Vol.
177,
June
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages 874-880
14, 1991
ESR
MEASUREMENT
Keizo
OF RADICAL
Takeshita,
Department of Health Showa University, Received
May 6,
CLEARANCE
Hideo
IN
Utsumi't
Chemistry, Hatanodai,
LUNG
OF
and Akira
MOUSE
WHOLE
Hamada
School of Pharmaceutical Shinagawa-ku, Tokyo 142,
Sciences, JAPAN
1991
hydroxy-TEMPO and carboxySUIWARY: Clearance of the nitroxide radicals, PROXYL, in whole-mouse lung was directly measured by --in vivo ESR. After injecting a nitroxide radical, distribution of the nitroxide radical all over the The ESR signal of hydroxy-TEMPO was reduced lung was confirmed by ESR imaging. in the lung and the clearance obeyed first-order kinetics, whereas the signal Comparison of the clearance rates of live of carboxy-PROXYL remained constant. and dead mice indicated the presence of 2 different clearance systems in the lung: loss of its paramagnetism in the lung, and transfer from alveolar to the 0 1991 AcademicPress, Inc. blood circulation system.
Hyperbaric cause
oxygen
lung
injuries
including sion
behavior Some
with enabled
in of
are
oxidants
great
usefulness
active
oxygens
ESR spectrometer
by the
and active
dioxide
suggested
that
lung
injury
using
lipids
after
gas inhalation
radicals,
oxygens
Recent
of --in
vivo
difficult (10).
in
nitrogen
--in
vivo
free
(1,2).
and tissue
and
ozone,
radicals,
This
conclu-
homogenates,
(1,3). experiments
and
To understand on radical
necessary.
measurement
is
have
may be involved
injury
an ESR technique. --in vivo
Reports
per-oxidation
lung
lung
gases,
experiments
of lipid of
pollutant
as edema.
on -~ in vitro
mechanism
the
oxygens,
on measurement the
such
active
is based
and
Thus,
are
development
radicals of
an L-band
of radicals
in whole
ESR, direct
detection
because nitroxide
of
insufficient radicals
*To whom correspondence should be addressed. Abbreviations used: hydroxy-TEMPO, 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl; PROXYL, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl. 0006-291X/91 $1.50 Copyight 0 1991 by Academic Press. Inc. All rights of reproduction in cur?: jbrm reserved.
874
and should
animals
ESR technique
has
(4-9).
Despite
the
radicals
or
of gas-phase sensitivity
are
commonly
be detectable
of the used
L-band
as probes
carboxy-
in
Vol.
in
177,
No.
vivo
ESR measurements.
radicals
2, 1991
are
suitable
and
macromolecules
the
biological In the
whole
BIOCHEMICAL
probes (11,12),
redox present
mouse
Recent
using
only
but
also
(14-16)
oxygen
and generation --in
vivo
of
in
live
nitroxide lipids
concentration active
compounds,
the
of membrane
(5,13),
oxygens
ESR measurement
in
(17).
the
lung
of
4-hydroxy-2,2,6,6and
(carboxy-PROXYL),
system
COMMUNICATIONS
that
dynamics
(hydroxy-TEMPO)
tetramethylpyrrolidine-1-oxyl clearance
the
monitor
nitroxide
tetramethylpiperidine-l-oxyl
radical
to
RESEARCH
demonstrated
to study
we performed the
BIOPHYSICAL
investigation
not
systems
study,
AND
3-carboxy-2,2,5,5-
as probes,
and
found
the
lungs.
