophotometric analysis of mult icomponent systems commonly employed in analytical chemistry 22. Thus

two matrix sets were established, one for 605 and 586 nm and another for 586 and 580 nm, and then solved

with the M A T R I X and INV programs for the Hew-

iett Packard HP-41CV calculator. For instance, the

concentration changes of Hb and H b O 2 at 605 nm in

relation to 586.1 nm can be characterized by:

ACHb = 7.570(AA~)5)- 1.235(AA5861) (9)

ACHbo2 =-7.570(AAs)5) + 2.235(AA586. 0 (10)

To aid in understanding the way in which the expres- sions are interrelated, we examined them under two conditions: fully reduced (concentration of Hb = 100% or 1) or fully oxygenated (concentration of

HbO 2 = 100% or 1). With this in mind, the absorb-

ance at each wavelength in equations 6, 7 and 8 can be determined and substituted into the expressions for Hb and HbO2. Thus, incorporating equation 6 and 7 into equation 9 we will have:

ACHb = 7.570(0.295 X 1)- 1.235(1.000X 1) (11)

=1

ACHbo2 = 0 (12)

Similarly, if only oxidized hemoglobin is present:

ACHb ---- 0 (13)

mfHbO2 = -7.570(0.163 X 1)+2.235(1.000X 1) (14)

=1

In equations 15-20 below, the 580 and 586.1 nm wavelengths are compared in a manner similar to that described in equations 9-14:

ACHb = 3.774(AAss6.1)-2.395(AAss0) (15)

ACHbo 2 = -2.774(AAss6.1) + 2.395(AAss0) (16)

If hemoglobin is totally reduced:

ACHb = 3.774(1.000xl)-2.395(1.158×1) (17)

=1

ACHbo2 = 0 (18)

and if hemoglobin is totally oxidized:

ACHb = 0 (19)

ACHbo2 =--2.774(1.000X1)+2.395(I.575X1) (20)

= 1

A pattern has emerged which can be used to derive a final expression for changes in the redox state of cyt aa 3. The values, and subsequent changes, of oxidized and reduced hemoglobin at the 605 and 586.1 nm wavelength pairs are equivalent to those for the 586.1 and 580 pairs as described by the equations. By simple subtraction of the dual wavelength pairs (605 vs 586.1 and 586.1 vs 580) for either hemoglobin state, changes in blood volume and saturation will be cancelled out. For example, subtracting equation 15

205

from 9 gives: 7.570(AA605)- 1.235(AA586.1)

-3.774(AA586.x) + 2.395(AA580)

0 = 7.570(AA605)- 5.009(AAss6.1) + 2.395(AA580)

(21)

According to this equation any change in blood vol- ume or saturation will equal zero net response. Therefore, in the living rat brain, changes that are observed with this expression should be predomi- nantly associated with cyt aa 3. As shown in the Re- sults section this was experimentally proven. The cyt aa 3 redox state changes can now be represented

by:

Acyt aa 3 = 7.570 (AA605)- 5.009(AA586.1) +

2.395(AA580) (22)

To maintain the signal changes within the present in- strument operating limits, the coefficients are nor- malized to that for 605 nm thus providing:

Acyt aa3 = 1.000 (AA605) - 0.662(AA586.1) + 0.316(AA580)

(23)

Experimental set-up Three series of experiments were performed: in

vitro, in situ and in vivo. The in vitro experiments were executed in a cuvette made of tygon tubing con- taining a crystalloid solution to which either fully ox- ygenated or fully deoxygenated blood were added. This was a closed system and the solution was con- stantly circulated via a roller pump. A simple oxyge- nator fashioned from a 10 ml pipette allowed rapid changes in the gas concentration of the circulating so- lution and an on-line electrode permitted continuous recording of the oxygen concentration. The in situ experiments were performed in isolated perfused rat head using essentially the same set-up described for in vitro as shown in Fig. 1. Final experiments were done in living rat heads in vivo.

