U.S. patent number 3,830,222 [Application Number 05/269,580] was granted by the patent office on 1974-08-20 for method and apparatus for observing rates of reaction of oxygen in living tissues.
Invention is credited to Britton Chance.
United States Patent |
3,830,222 |
Chance |
August 20, 1974 |
METHOD AND APPARATUS FOR OBSERVING RATES OF REACTION OF OXYGEN IN
LIVING TISSUES
Abstract
A system for providing flash photolysis activation of
CO-inhibited cytochrome oxidase in living tissue in the presence of
oxygen. In a typical procedure employing cardiac tissue this
initiates oxidation of reduced pyridine nucleotide (PN) and
flavoprotein (Fp), with a high rate of response. The fractional
extent of the photolysis response of PN and Fp indicates the
fraction of the total mitochondrial population containing
cytochrome a.sub.3 CO to which oxygen has diffused at the time of
the photolysis flash, thereby providing an indication of the
effectiveness of oxygen diffusion in the tissue without destruction
of the tissue. A double flash is used to evaluate the extent of
photolysis, one flash occurring a few seconds after perfusing the
tissue with oxygen, followed by another flash a few seconds later.
The readout is obtained on a storage oscilloscope, using a double
beam, time-shared photometer assembly with a compensating
photomultiplier. The flash lamps are triggered by a pulse from the
compensating photomultiplier, with a delay to fire the flash lamps
at an appropriate phase angle.
Inventors: |
Chance; Britton (Philadelphia,
PA) |
Family
ID: |
23027853 |
Appl.
No.: |
05/269,580 |
Filed: |
July 7, 1972 |
Current U.S.
Class: |
600/334;
356/39 |
Current CPC
Class: |
G01N
21/631 (20130101) |
Current International
Class: |
A61B
5/15 (20060101); G01N 21/63 (20060101); A61b
005/00 () |
Field of
Search: |
;128/2A,2R,2.1R,2.1Z
;424/9 ;356/39 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chance, B. et al. Arch. of Biochem. & Biophysics, Vol. 143, No.
2, April 1971 pp. 675-687. .
Chance, B. et al. IEEE Trans. On Bio-Med. Engineering, Vol. 2,
April 1970, pp. 118-121. .
Chance, B. et al. Review of Sci. Instruments, Vol. 42, No. 7, July
1971, pp. 951-957. .
Chance, B., Journ. Of Biological Chemistry, Vol. 202, No. 1, May
1953, pp. 407-416..
|
Primary Examiner: Howell; Kyle L.
Attorney, Agent or Firm: Gordon; Herman L.
Claims
What is claimed is:
1. Apparatus for determining the effectiveness of oxygen diffusion
in animal tissue comprising a photometer device, means to support a
sample of intact animal tissue in the field of observation of said
photometer device, a source of anoxygenating material including
oxidation-inhibiting material, means to perfuse the sample with
said anoxygenating material from said source, whereby to place the
sample in anoxic condition, a source of oxygenating material, means
to perfuse the anoxic sample with oxygenating material from said
second-named source, whereby oxidation of the anoxic sample will
then take place at a relatively inhibited rate, and means to excite
the sample with a pulse of photolyzing radiant energy shortly after
oxygenation of the anoxic sample commences, said pulse being of
sufficient strength to destroy the inhibiting effect of said
anoxygenating material, said photometer device including means to
continuously read out the degree of oxidation of the anoxic tissue
with time, whereby the destruction of said inhibiting effect will
provide a readout indicating the effectiveness of oxygen diffusion
in the tissue sample.
2. The apparatus of claim 1, and wherein the photometer device
comprises a fluorometer including means to excite the tissue sample
with radiant energy and means to measure the fluorescence of the
tissue caused by said last-named radiant energy.
3. The apparatus of claim 1, and wherein the photometer device
comprises a time-sharing system delivering time-spaced excitation
beams of different wavelength to the sample, generating
corresponding time-spaced optical responses of the tissue sample,
whereby responses to photolysis of a plurality of components in the
tissue sample may be sequentially measured.
4. The apparatus of claim 1, and wherein the photometer device
comprises a time-sharing cyclic fluorometer including means to
excite the tissue sample with time-spaced excitation beams of
different wavelength and means to measure corresponding time-spaced
fluorescence responses of the tissue sample, whereby the responses
of a plurality of components in the tissue sample may be
simultaneously measured.
5. The apparatus of claim 1, and means to excite the tissue sample
with a second pulse of photolyzing radiant energy subsequent to the
first pulse for evaluating the extent of photolysis.
6. The apparatus of claim 1, wherein the anoxygenating material
includes carbon monoxide.
7. The apparatus of claim 1, and wherein the photometer device
comprises a time-sharing system delivering time-spaced excitation
beams to the sample, generating corresponding time-spaced optical
responses of the tissue sample, said means to excite the sample
with the pulse of photolyzing radiant energy comprising a flash
lamp means adapted to be located adjacent the sample, and circuit
means to momentarily energize the flash lamp means in the interval
between a pair of said time-spaced excitation beams.
8. The apparatus of claim 7, and wherein the photometer device
includes means to generate a noise-compensating electrical pulse
coincidentally with each excitation beam, said circuit means
including means to energize said flash lamp means after a
predetermined time interval following a noise-compensating
electrical pulse.
9. The apparatus of claim 29, and a second photolysis flash lamp
means adapted to be located adjacent the sample, and wherein said
circuit means includes means to momentarily energize the second
flash lamp means after a second predetermined time interval
following the momentary energization of the first-named flash lamp
means, for evaluating the extent of photolysis produced by the
first-named flash lamp means.
10. A method of determining the effectiveness of oxygen diffusion
in intact animal tissue comprising the steps of perfusing a sample
of the intact tissue with an anoxygenating material which inhibits
oxidation of the tissue sample, then perfusing the sample with
oxygen to initiate oxygenation thereof, continuously measuring the
progress of oxidation with time, and then rapidly terminating the
inhibiting effect shortly after oxygenation has commenced by
illuminating the sample with a photolyzing flash of radiant energy
of sufficient intensity to destory the inhibiting effect to thereby
allow the sample to rapidly react with the oxygen, whereby to
obtain a readout in accordance with the extent to which the oxygen
has diffused in the tissue sample at the time of the photolyzing
flash.
