Download Brief Communication Direct Detection of Free Radicals in the

757
Brief Communication
Direct Detection of Free Radicals in the Reperfused
Rat Heart Using Electron Spin Resonance
Spectroscopy
Pamela B. Garlick, Michael J. Davies, David J. Hearse, and Trevor F. Slater
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The purpose of this study was to use a direct method, that of electron spin resonance (ESR)
spectroscopy, to demonstrate that reperfusion after a period of ischemia results in a sudden increase
in the production of free radicals in the myocardium. The isolated buffer-perfused rat heart was used
with N-tert-butyl-a-phenylnitrone (PBN) as a spin-trapping agent. Samples of coronary effluent were
taken and extracted into toluene for detection of radical adducts by ESR spectroscopy. After 15 minutes
of total, global ischemia, aerobic reperfusion resulted in a sudden burst of radical formation that
peaked at 4 minutes. When hearts were reperfused with anoxic buffer, no dramatic increase in radical
production was observed. Subsequent reintroduction of oxygen, however, resulted in an immediate
burst of radical production of a similar magnitude to that seen in the wholly aerobic reperfusion
experiments. The ESR signals obtained (aN = 13.60 G, aH = 1.56 G) are consistent with the spin-trapping
by PBN of either a carbon-centered species or an alkoxyl radical, both of which could be formed
by secondary reactions of initially-formed oxygen radicals with membrane lipid components.
(Circulation Research 1987;61:757-760)
R
eperfusion of the heart after a period of ischemia
can lead to the occurrence of potentially lethal
arrhythmias.1"* The spontaneous or drug-induced relief of coronary spasm and the restoration of
blood flow after cardiac surgery are two of the clinical
situations in which this problem may be encountered.
There is considerable controversy over the mechanisms
responsible for the induction of these arrhythmias and
a number of different factors have been suggested.
These include the stimulation of a- or/3-receptors,5 the
elevation of cyclic adenosine monophosphate (cAMP)
content,6 the release of lysophosphatides,7 the disturbance of.ionic homeostasis (particularly for Na + , K + ,
and Ca2+ ions),8-9 the metabolism of free fatty acids,10
and the heterogeneity of tissue injury and recovery (for
review, see Manning and Hearse4)- Recently, a new
arrhythmogenic factor has been proposed: the production of short-lived reactive oxygen radicals.""' 4 These
species could arise from various sources such as the
conversion of xanthine to hypoxanthine by xanthine
oxidase, the degradation of catecholamines, and mi-
From Cardiovascular Research, The Rayne Institute, St. Thomas'
Hospital, London, and Department of Biochemistry, Brunei University (M.J.D., T.F.S.), Uxbridge, Middlesex, UK.
Part of this work was presented at the American Heart Association
Meeting in Dallas in November 1986.
Supported in part by the British Heart Foundation, St. Thomas'
Hospital Research Endowments Fund, and the National Foundation
for Cancer Research. P.B.G. is an SERC Advanced Fellow.
Address for correspondence: Dr. P.B. Garlick, Cardiovascular
Research, The Rayne Institute, St. Thomas'; Hospital, London SE1
7EH, UK.
Received February 5, 1987; accepted June 3, 1987.
tochondrial electron transport. Evidence supporting
the proposed involvement of oxygen free radicals in
arrhythmogenesis has been obtained from experiments
on isolated perfused rat hearts. In these experiments,
interventions that are known to decrease the myocardial free radical content (e.g., anti-oxidant enzymes,
free radical scavengers, and spin-trapping agents) are
found to decrease the vulnerability of the heart to
reperfusion arrhythmias."' 14 Conversely, agents known
to promote free radical production13 increase the
vulnerability to such arrhythmias. All this evidence,
however, is indirect and as such may be circumstantial.
We have, therefore, used the technique of electron spin
resonance (ESR) spectroscopy to provide a direct
demonstration of free radical production during reperfusion and reoxygenation in the isolated rat heart.