Reflectance spectrophotometric monitoring A quadruple-beam spectrophotometer with a sin-

gle light source for 4 monochromators was used. The details of this instrument have been reported pre- viously by J6bsis and co-workers 10. Quarter inch op-

206

light

to signal processor and recorder

PM

to recorder

P2

to recorder

2 o

o ~ ° ; analyzer

Mixed

0

P1

! ~ i~t Flow control

Fig. 1. Schematic diagram of the perfusion system for the isolated perfused rat head preparation. Perfusion fluid (F) is suctioned by the first roller pump (P]) and enters through the side end of a pipette used as an oxygenator (O). The desired gas mixture enters through the end of the pipette and mixes with the fluid. The excess gas escapes from an exhaust port at the opposite end. A second roller pump (P2) is used for perfusion of the rat head. OE, 02 electrode; PT, pressure transducer; PM, photomultiplier. The arrows indicate the di- rection of fluid flow.

tical coupling rods (light guides) were used for both

light entry and pick-up from the skull. The first was

located below the left t empora l r idge and si tuation

flush against the skull surface, while the pick-up rod

was located slightly to the left of the sagittal suture. A

small amount of optical coupling gel was placed on

the end of the pick-up rod and then posi t ioned

against the skull surface and sealed with a small O

ring. This part icular posi t ioning ensures efficient in-

teraction of the light with the tissue, thus providing a

higher concentrat ion of cytochromes available for

monitoring.

For continuous moni tor ing of the cyt aa 3 redox

changes ei ther the differential spec t rophotometr ic

mode or the 3 wavelength reflectance mode was

used 4J0. The half intensity bandwidth of 1.5 nm at 2

= 586.1, 2 nm at 2 = 605 nm and 2 nm at 2 = 580 nm

was chosen.

Surgical preparation For in situ exper iments the isolated perfused rat

head model of And jus et al. 1 was used. Male Wistar

rats (250-400 g) were anesthet ized with sodium pen-

tobarbi ta l (Nembuta l 50 mg/kg i .p.) . A t racheostomy

was per formed followed by a venous femoral cannu-

lation for subsequent administrat ion of Nembuta l

and sodium heparln. The animal was placed in a

prone posit ion and hypervent i la ted with 30%

02:70% N2. A midline scalp incision was en la rged by

blunt dissection, skin margins were re t racted and the

underlying fascia and per ios teum were dissected

free. The skull was further exposed by detaching the

temporalis muscles from the temporal crest on both

sides of the skull. The remaining underlying fascia and muscle tissue was scraped free from the skull sur- face. Bleeding was controlled with Ethicon bone wax and cauterization. Physiological saline was used for irrigation and the exposed muscle tissue on each side of the skull surface was packed with cotton filled

sponges. The common carotid arteries were then identified and dissected free from the carotid sheath in each side. After ligation of the distal end of the

vessel in each site, a small incision perpendicular to the vessel was made, a polyethylene catheter (PE 50) inserted and proximal and distal ties secured. The

right common carotid artery was cannulated in the

same manner. The time required for cannulation of each carotid artery, during which circulation was in- terrupted, was generally less than 2 min. Just prior to the actual cannulation, 150-200 I.U. of sodium hep-

arin was administered intravenously; 2.5 mg of sodi- um pentobarbital was frequently administered at this

point. Upon insertion of each catheter within the ves- sel, a slow hypothermic infusion (23 + 1 °C) was ini- tiated. With the head being continuously perfused, the animal was then decapitated and the head secure- ly mounted in a stereotaxic holder to provide a steady preparation for optical monitoring. The muscle tis- sues surrounding the skull surface were finally re- packed with cotton filled sponges and the optical probes positioned.

Perfusion solutions The head was perfused with perfluorotributyla-

mine (FC-43) emulsions equilibrated with 95%

Oz:5% CO 2 for approximately 2.5 h prior to use. This provided a pCO 2 of approximately 36 mm Hg and a pH of 7.40 + 0.05 (mean + S.D.). The FC-43 solu- tion was maintained at hypothermic conditions (23.5 + 1 °C) during the equilibration period as well as during organ perfusion. When it was preferred to maintain the preparation in a reduced state, a crystal- loid solution was used as perfusate. This was a Kreb's

Henseleit solution to which MgCI 2 (0.043 g%) and hydroxyethyl starch (HES, 4 g%) were added. Both FC-43 and the crystalloid solution were found to be quite satisfactory in maintaining the preparation vis- ually free of edema up to several hours after the ter- mination of the experiments. Adequate perfusion time was allowed to remove blood from the cerebral

207

circulation before execution of the experimental pro-

tocol.