11. The method of claim 10, and wherein the oxidation-inhibiting
material is carbon monoxide.
12. The method of claim 10, and illuminating the sample with a
second photolyzing radiant energy flash shortly after the first
flash, for evaluating the extent of photolysis.
13. The method of claim 12, and wherein the first photolyzing flash
is applied approximately 1.5 seconds after the start of the
perfusion of the sample with oxygen and the second photolyzing
flash is applied between 1 and 2 seconds after the first flash.
14. The method of claim 10, and wherein the photolyzing flash is
applied approximately 1.5 seconds after the start of the perfusion
of the sample with oxygen.
Description
This invention relates to methods and means to study mitochondrial
electron transport, and more particularly to systems for
non-destructively determining the effectiveness of oxygen diffusion
in tissue.
A main object of the invention is to provide a novel and improved
system for obtaining fast activation of mitochondrial electron
transport in intact tissue perfused with oxygen, whereby to
identify the nature and magnitude of the metabolic energy load of
the mitochondria.
A further object of the invention is to provide an improved system
for studying the nature and magnitude of the metabolic energy load
of mitochondria, employing flash photolysis activation of
CO-inhibited cytochrome oxidase in the presence of oxygen so as to
initiate the oxidation of reduced pyridine nucleotide (PN) and
flavoprotein (Fp), with a high rate of response, the system
including means to provide an indication of the fraction of the
total mitochondrial population containing cytochrome a.sub.3 CO to
which oxygen has diffused at the time of the photolysis flash.
A still further object of the invention is to provide an improved
system as above described wherein a double flash is employed to
evaluate the extent of photolysis, one flash occurring a short time
after perfusing the tissue under study with oxygen and the second
flash occurring shortly thereafter.
A still further object of the invention is to provide a novel and
improved apparatus for the flash photolysis of intact tissue
perfused with oxygen to obtain information as to the extent to
which oxygen has diffused to the mitochondrial population.
Further objects and advantages of the invention will become
apparent from the following description and claims, and from the
accompanying drawings, wherein:
FIG. 1 is a diagram showing a system in accordance with the present
invention for measuring the effectiveness of oxygen diffusion in a
perfused heart.
FIG. 2 is a block diagram of the double flash circuitry employed
with the system of FIG. 1.
FIG. 3 is a diagram showing typical wave forms present at various
points of the circuit shown in FIG. 2.
FIG. 4 comprises graphs showing transitions from anoxia to normoxia
as indicated by flavoprotein (Fp) and pyridine nuclotide (PN),
graph A representing the condition wherein carbon monoxide is
absent and graph B representing the condition wherein carbon
monoxide is present.
FIG. 5 comprises graphs respectively illustrating the reaction of
cytochrome a.sub.3 with carbon monoxide in the anaerobic heart
(graph A), and the effects of flash photolysis upon cytochrome
a.sub.3 of the heart mitochondria in the substantial absence of CO
(graph B) and in the presence of substantial CO (graph C).
FIG. 6 comprises graphs providing an evaluation of the extent of
photolysis by the double flash technique, graph A representing the
results obtained when the interval between flashes is about 1.3
second and graph B representing the results when the interval
between flashes is about 0.6 second.
The increasing fund of knowledge regarding the properties of
respiratory pigments of isolated mitochondria coupled with new
techniques for their study in cells and tissues affords new
approaches to the study of four fundamental biochemical and
physiological relationships: (a) the nature and function of the
electron transport system in the intact tissue: (b) the interaction
between energy sources and energy sinks in a functional tissue such
as cardiac muscle; (c) the effectiveness with which oxygen is
transported to the intracellular mitochondrial spaces from the
extracellular capillary space; (d) the nature of the specific
activator of mitochondrial function in situ, ADP + Pi and/or
Ca.sup.2 .sup.+.
Particularly important to the problem of cell function is the
identification of metabolic states in mitochondria controlled by
the substrate, oxygen and energy levels. A transition between such
states that is significant in muscular activity is the
resting-active transition in which the energy demand upon the
mitochondrial energy conservation vastly alters the steady state
and kinetic properties of the electron carriers. (See B. Chance and
G. R. Williams, "The Respiratory Chain and Oxidative
Phosphorylation" in "Advances in Enzymology," F. F. Nord. ed.,
Interscience Publishers, New York, 1965, vol. XVII: pp. 65-134). In
previous studies repetitive stimulation of the isolated sartorius
muscle activated the resting (state 4) to active (state 3)
transition and increased the oxidation of reduced PN. (See B.
Chance and F. F. Jobsis, "Changes in Fluorescence in a Frog
Sartorius Muscle Following a Twitch," "Nature," 184: 195-196, 1959;
B. Chance and A. M. Weber, "A Steady State of Cytochrome b During
the Rest and After Contraction in Frog Sartorius," J. Physiol.,
169:263-277, 1963;C. M. Connelly and B. Chance, "Kinetics of
Reduced Pyridine Nucleotides in Stimulated Frog Muscle and Nerve,"
Fed. Proc. 13:29, 1954). Such transitions are observed in the
steady state level of reduced PN by spectrophotometry (see C. M.
Connelly and B. Chance, above cited) and fluorometry (see B. Chance
and F. F. Jobsis, above cited) and of cytochrome b by
spectrophotometry (see B. Chance and A. M. Weber, "Steady State
Changes of the Cytochromes Following Isometric Twitches," Fed.
Proc., 16:146, 1958; "Early Kinetics of the Cytochrome b Response
to Muscular Contraction," Ann. N.Y. Acad. Sci., 81:505-509, 1959;
F. F. Jobsis, "Spectrophotometric Studies on Intact Muscle, I.
Components of the Respiratory Chain," J. Gen. Physiol., 46:905-928,
1963). More recent studies of isolated mitochondria (see B. Chance,
D. F. Wilson, P. L. Dutton and M. Erecinska, "Energy-coupling
Mechanisms in Mitochondria: Kinetics, Spectroscopic and
Thermodynamic Properties of an Energy-transducing Form of
Cytochrome b," Proc. Natl. Acad. Sci. 66:1175- 1182, 1970) show
that the energy state of the mitochondria can be deduced from the
kinetics of respiratory enzymes in the anaerobic-aerobic transition
of cytochrome b, c and a.sub.3 caused by O.sub.2 pulses. (See B.