MATERIALSANDMETHODS Reagents. Hydroxy-TEMPO and carboxy-PROXYL were purchased from Aldrich Chemical Co., Inc.. The radicals were dissolved in isotonic buffer (50 mM sodium phosphate buffer, pH 7.4, containing 0.6% sodium chloride) in 5 mM concentration. Other reagents were reagent grade. Animals. Female, 4 week old, ddY strain mice (15-18 g) were purchased from Nippon Bio-Supp. Center, Co., Ltd., Tokyo and used throughout these experiments. Injection radical mouse lung. Mice were anesthetized by -of nitroxide --into intramuscular injection of 40 ~1 Nembutal (Abbott Laboratories) or killed by administration of 100 pl Nembutal into the peritoneal cavity, and placed ventral side up on a Teflon plate (40 mm x 150 mm x 1 mm). After cervical incision, the chest was pressed gently to squeeze the remaining air from the a polyethylene tube (0.8 mm i.d., 1.4 mm o.d.) was inserted into a lung, trachea, and 0.9 ml of radical solution was then injected into the lung. The trachea was then closed tightly so the solution would not leak. Measurement with a JEOL JES-RE-3X spectrometer --of ESR. L-band ESR was measured equipped with a JEOL L-band ESR unit (ES-LBlA) and a loop-gap resonator (33 mm i.d. x 24 mm long; JEOL L-Horizontal cavity). ESR spectra were recorded at 1.3 GHz, 1 mW, 100 kHz field modulation, 0.2 mT modulation width. ESR imaging. The magnetic field gradient was 1.6 mT/cm. For each image, 18 spectra at angular increments of 10' of the gradient direction were obtained (1024 data points/spectrum, 0.013 mT/point). Reconstruction of the image was performed by the Filtered Back-Projection method. This system has been previously described in detail (18).
RESULTSANDDISCUSSION An aqueous and
the
spectra
L-band of
due
Fig.
1 shows
The
hyperfine
signal
of nitroxide
ESR spectrum
nitroxide
level
tumbling
solution
motion
was observed,
the
injected but
spectra
constants of
at
radical
to respiration, typical
compounds
was injected
breast to
distinct
was
nitroxide indicating
mT)
and
molecules
mouse lung
immediately.
The
mice
contained
a high
noise
spectra
were
obtained
in dead
mice.
and carboxy-PROXYL
sharp in
few molecules 875
recorded
the
living
of hydroxy-TEMPO (1.72
into
the
triplet aqueous in
lines phase.
the biological
in
dead
indicate
mice. rapid
No superimposed membrane.
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
a
to-
0
1
a
I
loo
2.6 min
l.OmT
0
5
10
15
0L--
20
10
5
Time
15
20
Cmln)
of hydroxy-TEMPO (a) and carboxy-PROXYL (b) Fin. 1. L-band ESR spectra injected into mouse lung. L-band ESR spectra were recorded at the breast after 0.9 ml of 5 mM nitroxide radicals dissolved in phosphate-buffered Time after injection is saline was injected into dead mouse lungs. indicated at the left of each spectrum. Fin. mouse
2. Clearance curve of hydroxy-TEMPO (a) and carboxy-PROXYL (b) in lung. The experimental details are described in the legend of Fig. 1.
The peak la).
height
of the
The decrease
peak-to-peak
in
line
semi-logarithmic
the
width
clearance
of the
rate
constant
calculated
was essentially ring
compounds
(19-21), the
same tendency in
the
performed
using
hydroxy-TEMPO.
Fig.
the
with
of images
the did
to and
structural system of
rat
not
carefully exactly
slope of
lung
in
tissue
of living
mouse
injection,
(because
of its
stability),
coincide 876
images to the
after with
those
was
in
5-membered
tissue
homogenate
(24).
We observed on their
(9).
after
removed
The
spectrum
compounds
radicals
(b)
the
first-
experiments
radical
of nitroxide
since in
carboxy-PROXYL
stable
the
spin,
of hydroxy-TEMPO.
the
excised
(Fig.
indicating
of three
effect
parallel
time
relation
2a),
The nitroxide
3 a and b demonstrate
ribs
(Fig. radical.
be relatively
nitroxide
(a) and
time
height
lb and 2b).
with
was a linear
nitroxide
peak
carboxy-PROXYL
perpendicular
mouse
shapes
site
There
clearance
The
(Fig.
circulation the
directions;
the
decreased
be due to decreased
against
of the
(22-24),
in the
To delineate was
height
(mean+S.D.). -
cells
should
change.
has been reported
isolated
clearance
not
from
constant
spectrum
height
did
of peak
kinetics
mine1
peak
plot
order
0.107+0.014
hydroxy-TEMPO
ESR imaging instead
of
from
two
of nitroxide body axis.