In order to determine the signal interference re- lated to blood volume or saturation on the cyt aa 3 equation in isolated perfused head, it was necessary to maintain a constant redox state of cyt aa 3. For re- duced blood epxeriments, a reduced tissue state was

induced by perfusing with a fully deoxygenated crys- talloid solution. Increments of reduced blood were

then added to the perfusate. To examine the effects

of oxygenated blood on the cyt aa 3 expression, the head was perfused with fully oxygenated FC-43 in or-

der to keep cyt aa 3 in an oxidized state. Increments of oxygenated blood were then added to this perfusate. For live animal experiments, anesthetized rats un-

derwent femoral arterial and venous cannulations and a tracheostomy was then performed. The head

was prepared (muscle removed; skull intact) as de- scribed for the in situ experiments. Anesthesia was

maintained by periodic administration of small doses of Nembutal and body temperature was maintained at 36 °C. The effects of various insults on the cyt aa 3 redox state were tested as follows: transient interrup- tion of mechanical ventilation, left common carotid artery ligation, ventilation of the animal with various concentrations of oxygen and i.v. administration of

KC1. Also tested were the effects of slow exchange transfusion with oxygenated FC-43 (37 °C) on the cyt

aa 3 equation. In all of the above experiments the isobestic wave-

length for hemoglobin saturation (586.1 nm) was also monitored as a general indicator of relative blood

volume changes. It needs to be emphasized, howev- er, that the absorption changes at 586.1 nm may also

be related to cyt aa 3, c, cl, and b's and therefore may not always accurately estimate relative blood volume changes.

RESULTS

Fig. 2 depicts 3 representatives of a series of exper- iments performed in a cuvette-like system made of tygon tubing as described in the Materials and Meth- ods. As seen in Fig. 2A addition of Hb to the base crystalloid solution resulted in an increase in blood volume monitored at 586.1 nm. However, significant absorption changes were also observed at 605-620 nm and 605-590 nm which are supposed to represent

2(iS

@ ® ©

605-620 nm . . . . ~ . . . . . . . . . . . ~.-~ . . . . . . . . . . . . . . . Cytochrome oo 3 Equation .......................

605-590 nm ----,J// ,,x Absorbonce = 0.02

T /

,/ 586.1 nm 586.1 nm ,' I min

Increase OD t / J , Increase OD f ,,'

" A Absorbonce , :0.02 - -~ - --

I i

I mln

lO00] P02 I0001

P02 5001 ~ (mm Hg) 5001 ~ . . (mm Hg) / _ ~ - ~ - -~ _

° ~ t f o ~ t t l ..... f - - Hb 02 Hb02 HbO2 Remove Blood Hb

Fig. 2. Examples of a series of experiments performed in a cuvette-like system made of tygon tubing. The tubing is a closed system con- taining a crystalloid solution to which increments of either reduced (Hb) or oxygenated (HbO2) blood was added. Note that the addi- tions of reduced blood not only resulted in an increase in blood volume monitored at 586.1 nm, but also the 605-620 and 605-590 nm wavelength pair, supposed to represent cyt aa 3, show marked changes in absorption (A). Also, when oxygen was introduced to this so- lution, changes in 605-620 nm absorption occurred. In contrast, additions of either HbO 2 (B) or Hb (C) to the base solution had no ef- fect on the 3 wavelength cyt aa 3 equation.