Chance and M. Erecinska, "Flow Flash Kinetics of the Cytochrome
a.sub.3 - oxygen Reaction in Coupled and Uncoupled Mitochondria
Using the Liquid Dye Laser," Arch. Biochem. Biophys., 143:675-687,
1971). These experiments identify accelerated responses of the
flavoproteins and pyridine nucleotides when ADP and Pi are present
and even faster rates of these transitions when the energy is
needed for transporting calcium as well. In essence, the
mitochondrial respiratory chain contains sensitive indicators of
the extent and nature of the energy demand. Since one of the
principal problems of tissue bioenergetics in normal and abnormal
states is the functionality of mitochondria in situ (examples are
irreversible damage in stroke and shock, see A. G. B. Kovach, "The
Function of the Central Nervous System After Hemorrhage," J. Clin.
Path., 23:Suppl. (Royal Coll. Path.) 4:202-215, 1970), it seems
desirable to be able to apply the techniques developed for isolated
mitochondria directly to the intact tissue.
In mitochondrial experiments conducted with the system of the
present invention the metabolic transition employed was from anoxia
to normoxia in the rapid flow apparatus; oxygen pulses were
delivered to the anaerobic suspension of isolated mitochondria. The
oxidation time for Fp and PN was as short as 100 msec. When oxygen
pulses are delivered to perfused organs the time from anoxia to
normoxia may be 20-30 sec. (see B. Chance, J. R. Williamson, D.
Jamieson and B. Schoener, "Properties and Kinetics of Reduced
Pyridine Nucleotide Fluorescence of the Isolated and In Vivo Rat
Heart," Biochem. Z., 341:357-377, 1965; J. R. Williamson and D.
Jamieson, "Metabolic Effects of Epinephrine in the Perfused Rat
Heart. I. Comparison of Intracellular Redox States," Tissue
pO.sub.2 and Force of Contraction, Mol. Pharmacol., 2:191, 1966)
and even with the intact organ circulated in vivo, recovery from
ischemia in the liver requires 2 to 3 sec. (see B. Chance and B.
Schoener, "A Correlation of Absorption and Fluorescence Changes in
Ischemia of the Rat Liver In Vivo," Biochem. Z., 341:340-345, 1965)
due to diffusion limitation in the tissue.
Spontaneous or evoked contractility affords fast perturbation of
the metabolic state of skeletal and cardiac muscle and causes a
fast oxidation of PN due to the arrival of ADP (or Ca.sup.2 .sup.+)
at the mitochondria. (See B. Chance, G. Mauriello and M. Aubert,
"ADP Arrival at Muscle Mitochondria Following a Twitch In Muscle as
a Tissue," K. Rodahl and S. Horvath, eds., McGraw Hill, New York,
1962, pp. 128-145). This perturbation has the great advantage that
diffusion time from the myofibrils to the mitochondria is short
(<100 .mu.sec.) and in particular cases mitochondria responses
200 msec. after a single muscle twitch are observed.
The diffusion limitation in the anaerobic-aerobic transition can be
avoided by methods that perturb the biological system after
diffusion equilibrium of the relevant metabolites has been
established. Specific perturbation of single enzymes is not
possible with available relaxation methods which alter only the
fundamental variables of temperature, pressure, volume, etc. For
example, a temperature perturbation alters the enzyme activities
and metabolic flux rates on a wide range of components rather than
a specific desired component. An example of a temperature
perturbation is afforded by a laser induced temperature jump (about
10.degree.) applied to a toad sartorius muscle. (See B. Chance, B.
Schoener and D. DeVault, "An Attempt to Apply the Temperature Jump
Technique to Enzyme Reactions in Tissues," Science, 144: 561,
1964). A biphasic decrease of the fluorescence of NADH was
observed. The fast decrease was due to a decrease in quantum
efficiency and the slow decrease was due to a temperature-induced
change of metabolism that reached a peak at 200 msec. This type of
perturbation was exploited further in cell suspensions (see I-Y.
Lee and B. Chance, "Method for Creating Rapid Cellular Temperature
Perturbation," Anal. Biochem., 29: 331-338, 1969) in which xenon
flash and joule heating were employed. The 5.degree. temperature
jump reached equilibrium in about 400 msec., and caused an
activation of the enzymes of glycolysis as evidenced by an
increased NADH oxidation. However, the response could not be
identified with a single enzyme or chemical species. One way to
obtain a more specific perturbation is the electrophoretic
injection of substrate into the cytosol of a single cell which
gives a fluorescence increase due to NAD or NADP reduction with a
half-time of a few hundred msec. (See E. Kohen, C. Kohen and B.
Thorell, "Rapid Microfluorimetry of Enzyme Reactions in Single
Living Cells," Biochim. Biophys. Acta, 234:531-536, 1971).
While the temperature jump perturbations caused extensive
transients in the glycolytic metabolism because the temperature
coefficients of the steps are large and non-identical, the
successive steps of electron transport in mitochondria have nearly
identical temperature coefficients and thus little effect can be
observed with temperature perturbation. However, a large and
specific activation of mitochondrial electron flow is caused by
flash photolysis of cytochrome a.sub.3 in the presence of
oxygen.
In brief, the technique involves carbon monoxide inhibition of the
anaerobic mitochondria, their rapid mixture with oxygen in the
regenerative flow apparatus, illumination with a photolyzing light
of sufficient intensity to break the cytochrome a.sub.3 -CO bond
(see B. Chance, "The Carbon Monoxide Compounds of the Cytochrome
Oxidase," J. Biol. Chem., 202:407-416, 1953), and the use of a
sufficiently rapid optical readout system to follow the rapid
oxidation of cytochrome a.sub.3 + a and cytochrome c. Under these
conditions, the observed reaction rates are limited neither by the
speed of photolysis of the CO compound nor by the mixing of oxygen
with the cytochrome oxidase; the intrinsic rates of the electron
transport reactions are directly measured. In addition to electron
transport activation, ATP formation, ion pumping and substrate
transport are rapidly initiated and thus this technique can be of
great value in relating the response time for activation of these
functions with overall physiological activities. The kinetics of
activation of electron transport can be read out not only in terms
of cytochromes of types c, a and a.sub.3 but also from the
energy-dependent cytochrome b.sub.T (see B. Chance, D. F. Wilson,
P. L. Dutton and M. Erecinska, "Energy-coupling Mechanisms in
Mitochondria: Kinetics, Spectroscopic and Thermodynamic Properties
of an Energy-transducing Form of Cytochrome b," Proc. Natl. Acad.