Fig.
3c shows
measurement.
Although
of the
the
lungs,
the clarity
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
a
b
Fig. 3. ESR image of nitroxide radical injected into mouse lung dissected mouse (c). ESR imaging was performed from two directions ml of 15 mM carboxy-PROXYL dissolved in phosphate-buffered injected into mouse lungs; from the ventral side (a)and from the body axis (b). After ESR measurement, the chest of the incised and the ribs were carefully removed (c).
of the
images
indicate
that
nitroxide
radical
(a, b) and after-O.9 saline was tail along mouse was
was distributed
all
over
the
lung. The
clearance
circulation from
were
the
times difference
the
To evaluate the
and the
a living
4).
Since
declined,
organ
was
heartbeat
two
phases phase
clearance
constants
was
than
2 times
min -I
The
and/or in the
radical
magnetic
field
linear,
II are
defined
to for
than
shown
in Table
as large
3 min
as that 877
the
between
dead
lungs
of living
mice,
The clearance
curve
the
point
inflection
measured
and
live
and
(Fig.
then
soon
I (up to 3 min after
injection). clearance
or dead.
was observed point
phase
after
l.The
from
6
decrease
convenience;
(later
arise
than
mouse was alive
was traced.
the
more
the
and an inflection
was vigorous
blood
(calculated
are
might
into
(I=+l)
in the
min -'
constants
whether
was injected
were
and 0.051
(9)
discrepancy
solution
not
and carboxy-PROXYL
respectively.These
difference
the
be 0.71
examined
at a low mouse
hydroxy-TEMPO
obtained.
hydroxy-TEMPO
injection)and
more
values
the
signal
in
to
in ref.8),
present in
of
reported
half-life
the
mice,
constants
The calculated constant
immediately
in after
phase death
I
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
100 -i 80 -' 60 I "m 40 B
30
z rd 9, ar
20
10
0
5
10
15 20
Time
(min)
curve of hydroxy-TEMPO injected Fig. 4. Clearance living mouse. The experimental details are described 1 except that the mouse was living.
(heartbeat-free should
result
one and/or in
the
3 min
after
amount
of
injected another
The
(i)
of
was next
radicals
into
radical
was detected
in the
no heartbeat.The
results
hydroxy-TEMPO
Table
1.
in
indicate
reported
that, into
the
Clearance
transfer
living
rapid
constants mouse
blood,
vessels
1.5
h after
Clearance Numbers in of experiments.
and
delivered
of hydroxy-TEMPO
hydroxy-TEMPO
in
0.22220.055
(3)
(Phase
II)
0.143+0.011
(3)
0.107+0.014
(3)
0.075+0.006
(3)
constants parentheses
are
presented
indicate
878
as
numbers
to mice part
I)
death
no nitroxide
mouse,
lung
death
mean+S.D.
into
eye of mice
was injected
clearance
a dead
a significant
but
(Phase
after
Immediately
the
lung,
k (min -l) Anesthetized
in
by diffusion
from
in a living
of
than
mouse
hydroxy-TEMPO
mouse
blood
living
lung
of
the
the
followed
hydroxy-TEMPO
was transferred have
We
into
after
a live
was collected
detected
blood
in
vessels
of
When blood
was
in
blood
The possibility
examined.
observed
activity
of hydroxy-TEMPO
nitroxide
domain.
clearance
clearance
system.
injection
rapid
higher
transfer
circulation vessels
with
from (ii)
blood
radical
state).
into the lungs of a in the legend of Fig.
of the to in
the
Vol.