Cytochrome 003 Equation

Oxidation f

586.1 nrn Increase OD t

I rain I I

j ~

A Absorbance

:0 .02 1

j -

I000]

P0z 500] (mm Hg)

0 J . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

400 Perfusion Pressure I

(turn Hg) 200]" " ,,=2 - ~: : ~1 ;1" ,i .... fill J i U-!J~ - :- .... - ...... ' j~H!IiiIIII ,. ..... :~:T II._~ ~.' I

OJ I I f t f Hb Hb Hb N02S204 Hb

Fig. 3. Effects of addition of Hb on the cyt a% equation in a fully reduced isolated perfused rat head preparation. Cyt aa 3 was mon- itored using reflectance spectrophotometry. The head was free of blood and first perfused with a crystalloid solution containing sodi- um dithionite (note the perfusate pO 2 remains zero throughout the experiment). Increments of reduced blood were then added to the perfusate. Despite marked increases in the relative blood volume signal the cyt aa 3 equation signal remains constank

cyt aa 3. When oxygen was in t roduced later , 605-590

nm remained unchanged while a significant absorp-

t ion change was observed at 605-620 nm. In con-

trast, addi t ion of ei ther H b O 2 (Fig. 2B) or Hb (Fig.

2C) to the base solution had essentially no effect on

the 3 wavelength cyt aa 3 equation.

Figs. 3 and 4 are representa t ions of a series of ex-

per iments per formed in isolated perfused rat head.

In Fig. 3 the head was being perfused with a deoxyge-

nated crystalloid solution in order to maintain the cyt

aa 3 in a reduced state. Addi t ions of Hb increased the

586.1 nm signal but had no effect on that o fcy t aa 3. In

Fig. 4 the head was being perfused with a fully oxy-

genated FC-43 solution to hold cyt aa 3 in an oxygen-

a ted state. Before addit ions of b lood the effects of

t ransient perfusion interrupt ion on cyt aa 3 as well as

on 586.1 nm were observed as shown in Fig. 4A. This

209

type of insult resul ted in reduct ion of the cytochrome

moni tored by ei ther method. In Fig. 4B the effects of

H b O 2 addit ions to this prepara t ion are tested. In-

creases in b lood volume (586.1 nm) after the addi t ion

of H b O 2 induced no change in the cyt aa 3 equat ion

while corresponding changes are observed at

605-590 nm.

Fig. 5 depicts the response to slow exchange trans-

fusion with oxygenated FC-43 solution in a live ani-

mal. In each trial, 10 ml of blood was continuously

exchanged with FC-43. The effects of such an ex-

change transfusion on 586.1 nm, 605-625 nm and

the cyt aa 3 equat ion were continuously moni tored . If

the equat ion is accurate such exchanges should have

no effect on cyt aa 3 as long as oxygen availabili ty to

the tissue is mainta ined by FC-43. As seen in Fig. 5

two trials of exchange transfusion had no effect on

® Cytochrome 003 Equation ~ ~ Oxidation t

6 0 5 - 5 9 0 nm Oxidation t

I min I

586.1 nm Increase OD t

I000]

P02 500] (ram Hg)

O" 40O

Perfusion Pressure ] (mm Hg) 2001

O"

®

z& Absorbance :o.o2 T

II II _ . 1 . . . .

I t Pump Off HbO2 Hb02

Fig. 4. A is an example of the response of cyt aa 3 to mitochondrial insults induced by transient perfusion interruption in an isolated per- fused rat head preparation using reflectance spectrophotometry. The head was perfused with an oxygenated FC-43 solution. Cyt aa 3 redox state was monitored using both the equation as well as the 605-590 nm wavelength pair. Perfusion cessation resulted in reduc- tion of cyt aa 3 monitored by either the dual or triple wavelength expression. B is the same experiment shown in A to which increments of HbO 2 are added. Note that the cyt aa 3 equation signal remains stable despite significant changes observed in the 586.1 nm signal. Also note marked changes in 605-590 nm signal when HbO 2 is added to the perfusate.