Sci. U.S., 66:1175-1182, 1970; D. F. Wilson and P. L. Dutton,
"Energy Dependent Changes in the Oxidation-reduction Potential of
Cytochrome b," Biophys. Res. Comm., 39:59-64, 1970) and the
fluorescent electron transport components, oxidized Fp and reduced
NAD. The rate of the response can be a sensitive indicator of the
mitochrondrial phosphate potential (see B. Chance and G. Hollunger,
"The Interaction of Energy and Electron Transfer Reactions in
Mitochondria. III. Substrate Requirements for Pyridine Nucleotide
Reduction in Mitochondria," J. Biol. Chem., 236:1555-1561, 1961) or
energy charge (see D. E. Atkinson and G. M. Walton, "Adenosine
Triphosphate Conservation in Metabolic Regulation," J. Biol. Chem.,
242:3239-3241, 1967).
With flash photolytic techniques in general, it is essential that
the mixing time for the a.sub.3 -CO compound with oxygen be shorter
than the time for spontaneous dissociation of the a.sub.3 -CO
compound. While this is easy to achieve in the regenerative flow
apparatus, it can become of critical importance in tissues where
the mixing of oxygen with the CO inhibited oxidase depends upon the
effectiveness of perfusion and the speed of diffusion from the
capillaries to the tissue mitochondria. From previous experimental
results on the relatively slow recovery of tissues from anoxia, it
is clear that the rate of dissociation of carbon monoxide from
cytochrome a.sub.3 at 20.degree. (see B. Chance, "The Carbon
Monoxide Compounds of the Cytochrome Oxidase," J. Biol. Chem.,
202:407-416, 1953) is fast enough so that some difficulties might
be encountered. The system of the present invention provides for
the resolution of these problems and provides a satisfactory
experimental approach to flash photolysis of cytochrome a.sub.3 -CO
in the presence of oxygen in cardiac tissue, with the readout of
the kinetics of electron transfer and tissue bioenergetics in terms
of the fluorescent Fp and reduced PN components of the
mitochondria.
In typical studies employing the system of the present invention
the rat heart was selected because of its physiological and
biochemical characteristics. The high volume ratio of mitochondria
(0.34) together with the relatively small contribution of tubular
membrane to the total sarcolemma also make it an experimental
material suitable for evaluation of mitochondrial function in
contraction-relaxation processes. Thus, the system is useful for
further investigation of the role of mitochondria in muscular
function and for the further study of the kinetics of enzyme
reactions in tissues.
A typical illustration of the apparatus of the present invention as
employed for recording fast fluorescence changes in the perfused
heart is provided by FIG. 1. The general characteristics of the
time sharing fluorometer for Fp and PN, generally indicated at 30,
have been described previously (see B. Chance, D. Mayer and L.
Rossini, "A Time Sharing Instrument for Direct Readout of
Oxidation-reduction States in Intracellular Compartments of Cardiac
Tissue," IEEE Transactions on Bio-Medical Engineering, 2:118-121,
1970, and B. Chance, N. Graham and D. Mayer, "A Time Sharing
Fluorometer for the Readout of Intracellular Oxidation-reduction
States of NADH and Flavoprotein," Rev. Sci. Instr., 42:951-957,
1971). Modifications required for flash photolysis in the perfused
heart are indicated by the diagram. These modifications comprise
the provision of a suitably mounted first flash device 31, for
example, a Xenon flash assembly arranged to deliver a flash beam 32
to the surface of the heart 33 under study and the provision of a
suitably mounted second flash device 34, for example, a liquid dye
laser assembly, arranged to deliver a flash beam 35 via a suitably
angled mirror 36 to the heart surface area under study. The heart
can be perfused with either oxygen or nitrogen plus carbon monoxide
by shifting between reservoirs equilibrated with the appropriate
gas mixture. Fluorescence excitation is applied normally to the
heart by an intermittent beam 37 so that the specular reflection
travels back along the incident path and not into the fluorescence
detector, and the scattered light does not change appreciably with
the motion of the heart. In some cases, the heart may be supported
upon a glass surface, for example, a suitably supported small
funnel 38 which collects the perfusate which helps thereby to damp
the gross movement of the heart. Fluorescence emission is observed
at a beam 39 at an angle of 30.degree. through the secondary filter
40. Each 180.degree. turn of the disk 41 holding the excitation and
emission filters brings them into alignment for reduced PN (366 nm
and 480 nm, respectively) and for oxidized Fp (460 nm and 580 nm,
respectively).
The fluorometer observes a portion of the surface of the heart 33
between 2 and 4 mm in diameter and on the left or right ventricle
as may be desired. The depth of penetration has been estimated in
experiments in which successive layers of tissue are piled one upon
the other and the increase of the fluorescence signal is observed.
The "endpoint" of the fluorescence increase is identified with the
end point of the penetration of the excitation and emission
wavelengths. For PN (366 nm excitation, 460 nm emission) the value
is 0.36 mm. For Fp (excitation 460 nm, 570 nm emission) the value
is 0.84 mm. Thus the fluorescence recording is from about the first
third of the thickness of the wall of the heart (about 3 mm.).
Absorption measurements may be obtained by employing an absorption
detector 42 in the manner disclosed in B. Chance, D. Mayer and L.
Rossini, "A Time Sharing Instrument for Direct Readout of
Oxidation-reduction States in Intracellular Compartments of Cardiac
Tissue," IEEE Transactions on Bio-Medical Engineering, 2:118-121,
1970.
Fluorescence change in response to a normoxic-anoxic cycle may be
recorded from the left and right ventricles with satisfactory
results, although somewhat larger signals are obtained from the
left ventricle (about 50 percent), which technique has been
employed in most experiments.
The two light sources 31 and 34 afford photolysis for the carbon
monoxide-inhibited cytochrome system. The xenon flash lamp 31 (2
msec flash, 28 J white light) (Braun F 270) is filtered through a
430 .+-. 10 nm filter 43 so that interference with the PN readout
at 480 nm is diminished. A reflector in the flash lamp plus a
lucite light cone increases the efficiency of illumination. Two
xenon flash lamps and cones may be employed.