Materials and Methods
Male Wistar rats (280-320 g) were lightly anesthetized with ether and after administration of heparin (200
U), the hearts were excised and perfused (65 cm H2O
pressure) in the Langendorff mode15 using a flowthrough system. The perfusion medium was KrebsHenseleit buffer16 gassed with 95% O 2 -5% CO2 (for
aerobic perfusion) or 95% N 2 -5% CO2 (for anoxic
perfusion). The buffer contained a substrate (11 mM
glucose) and a spin-trap (N-tert-butyl-a-phenylnitrone, PBN [Aldrich], at 3 mM). It was essential to
include a spin trap because of the short half-lives of radicals generated in the reperfused myocardium. l71! PBN
was chosen since it is known to form relatively
long-lived radical adducts with certain types of rad-
Circulation Research
758
icals1 and has been shown to be compatible with normal myocardial function at low concentrations."
Ph-CH=
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N -'Bu + X->-Ph-C H -
N -'Bu
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Experiments have shown that PBN can enter hepatocytes, brain, spleen, and heart cells2021 and that spin
adducts of PBN can cross hepatocyte membranes20; we,
therefore, assume that both PBN and its spin adducts
can cross myocardial membranes.
The perfusion apparatus was kept in darkness
throughout the study to prevent any photolytic degradation of PBN. After a 35-minute stabilization period
of control aerobic perfusion, the hearts were subjected
to 15 minutes of total, global ischemia, induced by
cross-clamping the aortic input line. This was followed
by reperfusion, which was either aerobic for the entire
period (30 minutes) or anoxic for the first 10 minutes
and then aerobic for the subsequent 15 minutes.
The preparation of samples for ESR measurements
was the same for both protocols. Coronary effluent
fractions were collected at one-minute intervals and 5
ml aliquots were taken from each fraction and added
to 0.75 ml of toluene (4° C) and vortex-mixed for 20
seconds. After centrifugation (800g, 5 minutes), the
toluene layer was aspirated and its ESR spectrum
recorded at 200 K, on a Bruker ER 200D spectrometer,
equipped with 100 kHz modulation and a Bruker ER
411 variable temperature unit. Hyperfine coupling
constants were measured directly from the field scan
using 10 gauss marker signals for calibration.
Results
Figure 1 shows typical spectra recorded from oxygenated buffer before passage through the heart (A),
coronary effluent collected during the last minute of
aerobic stabilization (B), coronary effluent collected
during the second minute of aerobic reperfusion (C),
and coronary effluent collected during the 15th minute
of aerobic reperfusion (D). It is clear from these spectra
that signals were not detectable in samples of fresh
buffer or in effluent samples collected either prior to
ischemia or after a long period (15 minutes) of
reperfusion. It was only during the initial moments of
(aerobic) reperfusion that signals were detected (Figure
1C). During this period, the signals observed were
always similar (Figure 2) and differed from each other
only in intensity.
The spectrum shown in Figure 2 is typical of that
obtained from a nitroxyl radical adduct and has
parameters a N = 13.60 G and a ^ l . 5 6 G. This is
consistent with the spin-trapping by PBN of either a
carbon-centered species or an alkoxyl radical17 both of
which would be produced by secondary reactions of
short-lived oxygen radicals with membrane lipids.
Since the intensities of the ESR peaks observed are
directly proportional to the free radical content of the
A
Vol 61, No 5, November 1987
' ^ / ^ ^ ^
10 gauss
B
Increasing magnetic field
•
FIGURE 1. Electron spin resonance spectra of spin adducts
extracted into toluene from oxygenated buffer (A), coronary
effluent collected during the last minute of aerobic stabilization
(B), coronary effluent collected during the second minute of
aerobic reperfusion (C), coronary effluent collected during the
fifteenth minute of aerobic reperfusion (D). Spectrometer
conditions: Gain 2 x 10*; modulation amplitude 1.6 gauss; time
constant2 seconds; scan time2,000 seconds;field3,380gauss;