210

C Ylochrome oa 3 Equation

Oxidation I

605-625nm Oxidotionl

,~Absorbance ~ : 0.05 - - T

586.1 nm increase OD I

Arterial Blood Pressure (mm Hg)

~ Absorbance ,t =0.1 7--

I rain I I

OJ I Begin

t t End Begin

Exchange Transfusion

t End

Fig. 5. An example of the results of stow exchange transfusion with oxygenated FC-43 solution on cyt aa 3 monitored by the equation and 605-625 nm, using reflectance spectrophotome- try on a live rat head (skull intact, muscle removed). In each tri- al 10 ml of blood was continuously exchanged with FC-43. Blood removed during these exchange transfusions is marked by large decreases in the 586.1 nm signal. During these trials the cyt aa 3 equation remains constant while 605-625 nm shows significant changes.

the cyt aa 3 equation and only decreased the 586.1 nm signal. In contrast, significant changes occurred at

605-625 nm underscoring the unreliability of this

wavelength pair in reflecting cyt aa 3 redox changes.

It is noteworthy that the 620 and 625 nm wavelengths

can be used interchangeably as reference, since there

is essentially no difference in hemoglobin absorption

at these wavelengths in situ.

Fig. 6 represents the response to various brain in- sults in a live animal. In the first trial (Fig. 6A) mech-

anical ventilation was temporarily interrupted. Cyto-

chrome aa3 redox changes occur in 3 distinct phases. The first small transient oxidation is probably due to hypercapnic/hypoxic vasodilation. Marked rapid re-

duction then begins and continues until oxygen is made available by turning on the ventilator. Finally a

rapid oxidation of cyt aa3 occurs which exceeds the baseline level (oxidative overshoot) and gradually returns to baseline over approximately 2 min. Note that the kinetics of oxidation are 3 times faster than

those of reduction. In regard to 586.1 nm, at first there is a rise in this signal indicating an increase in relative blood volume during ventilation interrup-

tion. When ventilation is reinstituted a gradual fall

slightly below the baseline level is observed. As seen

in this figure there is a shoulder during the first phase

in the rise of 586.1 nm which most likely reflects the

contamination of cyt aa 3 redox changes. Further-

more, the pattern and kinetics of cyt aa 3 and 586.1

nm show clear independence and signal separation.

Three other types of insults were also induced in

the experiment shown in Fig. 6. Fig. 6B shows the ef- fect of transient ligation and release of the left com-

mon carotid artery in the cyt aa 3 and 586.1 nm sig-

nals. During the entire period of ligation there was a

transient reduction of cyt aa 3 which soon changed to-

ward a transient oxidative overshoot before return- ing toward baseline. When the clamp on the carotid

artery was released, a second transient oxidative

overshoot occurred. In contrast to ventilation inter-

ruption (Fig. 6A), reduction of cyt aa 3 began immedi-

ately after carotid clamping with no transient oxida-

tion observed. In regard to the 586.1 nm signal, fol-

lowing carotid artery clamping there was first a slight

brief fall before the forthcoming compensatory vaso-

dilation (or recruitment of the anastomotic blood vessels) which resulted in an increase in relative

blood volume. This signal gradually returned to base-

line except for a brief interruption due to the release of the clamp. Fig. 6C and D depict cyt aa 3 and 586.1

nm signal responses to ventilating the rat with 95%

02:5% CO 2 and i.v. administration of KC1, respec- tively.

Fig. 7 is an example of a series of experiments per-

formed in order to examine the effects of various con-

centrations of inspired oxygen on the cyt aa 3 redox

state. As seen in this figure, small changes in the oxy-

gen concentration affected the redox state of cyt aa 3.

Note the signal separation between 586.1 nm and cyt

aa3 when the animal is ventilated with 30% 0 2, 100%

02, and 95% 02:5% CO2. In response to each of these sequential manipulations there is an oxidative

response of the cyt aa3 signal. In contrast, a decrease in the 586.1 nm signal occurs in transition from 30 to

100% 02 while in response to 95% 02:5% CO 2 there is an increase in this signal.

DISCUSSION

In this paper we have demonstrated the means to reliably and accurately monitor brain cyt aa 3 in viva.