Photolysis with the liquid dye laser 34 (General Laser GL-1,000)
(585 nm, 100 mJ intensity) is via the small mirror 36. The laser
affords this power output at an operating voltage of approximately
14 kV when employing a Rhodamine 6G dye. The laser beam is highly
parallel and of roughly 1 cm diameter. Thus, the heart is uniformly
illuminated by the laser beam 35.
The heating of the heart from the 0.1 mJ laser flash is readily
calculated since 4 J are required to raise 1 ml of H.sub.2 O by
1.degree.C. Two calculations can be made. The first one assumes
that all of the laser light is uniformly absorbed in the
approximately 1 cc volume of the heart. The temperature rise under
these conditions is 0.1/4 or 0.025.degree. . A second calculation
assumes that the laser light is all absorbed in a square 5 mm on a
side and 3 mm thick, the thickness of the heart wall. The
temperature rise would then be 0.3.degree.. A third calculation
assumes that only the mitochondria are heated within this volume of
tissue since the mitochondria comprise about 1/3 of the total
tissue volume, and the temperature rise would be 1.degree.. The
possibility that the laser damages the mitochondria as suggested by
experiments at higher powers is unlikely; repeated flashes cause no
decrease of mitochondrial signals. A response to increased
temperature to be expected is the decrease of PN fluorescence at
about 1 percent per degree due to the decrease of quantum yield; no
significant effect was seen. The kinetic responses do not depend
upon which ventricle is illuminated and thus a specific effect upon
the pacemaker cells is unlikely. This viewpoint is supported by
studies with electrically driven hearts.
The xenon flash lamp with the 430 nm filter 43 causes about the
same photolysis as the liquid dye laser; the extinction coefficient
of cytochrome oxidase is approximately 10 times greater at 430 nm
that at 585 nm.
A double flash is useful to evaluate the extent of photolysis. One
flash occurs a few seconds after perfusing the tissue with oxygen
and another flash may be applied several seconds later. The two
flash lamps 31 and 34 are triggered by an appropriate synchronizing
circuit such as shown in FIG. 2, presently to be described. Two
lucite cones can be employed to direct the light from these lamps
upon the same area of the heart.
In addition to the circuitry for the time sharing fluorometer, the
photolysis lamps 31 and 34 are also synchronized with the rotating
disk 41 so that their flashes are triggered during an interval when
the photomultiplier 45 is not illuminated. The xenon flash occurs
following the Fp measurement since the 580 nm emission filter 40
protects the photomultiplier 45 from the blue flash which decays
substantially in the 8 msec interval between fluorescence
measurements.
In the case of the liquid dye laser 34, the 0.4 .mu.sec flash is
terminated in the 8 msec interval between measurements. The 585 nm
light does not interfere with the 450 nm PN measurement but does
interfere with Fp measurement at 580 nm. The laser is flashed just
after the aperture for Fp measurement is closed and thus about 14
msec is available for recovery of this channel prior to the first
post-flash Fp measurement. In this way the time sharing system not
only alternates the filters for the fluorescence measurement but
also acts as a phosphoroscope to guard the measuring
photomultiplier 45 against overload during the flash photolysis of
the cytochrome oxidase-CO compound.
If some flash artifact occurs, the recovery of the photomultiplier
output is short compared to the 75 to 100 msec rise time of the
metabolic responses of Fp and PN.
A trigger for the synchronizing circuit can conveniently be derived
from either one of the two pulses from the compensating
photomultiplier, shown at 44. Such a pulse triggers the delay
circuit which provides a relay closure for firing the flash lamps
at an appropriate phase angle. A Tektronix Storage Scope, Model 564
is provided with a suitable pre-trigger adjustable from 0.5 to 3
sec. with respect to the photolysis flash. In usual operation, the
oscilloscope trace may be triggered at the time the oxygen
saturated perfusate is turned on. The operation thereafter is
automatic.
FIG. 2 is the block diagram of the typical double flash circuitry
which may be employed, and FIG. 3 shows the key waveforms for the
circuit.
The signal 18 from the compensation photomultiplier 44 is squared
up by Schmitt trigger 1, resulting in waveform 19. Monostable 2
allows phase adjustment so that both flash lamps 31 and 34 can be
flashed at a given point with respect to waveform 18. Monostable 3
generates a 20 ms gate shown as waveform 20 and is applied to the
toggle inputs of flip-flops 4 and 14 as the clock or synchronous
input. Pushbutton switch 6 initiates the flashing sequence.
Flip-flop 7 provides a suitable trigger to monostable 8 and the
storage oscilloscope 25. Thus, a sweep is started. Monostable 8
delays the flashing sequence from 0.5 to 3 sec. to permit storage
of baseline on oscilloscope 25. After said delay monostable 9 is
triggered, which results in waveform 22. The negative going edge of
waveform 21 is phased to occur approximately 1 ms after the Fp
pulse signal of waveform 18. The first negative transition of
waveform 21 after waveform 22 has gone negative causes waveform 23
at the output of flip-flop 4, which is suitably shaped by drive
circuit 5 to trigger xenon lamp 31 or liquid dye laser 34. Inverter
10 keeps the J and K inputs of flip-flop 4 in 180.degree. phase
opposition. Monostables 11 and 12 delay the flashing of liquid dye
laser 34 from 0.5 to 3 sec. Inverter 13 and flip-flop 14 act to
trigger liquid dye laser 34 coincidentally with the negative
transition of waveform 20, resulting in waveform 24, which is
shaped by drive circuit 15 to trigger lamp 34, thus completing the
double flash cycle.
A 1800 RPM synchronous motor 46 is employed. The rotating disk 41
is mounted directly on the shaft of the motor.
The display oscilloscope 25 is a Tektronix, Model 564, using a four
channel vertical amplifier and a time base used in the external
trigger input mode.
In typical experiments, after suitable preparation rats were
decapitated, the heart rapidly excised and securely attached to a
"Y" glass cannula (15 gauge equivalent) at 48 via the aortic root.
It is essential to avoid a prolonged anoxia between the interval of
excision of the heart and the perfusion with oxygen. The estimated
interval for most preparations is about 45 seconds. A Langendorf
perfusion apparatus was employed and the perfusate was not
recirculated. The perfusion pressure was 50 .+-. 10 mm Hg and the
temperature was 24.degree.. The flow rate was 12-17 ml/min. (For
further details see R. B. Fisher and J. R. Williamson, "The Oxygen
Uptake of the Perfused Rat Heart," J. Physiol. (London) 158:86-101,
1961). The perfusate was Krebs Ringer bicarbonate (see H. A. Krebs
and K. Henseleit, "Untersuchungen Uber Die Harnstoffbildung Im
Tierkorper," Hoppe-Schler's Z. fur Physio-Chemie, 210:33-37, 1932)
containing half the usual calcium concentration.