scan 100 gauss; power 13 dB; frequency 9.48 GHz; temperature
200 K.
coronary effluent samples, a time-course of spin-adduct
formation can be constructed (Figure 3). This was
achieved by measuring the height of the central field
line of each spectrum and expressing it as a percentage
of the control height (obtained from the spectrum of the
last minute of aerobic stabilization, shown .in Figure
IB). It is evident from Figure 3 that during aerobic
reperfusion, there is a burst of PBN-spin adduct formation that peaks during the fourth minute of reperfusion (t = 54 minutes from start of experiment); this
would suggest that immediately preceding this, there
is a burst of free radical production in the myocardium. This sudden increase in free radical content is not
observed when the hearts are reperfused initially with
anoxic buffer. Subsequent switching to aerobic buffer,
after 10 minutes of anoxia, results in an immediate
burst of free radical production (t = 61 minutes from
start of experiment) of a similar magnitude to that seen
in the wholly aerobic reperfusion experiments.
Discussion
In both sets of experiments, we have demonstrated
the presence of nitroxyl radical adducts in the coronary
effluent of the reperfused heart. From the anoxic
experiments, we conclude that oxygen is essential for
the production of these radicals. We believe that during
Garlick et al
Free Radicals in the Perfused Rat Heart
759
FIGURE 2. Electron spin resonance spectrum of PBN spin
adducts in the coronary effluent
during the third minute of reperfusion, after extraction into toluene. Spectrometer conditions are
as given for Figure 1.
INCREASING MAGNETIC
10 gauss
FIELD
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the initial moments of reperfusion, oxygen-centered
species such as superoxide or hydroxyl free radicals are
generated. The most probable sources of these oxygen
radicals are the xanthine oxidase pathway, the electron
transport chain and catecholamine oxidation. Leukocytes are unlikely to represent a significant source
since there are virtually no white cells present in buffer
perfused hearts. Subsequent reaction of these oxygen
radicals with various cellular components (e.g., membrane lipids), would then give rise to the radical which
is trapped by PBN. The results of the anoxic experiments also confirm that the PBN radical adducts were
not produced by metabolism of the spin trap during the
ischemic period with subsequent washout during reperfusion. A preliminary report in 198422 described a
similar PBN radical adduct in lipid extracts of myocardial tissue after 15 minutes of reperfusion with the
spin trap. In this study, however, samples were only
s
240
-
220
-
collected at a single time point and no additional
experiments were performed to verify that the radical
species were only generated during aerobic reperfusion. Other preliminary reports23"23 have described
studies in which ESR has been used to observe free
radicals in frozen myocardial tissue in the absence of
a spin trap. Quantification of the observed radicals has
shown the importance of oxygen23 and also the problems associated with the artifactual generation of
radicals during the preparation of the frozen tissue
samples.26
In conclusion, by using ESR spectroscopy, we have
demonstrated that free radicals are produced in significant amounts in the isolated rat heart during aerobic
reperfusion. The immediate extrapolation of these
results (obtained in an isolated heart) to the clinical
situation should be viewed with some caution due to the
absence of blood in our model system.
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FIGURE 3. Time course of PBN spin adduct
formation during aerobic and anoxic reperfusion of the ischemic rat heart. All hearts were
perfused with aerobic buffer for a 35-minute
stabilization period, prior to the onset of
ischemia, o, reperfusion with oxygenated buffer
(n = 6);*, initial reperfusion with anoxic buffer
(n = 5), switching to oxygenated buffer after JO
minutes (t = 60). The bars represent the standard errors of the means.
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Circulation Research
760
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KEY WORDS
fusion
free radicals • ESR spectroscopy
• reper-
Direct detection of free radicals in the reperfused rat heart using electron spin resonance
spectroscopy.
P B Garlick, M J Davies, D J Hearse and T F Slater
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Circ Res. 1987;61:757-760
doi: 10.1161/01.RES.61.5.757
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