211

Cytochrome 003

Equation Oxidation t

586.1 nm Increase OD f

Arterial Blood Pressure (ram Hg)

4001 2°i]

Ventilator

® ® © @

,~ Absorbonce .. I Z~ Absorbonce - - ' ' = 0 . 0 8 =0.1

t

~ _ - i - L t L - I ~

t t t t t t Off On On Release 95=/0 02 Add KCI

Left Carotid Clamp 5% C02

Fig. 6. Response of the cyt aa 3 equation and relative tissue blood volume (586.1 nm) to various brain insults in an anesthetized rat head (skull intact, muscle reoved), using reflectance spectrophotometry. In A the effects of interruption of mechanical ventilation is shown. B is representative of left common carotid artery ligation. In C and D the effects of ventilating the animal with 95% 02:5% CO 2 and the administration of KCI are demonstrated. Compare the pattern and kinetics of cyt aa 3 redox changes and relative tissue blood vol- ume 586.1 nm signal. See Results for description of this figure.

The method is non-invasive and produces a strong

signal related to this cytochrome. It needs to be em-

phasized that al though the basic principle used in this

equat ion can be appl ied to other organs, the absorp-

tive constants using reflectance spec t rophotomet ry

in vivo may not be valid for organs o ther than rat

head.

Despi te the lack of adequate compensat ion for

b lood volume and saturat ion, dual-wavelength spec-

t rophotomet ry has the advantage of compensat ing

for light scattering changes as well as for instabili t ies

re la ted to the photomul t ip l ier high voltage or lamp.

It is fortuitous that the factors incorpora ted into our

t r iple-wavelength equat ion are such that this com-

pensating quality is not significantly hampered . For

instance, in isolated perfused rat head exper iments

despite significant changes in blood volume and satu-

ration, the cyt aa 3 equat ion remained unaffected. In

live animals, changes in blood volume and oxygen-

a ted state are basically responsible for variat ions in

light scattering. Similarly, instability in the photo-

mult ipl ier high voltage 10 t imes greater than that

Cytochrome o03 ~ _ ~Absorbance - L ~ Equation ~ - : 0.08 --F "

Oxidation I ~ ~

I min /', Absorbonce : 0.05 - -

586.t nm ~ ~ / [ Increase OD I ~ -

r t r t i I ~ T Room Air 13.8% 02 8.8% 02 13.8% 02 Room Air 30% 02 100% 02 95% 02 30% 02

5% C02

Fig. 7. An example of the effects of the administration of various concentrations of oxygen on the cyt aa 3 redox changes using the equa- tion and the 586.1 nm signal. Note the signal separation of 586.1 nm and cyt aa 3 in transition from ventilation with 30% 02 to 100% 02, and then to 95% 02:5% CO_~.

212

routinely observed in our instrument was necessary to cause a significant deflection in the cyt aa3 signal.

None of the 3 wavelengths selected for the equa- tion are within the range of the major bands of cyt c,

c i and b's. However, there is a small contribution of

the absorption spectra of these cytochromes at 586. I

and 580 nm. In order to estimate the amount of over-

lap, we used the existing literature for isolated mito-

chondria in vitro. Redox changes in cyt c I will under-

estimate the redox state determined by the cyt aa 3

equation by 1.9% Is while those of cyt c and b's will

overestimate by 1.3 and 4.2%, respectively 0,1i. Since

under biological conditions reduction or oxidation of

cyt aa 3 is usually associated with corresponding changes in the other cytochromes, the net effect can

maximally be a 3.5% overestimation of the cyt aa 3 re-

dox state monitored by the equation. In reality, the

overestimated value may even be much less since

changes in the cyt b T redox state require forcing func- tions to an extreme degree.

The approach to monitoring brain cytochrome oxi- dase in living subjects in the presence of blood de-

scribed here is similar to the in vitro spectrophotome-

tric analysis of mult icomponent systems commonly

used in analytical chemistry. In this approach it is im-

portant not to deviate from the B e e r - L a m b e r t - B o u -

guer Law. All the numbers incorporated into the

equation are measured values obtained first from cu-

vette experiments in vitro and then isolated perfused

heads in situ. The only assumption made is that the

light path length for one particular preparation re-

mains virtually constant. If the light entry and pick-

up rods are firmly stabilized over the skull there is no reason to expect otherwise. For smaller intracranial

changes, such as possibly induced by cerebral swell-

ing or shrinking, we assume that the effects are shared equally by all 3 wavelengths.