0.1 mM NaNO.sub.2 was added to the perfusate in order to oxidize
the tissue myoglobin and any residual hemoglobin and thereby avoid
absorbancy changes due to these oxygen carriers in the aerobic
anoxic cycles.
In order to alternate aerobic and CO anaerobic perfusates, 95%
O.sub.2, 5% CO.sub.2 or 67% CO, 28% N.sub.2, 5% CO.sub.2 was
equilibrated with the perfusate in duplicate vessels each at the
same height. FIG. 1 illustrates the use of stopcocks 47 for
changing the perfusion media. The dead volume between the stopcocks
and the heart was about 0.1 ml. Thus the new perfusate arrived at
the heart in 0.1 sec. at the flow rates of 10 ml/min. The duration
of carbon monoxide exposure was 1 min. and a 5 min. aerobic
recovery interval was afforded. When prolonged intervals of anoxia
were employed, the heart was supplemented with 1-10 mM dextrose to
maintain a strong heart beat.
Ventricular pressure was measured directly with a 20 gauge needle
in the left ventricle and a Sanborn pressure transducer and D.C.
amplifier. The tension was measured from a hook at the apex of the
heart combined with a strain gauge 49. The measurements were not
localized to the exact region of fluorometric observation but did
afford a control of the generalized physiologic function of the
heart.
The experiments described here are based upon studies of many rat
hearts and the graphs shown in FIGS. 4, 5 and 6 are representative
of the results obtained in the vast majority of these
experiments.
A successful experiment requires rapid reoxygenation of the tissue
following an anoxic episode. In hearts from the Holtzman strain,
this was regularly observed and responses from Fp and PN were
obtained as indicated in FIGS. 4 and 6. It appears that the tissue
circulation in the area under observation is somehow impaired by a
brief episode of anoxia. Thus, the first response of the heart to
anoxia was carefully monitored. In addition, special precautions
were taken to avoid an anoxic episode in the interval between
excision of the heart and transfer to the perfusion apparatus.
In actual operation, after suitable calibration of the fluorometer,
the heart is initially put in anoxic condition with 67% CO, 28%
N.sub.2, 5% CO.sub.2. The perfusate is then changed to 95% O.sub.2,
5% CO.sub.2 by altering the fluid connections and at the same time
the sweep is triggered. The photolysis flash occurs 0.5 to 1.0 sec.
after the recovery from anoxia is detectable. The photolysis flash
then activates the respiratory carriers and the metabolic state of
the tissue can be read out in terms of their rate of oxidation. The
system is allowed to re-establish its normoxic state and an
interval of 5 min. elapses before the next episode of toxic
anoxia.
The apparatus illustrated in FIG. 1 can be employed for
spectroscopic measurements as well, as above mentioned. 445 and 455
nm interference filters are inserted into additional holes in disk
41. A photomultiplier in unit 42 views the light transmitted
through a single thickness of the heart wall via a 1 mm diameter
light pipe.
Optimal results for experiments such as above described require a
careful evaluation of the parameters. Ideally 100 percent saturated
cytochrome a.sub.3 -CO is completely photolyzed by the light flash
to give 100 percent reduced a.sub.3 which combines with excess
O.sub.2 to give 100 percent oxidized a.sub.3. This ideal is only
imperfectly realized; the amount of photolysis depends on the
interaction of four factors: (1) the CO concentration of the
perfusion; (2) the extent of pre-flash photolysis; 3 (3) the extent
of flash photolysis, and (4) the oxygen concentration. Whereas in
the dark a few tenths of a percent of CO will satisfy the
requirements for initial CO saturation, in view of the high
affinity for CO (10.sup.-.sup.6 M), the photolysis caused by the
fluorescence excitation shifts the CO affinity to lower values due
to an increase of k.sub.6 ' in equation 6 mentioned in B. Chance,
Jour. Biol. Chem., 202: 407, 1952, and causes "pre-flash
photolysis." The extent of pre-flash photolysis is diminished by
high CO (k.sub.5 .sup.. i is increased; see equation 5 of the
last-named reference). A high fluorescence excitation intensity is
required for the fast readout of fluorometric signals, thus a
signal-to-noise ratio in excess of 10 may result in a pre-flash
photolysis of up to 25 percent of the total oxidation reduction
change. Flash photolysis of cytochrome a.sub.3 was over 95 percent
complete with the laser source or the xenon flash source, taking in
this case 100 percent to be that obtained with the double flash
technique above described and to be further discussed below. The
oxygen concentration in the perfusate and the diffusion velocity of
oxygen in the tissue should be high enough so that the oxygen
gradient moves through most of the a.sub.3 -CO molecules in the
field of observation in the interval between initiating the oxygen
perfusion and the flash photolysis. Under these conditions the
oxidation rates of PN and Fp will be indicative of the ADP + Pi and
Ca.sup.2 .sup.+ levels in the cytosol.
The interaction of these factors above mentioned leads to four
categories of cytochrome a.sub.3 molecules at the time of
photolysis. First, cytochrome a.sub.3 -CO molecules which are in
the presence of a sufficiency of oxygen, that is, an oxygen
concentration which upon photolysis will give a significant
oxidation of Fp and PN. Second, cytochrome a.sub.3 molecules that
are in a deficiency of oxygen such that no significant response of
Fp and PN is observed on photolysis. Thirdly, reduced cytochrome
a.sub.3 molecules from which CO has dissociated but which oxygen
has not yet reached. Fourth, oxidized cytochrome a.sub.3 molecules
from which CO has already dissociated and with which oxygen has
already reacted prior to the time of the photolysis flash.
The populations of molecules in these categories will vary with
time. For example, the fourth category is increased rapidly upon
photolysis, and those in the third category will recombine with CO
if oxygen does not arrive in a short time. The molecules in
category 2 are of considerable interest because these can acquire
category 1 status as the oxygen diffusion gradient moves through
the tissue, which is in turn related to the effectiveness of tissue
oxygenation. The category 1 molecules will respond rapidly to
photolysis if the mitochondria are in a region of high Ca.sup.2
.sup.+ (about 10.sup.-.sup.4 M).