The pattern and the kinetics of cyt aa 3 redox re- sponses varied according to the nature of the injury.

When the respirator was transiently stopped (Fig. 6A), the compensatory hypoxic/hypercarbic vasodi-

lation first exceeded the rate of fall in oxygen avail- ability resulting in a brief oxidation before reduction

of cyt aa 3. When the respirator was turned on rapid oxidation of cyt aa 3 occurred which exceeded the baseline value (oxidative overshoot) for a short peri- od of time. The kinetics of oxidation after transient anoxia were approximately 3 times faster than those

of reduction. The enhanced tissue oxygen delivery

due to the autoregulation of cerebral blood flow or so-called 'luxury perfusion" probably plays an impor-

tant role in this oxidative overshoot. Howe~cr, it has

also been demonstrated that in response to hypoxic

hypoxia cyt aa 3 activity alters and continues to utilize m o r e oxygen 12.14-17.-'1 .

In regard to ligation of the left common carotid ar-

tery this insult induced a small and transient reduc-

tion of cyt aa~ followed by a brief oxidative overshoot despite continuation of the arterial clamping. When

the arterial clamp was released, further oxidation of

cyt aa 3 was observed (Fig. 6B). This observation is consistent with the presence of a large number of col-

laterals that exist in rat brain circulation-. In re-

sponse to the carotid arterial ligation the 586.1 nm

wavelength first showed a small brief decrease which

then was followed by a large increase due to cerebral

vasodilation (compare cyt aa 3 signal changes with that of 586.1 nm). The initial fall in cerebral perfu-

sion indicated by a fall in the 586.1 nm signal might

have caused a release of a substrate (S) responsible

for later vasodilation. As expected, in response to the

administration of 95% O~:5% CO:, the cyt aa3 and

586.1 nm signals markedly increased. On the other

hand, the cardiac arrest associated with the adminis-

tration of KCI resulted in the reduction of both the

cyt aa 3 and 586.1 nm signals. It is noteworthy that the

586.1 nm signal, although largely reflecting the rela-

tive blood volume changes, contains some contami-

nation of cyt aa~ as well as other cytochromes.

As shown in Fig. 7 the degree of oxidation of cyt

aa 3 varies directly with oxygen concentration around the normal value. For instance, increasing the oxy-

gen concentration from the room air value to 30 and

100% resulted in corresponding oxidation of cyt aa~.

A decrease in oxygen concentration from the room air value to around 14% resulted in reduction of this cytochrome. These observations are in agreement

with findings made by Rosenthal et al.l'~ in cat cere-

bral cortex and suggest the presence of a diffusion barrier for oxygen in the brain.

ACKNOWLEDGEMENTS

We thank Dr. Frans F. J6bsis for useful discussion.

Supported by a National Institute of Health Grant (GM-29531).

REFERENCES

1 Andjus, R.K., Kunikazu and Sloviter, H., An isolated per- fused rat brain preparation, its spontaneous and stimulated activity, J. Appl. Physiol., 22 (1967) 1033-1039.

2 Austin, G., Schuler, W., Hangen, G., Willey, J., La Man- na, J. and J6bsis, F.F., Brain metabolism in the cat during brief transient ischemia. In G. Austin (Ed.), Proceedings of The Thirteenth Annual Meeting of the Federation of Western Societies of Neurological Science. Contemporary Aspects of Cerebrovascular Disease, Professional Information Li- brary, Dallas, TX, 1976, pp. 46-58.

3 Chance, B., Cellular oxygen requirements, Fed. Proc. Fed. Am. Soc. Exp. Biol., 16 (1957) 671-680.

4 Chance, B., Rapid and sensitive spectrophotometry. III. A double-beam apparatus, Rev. Sci . Instr., 22 (1951) 634-638.

5 Chance, B. and Williams, G.R., The respiratory chain and oxidative phosphorylation, Adv. Enzymol., 17 (1965) 65-134.

6 Erecinska, M., Oshino, R., Oshino, N. and Chance, B., The b cytochromes in succinate-cytochrome c reductase from pigeon breast mitochondria, Arch. Biochem. Bio- phys., 157 (1973) 431-445.