Categories 1 and 2 are identified with a sufficiency and deficiency
of oxygen. This can be quantitated as follows: molecules are in
category 2 if on photolysis they produce a cytochrome oxidation
rate slower than the observed oxidation of Fp (half-time, t.sub.1/2
= 60 msec.). Since the second order velocity constant for
cytochrome a.sub.3 with oxygen is 3 .times. 10.sup.7 M.sup.-.sup.1
sec.sup.-.sup.1 (see B. Chance and M. Erecinska, "Flow Flash
Kinetics of the Cytochrome a.sub.3 -- oxygen Reaction in Coupled
and Uncoupled Mitochondria Using the Liquid Dye Laser," Arch.
Biochem. Biophys., 143:675-687, 1971) a half-time of 60 msec. would
be obtained at less than 1 .mu.M tissue oxygen; cytochrome a.sub.3
-CO molecules photolyzed in less than 1 .mu.M oxygen are therefore
in category 2.
The partitioning of molecules between category 1 and category 4
depends on the rate at which cytochrome a.sub.3 -CO molecules
dissociate in the time interval between the entry of oxygen into
the tissue and the flash photolysis. It is for this reason that the
coronary perfusion and the oxygen diffusability in the tissue are
important variables. In addition, the photolysis of the cytochrome
a.sub.3 -Co compound by the measuring light becomes of critical
importance and the rate of this pre-flash photolysis is suitably
decreased by a decrease of the fluorescence excitation
intensity.
The fluorescence excitation intensity is decreased in the typical
arrangement of FIG. 1 by the use of a number of suitable filters 50
in the beam from the excitation source. The filters may comprise
blue Corning glass filters, such as Corning Model CS-5-60 (5543)
which transmit 10 percent of the incident light at 366 nm and 45
percent at 460 nm, corresponding to absorbancy increments of 0.10
and 0.26 per filter. Three or four filters may be employed. The
signal-to-noise ratio is lower at the lower excitation intensity.
Such a low signal-to-noise ratio is satisfactory in many
experiments, but higher signal-to-noise ratio and more rapid speed
of response can be obtained with fewer filters to attenuate the
source light. In such cases the number of category 1 molecules is
less but the speed of the Fp and PN oxidation is more clearly
recorded.
It has been found that fluorescence excitation obtained through 3
to 4 filters is sufficiently small that a maximal response of PN
and Fp is obtained, corresponding to 62 and 50 percent of the total
normoxic-anoxic change. At present the difference between the
category 1 cytochrome a.sub.3 -CO molecules assayed by the Fp and
PN responses is not regarded to be significant. The fact that these
responses do not rise to 100 percent as the fluorescence excitation
intensity decreases is due to the dissociation of cytochrome
a.sub.3 -CO which is thermally activated and occurs without
illumination. Thus, 100 percent response could only occur if the
oxygen diffusion gradient reached the mitochondria very
rapidly.
Representative graphs shown in FIGS. 4, 5 and 6 illustrate typical
responses of the perfused heart. Usually a statistical analysis is
unnecessary since each experiment contains a prior control or
allows a recovery of the heart to its initial state, following
which the experiment is repeated. Thus, the illustrative graphs
each represent one of a series of repetitions of the particular
test.
In order to identify the a.sub.3 -CO compound in the cardiac tissue
and to afford a basis for the application to cardiac tissue studies
on the a.sub.3 -CO compound in isolated mitochondria, FIG. 5
illustrates dual wavelength absorbancy measurements at 445-455 nm
made through the wall of the heart into a light pipe and then to
the measuring photomultiplier of unit 42. In graph A the heart is
initially under normoxic conditions and is perfused with nitrogen
saturated medium. The consequent anoxia causes an upward deflection
of the trace corresponding to increased absorbancy at 445 nm.
Following complete reduction of cytochrome a.sub.3, perfusion with
0.6 percent CO is begun and at this low CO concentration
approximately 6 minutes are required to reach the saturation value
of cytochrome a.sub.3 -CO. The absorbancy change with CO is about
half of the oxygen-nitrogen change identifying the formation of
cytochrome a.sub.3 -CO. On addition of oxygen, the a.sub.3 -CO
compound is converted to the oxidized form as indicated by the
downward deflection of the trace to the original base line.
Evidence of flash photolysis of cytochrome a.sub.3 -CO is afforded
by graphs B and C of FIG. 5. In anoxia (FIG. 5B) the xenon flash
causes no absorbance change. In the presence of 20% CO (FIG. 5C)
the cytochrome a.sub.3 -CO compound rapidly photolyzes and gives an
abrupt absorbancy decrease at 445 nm. After the flash, CO rapidly
recombines with cytochrome a.sub.3. If however oxygen were present
as in FIG. 6, oxidation of cytochrome a.sub.3 and the mitochondrial
components would follow. In these experiments residual hemoglobin
and tissue myoglobin are converted from the ferrous to the ferric
forms by nitrite perfusion and thus, interference with their CO and
oxygen compounds is negligible.
The kinetics of recovery from anoxia are indicated in FIG. 4A for
nitrogen anoxia and CO anoxia. The oxidation-reduction changes are
read out from the fluorescence of reduced PN and oxidized Fp. The
cardiac tissue is rendered anoxic by perfusion with nitrogen
saturated medium. At the beginning of the trace the heart is
perfused with 95 percent oxygen and after the dead volume is
cleared out and a significant oxygen concentration has accumulated
in the tissue, increased fluorescence of oxidized Fp and decreased
fluorescence of reduced PN are observed.
The experiment is repeated in FIG. 4B with CO replacing 67 percent
of the nitrogen; both traces exhibit greater amplitude. After
perfusion with oxygen saturated medium, rapid oxidation of reduced
PN and Fp occurs as in the previous experiment. The half-time (1.5
sec.) for the normoxic-anoxic transitions are approximately the
same in FIGS. 4A and 4B. The amplitude and hence the initial slopes
are, however, different. In nitrogen anoxia there is partial
oxygenation of the surface of the heart and a diminished amplitude
of the oxidized-reduced changes. In the presence of carbon
monoxide, Fp and PN are more highly reduced. Nevertheless, the
anoxic-normoxic transition proceeds rapidly; illumination of the
heart by the fluorescence excitation causes photolysis of the
cytochrome a.sub.3 -CO compound, and consequently rapid oxidation
of Fp and PN.
An adequate flash photolysis intensity is needed to photolyze and
thus to assay the molecules in category 1, particularly for studies
of oxygen diffusion. However, the rate of PN and Fp oxidation does
not depend upon the degree of photolysis as shown by experiments on
isolated mitochondria. The extent of flash photolysis may be
evaluated in two ways: (1) by a step-wise variation of the
intensity of a single flash lamp, and (2) by a sequence of two
flashes. The first method is simple to employ but does not identify
100 percent photolysis. A pair of flashes, such as by laser 34
followed by xenon unit 31, spaced at a small time interval
identifies 100 percent photolysis when the second flash causes no
response.
In FIG. 6, two 28 J flash lamps were employed. Each was equipped
with blue filters giving maximal transmission near 430 nm. The
increment of photolysis afforded by the second flash following the
first (FIG. 6B) by 0.6 sec. is small (less than 5 percent). When
the second flash followed by the first by 1.3 sec. (FIG. 6A) a
measurable increment of photolysis is observed on the PN trace
(about 30 percent). Thus, photolysis is practically complete with
the first flash of the first 28 J lamp. In many cases, a single
laser flash verified these results. The increment of photolysis
observed with the second lamp is due to a further progress of the
oxygen diffusion outwards from the capillaries.
Experiments have shown different responses to early and late
flashes. With an early flash, for example, 0.5 sec. after the start
of oxidation, only a portion of the total amount of Fp and PN is
oxidized. However, in an experiment wherein the delay was 1.5 sec.
and the pre-photolysis oxidation reaction had proceeded about 50
percent to the normoxic steady state, the flash completed the
oxidation of Fp and nearly completed the oxidation of PN. Thus,
about 1.5 sec. after the start of oxidation no cytochrome oxidase
molecules remain in categories 2 and 3, all are in categories 1 or
4, and negligible further oxidation occurs after the flash
photolysis has substantially depleted the category 1 molecules.
Recordings on varying time scales (1.0, 0.5 and 0.2 sec/div.)
indicate the time for the photolysis response to reach 90 percent
for PN and Fp was similar, and a value of 100 msec .+-. 20 msec was
obtained in a sample of 10 experiments.
In order to further test for factors which influence tissue
oxygenation at the cellular level, the effect of nitrite on the
nature of the response was studied. When nitrite is absent
ferromyoglobin would be expected to be assisting in the tissue
oxygenation. When nitrite is present not only would the myoglobin
be oxidized in the met-form but also vasodilation of the heart
would be expected. The jump of PN and Fp on photolysis in the
presence of nitrite was larger than in the absence of nitrite. A
second flash followed the first flash in both these cases to ensure
that photolysis was substantially complete. Apparently the
vasodilation is significant but no significant contribution of
heart myoglobin to tissue oxygenation was identified under these
conditions.
The relationship between external calcium and the respiratory
activity of the perfused heart suggests that higher calcium would
cause an increased mitochondrial activity. An example of a 4-fold
increase of calcium (0.9-3.6 mM) was studied. Following perfusion
with the higher level of calcium not only was the Fp response
approximately doubled, but the further oxidation of Fp and PN
proceeded more rapidly. Calcium addition increased the rate of
oxidation of PN and Fp and increased the size of the photolysis
jump.
Usually the perfusate was supplemented with 10-20 mM dextrose in
order to maintain glycolytic activity at a maximum rate, and
thereby afford a minimal depletion of energy reserves in the anoxic
interval preceeding the flash photolysis. Such supplements of
substrate may be unnecessary for the initial anoxic cycles but are
of considerable importance after half a dozen anoxic episodes. The
striking effects of substrate supplementation are demonstrated in
experiments where the beat rate of the heart in the anoxic interval
is slow. Under these conditions, substrate supplement increases
cytoplasmic ATP so that sufficient energy for mitochondrial calcium
uptake is available and photolysis response is observed in the
anoxic-normoxic transition.
It can be concluded that photolysis of cytochrome a.sub.3 -CO in
the presence of O.sub.2 provides a pulsed activation of electron
transport that rapidly oxidizes the components of the respiratory
chain ranging from the very rapid responding components, a.sub.3,
a, c, c.sub.1, to the more slowly responding components, cytochrome
b, the flavoproteins, quinones and pyridine nucleotides.
Depending on the information desired, different components of the
chain afford a suitable readout. The fast reactions of electron
transfer of cytochromes a, c, and c.sub.1 are slightly affected by
the presence of extra-mitochondrial calcium and the slower kinetics
of cytochrome b, flavoprotein, quinone and pyridine nucleotide are
more sensitive. The readout of flavoprotein and NADH can be made by
fluorometry which is directly applicable to tissue surface as
opposed to transmission measurements which are required for
cytochrome b and quinone, and for which both sides of a tissue
appropriate thickness must be available. In the case of the
perfused heart, the total thickness is too great for satisfactory
absorbancy measurements of the cytochromes and thus it is necessary
to penetrate the heart wall with the light pipe to obtain the
single thickness of about 3 mm for absorbancy measurements. Since
this puncture is not necessary for surface fluorometry, a better
biological and physiological condition is possible.
The principal result from the experiments conducted is related to
mitochondrial function in cardiac bioenergetics. The result is read
out from the Fp and PN kinetics in response to carbon
monoxide/oxygen transitions followed by the flash photolysis.
The above points to a new method of determining oxygen diffusion
from the capillary bed to the mitochondria and suggests that a
suitable criterion is the percentage of the total Fp or PN
oxidation which can be observed (a) under conditions where the
photolysis flash is of sufficient intensity to afford complete
breakdown of the category 1 molecules and (b) where the measuring
light is so diminished that the photolysis jump is maximal. The
calculation of the actual diffusion rates depends upon computer
simulation of the geometries, reaction kinetics and stoichiometries
involved.
While a specific embodiment of a system for providing flash
photolysis activation of CO-inhibited cytochrome oxidase in tissue
in the presence of oxygen has been disclosed in the foregoing
description, it will be understood that various modifications
within the spirit of the invention may occur to those skilled in
the art. Therefore it is intended that no limitations be placed on
the invention except as defined by the scope of the appended
claims.
* * * * *