7 Greene, E.C., The Anatomy of The Rat, Hofner, New York, 1935, 252 pp.

8 Hempel, F.G., J6bsis, F.F., La Manna, J.C., Rosenthal, M.R. and Saltzman, H.A., Oxidation of cerebral cyto- chrome a,a 3 by oxygen plus carbon dioxide at hyperbaric pressures, J. Appl. Physiol., 43 (1977) 872-877.

9 JObsis, F.F., Basic processes in cellular resiration. In W.O. Fenn and H. Rahn (Eds.), Handbook of Physiology, Sec- tion 3, Respiration, Vol. 1, Washington, American Physio- logical Society, 1964, pp. 63-124.

10 J6bsis, F.F., Keizer, J.H., La Manna, J.C. and Rosenthal, M., In vivo reflectance spectrophotometry of cytochrome a,a 3 in the intact cerebral cortex of the cat, J. Appl. Physi- ol., 43 (1977) 858-872.

11 Kamen, M.D., Dus, K.M., Flatmark, T. and de Klerk, H., Cytochrome c. In T.E. King and M. Klingenberg (Eds.), Electron and Coupled Energy Transfer in Biological Sys- tems, Vol. 1A, Dekker, New York, 1970, pp. 243-324.

12 Kariman, K., Hempel, F.G. and J6bsis, F.F., In vivo com-

213

parison of cytochrome a,a 3 redox state and tissue P O 2 in transient anoxia, J. Appl. Physiol., 55 (1983) 1057-1063.

13 Kariman, K., Hempel, F.G., J6bsis, F.F., Burns, S.R. and Saltzman, H.A., In vivo comparison of cerebral tissue pO 2 and cytochrome a,a 3 reduction-oxidation state in cats dur- ing hemorrahagic shock, J. Clin. Invest., 68 (1981) 21-27.

14 Kariman, K., J6bsis, F.F. and Saltzmanm H.A., Cyto- chrome a,a 3 reduction: early indicator of metabolic recov- ery from hemorrhagic shock in rats, J. Clin. Invest., 72 (1983) 180-191.

15 Konig, R.W., Schilder, L.T.M., Tervoort, M.J. and Van Gelder, B.F., The isolation and purification of cytochrome c I from bovine heart, Biochim. Biophys. Acta, 621 (1980) 283-295.

16 Lemberg, M.R., Cytochrome oxidase, Physiol. Rev., 49 (1969) 48-121.

17 Mela, L., Mitochondrial function in cerebral ischemia and hypoxia: comparison of inhibitory and adaptive responses, Neurol. Res., 1 (1979) 51-63.

18 Piantadosi, C.P., Sylvia, A.L. and J6bsis, F.F., Cyanide-in- duced cytochrome a,a 3 oxidation-reduction responses in rat brain in vivo, J. Clin. Invest., 72 (1983) 1224-1233.

19 Rosenthal, M., La Manna, J.C., JObsis, F.F., Levasseur, J.E., Kontos, H. and Patterson, J.L., Jr., Effects of respi- ratory gases on cytochrome a in intact cerebral cortex: is there a critical pO2? Brain Research, 108 (1976) 143-154.

20 Sies, H. and Brauser, B., Analysis of cellular electron transport systems in liver and other organs by absorbance and fluorescence techniques, Meth. Biochem. Anal, 26 (1980) 285 - 325.

21 Smialek, M. and Hamberger, A., The effect of moderate hypoxia and ischemia on cytochrome oxidase activity and protein synthesis in brain mitochondria, Brain Research, 17 (1970) 369-371.

22 Sternberg, J.C., Stillo, H.C. and Schwendeman, R.S., Spectrophotometric analysis of multicomponent systems using the least squares method in matrix form, Anal. Chem., 32 (1960) 84-90.

23 Sylvia, A.L. and Rosenthal, M., The effect of age and lung pathology on cytochrome a,a 3 redox levels in rat cerebral cortex, Brain Research, 146 (1978) 109-122.

24 Van Assendelft, O.W., Spectrophotometry of Hemoglobin Derivatives, Thomas, Springfield, IL, 1977, 55 pp.