177,
No.
mouse
2, 1991
circulation
radical
system
clearance
in
The clearance
Even
the
should
not
measured death
neglect
slowly is
in
organs.
cells
and
sliced
lung
This
the
is
redox
first
It
radicals
system is
in
phase
above.
The
measured
but
clearance
the
we rate
immediately in
II.
after
lung
itself
mouse.
et al.
(19)
homogenate
compared
the
nitroxide
was
quite
homogenate
also
reported
compared
for the
min-1
extremely
slow
to homogenates rate
compounds.
indicates
was slight.
was 0.0015+0.0024
reduction
different
strongly
that
radical
Our results
in the
lung, to
The
isolated
of
between
lung
dependence cells
importance
The present
has been what work
was observed
reflect
may offer
which know
clearance must
ESR measurement
lung.
9 experiments
in lung (24)
of lung
of
than
of
for
in
vivo
animals. time
interesting
in the
from
various
This
whole
whole
the
I and the
clearance,
activity
activity
Couet
al.
using
body. --In vivo
time.
the
the
that
clearance
reduction
on structure
with
of the
whole
the
et
tissue.
measurement
radical
mentioned than
phase
was observed
to the
was slower
radicals
Kveder
rate
between
heartbeat
possibility
homogenate.
tissues
reduction
may enhance
mouse.
Feeble
obtained
of nitroxide
other
in blood
was intermediate
that
COMMUNICATIONS
death. that
lung
RESEARCH
clearance
contribute
first
constant
(mean2S.D.) reduction
might
suggesting
noteworthy
The clearance
II
1).
death
after
BIOPHYSICAL
The rapid
of phase
the
l),
AND
of a living
(Table
1.5 h after
declines
lung
heartbeat
(Table
It
the
state poor
(9).
constant
heartbeat-free
lung
BIOCHEMICAL
aspects
in
the
of clearance
in
the
more important difficult
happens seems
directly
during
to be the
information to obtain the
first
action step
about
up to this of
free
in gaining
answer.
ACKNOWLEDGMENTS We thank Mr. Y. Saeda for experimental assistance and Dr. A. Simpson for editing the manuscript. This work was supported by the Scientific Research Promotion Fund from Japan Private School Promotion Foundation, a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
REFERENCES 1. Menzel, II, pp.
D.B. (1976) In Free Radicals in 181-202. Academic Press, New York
879
Biology (W.A. and London.
Pryor,
Ed.),
Vol.
Vol.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Doelman, C.J. and Bast, A, (1990)F ree Radical Biol. Med. 9, 381-400. Plaa,G.L., and Witschi,H. (1976) Annu.Rev.Pharmacol.Toxicol. 16, 125-141. Nishikawa, H., Fujii, H., and Berliner,L.J. (1985) J. Mag. Res. 62, 79-86. Subczynski, W-K., Lukiewicz, S., and Hyde, J.S. (1986) Magn. Reson. Med. 3, 747-754. Bacic, G., Nilges, M.J., Magin, R.L., Walczak, T., and Swartz, H.M. (1989) Mag. Reson. Med. 10, 266-272. Ishida, S., Kumashiro, H., Tsuchihashi, N., Ogata, T., Ono, M., Kamada, H-, and Yoshida, E. (1989) Phys. Med. Biol. 34, 1317-1323. Ferrari, M., Colacicchi, S., Gualtieri, G., Santini, M.T., and Sotgiu, A. (1990) Biochem. Biophys. Res. Commun. 166, 168-173. Utsumi, H., Muto, E., Masuda, S., and Hamada, A. (1990) Biochem. Biophys. Res. Commun. 172, 1342-1348. Utsumi, H., Masuda, S., Muto, E., and Hamada, A. In Oxidative Damage & Repair: Clinical, Biological and Medical Aspects (K.J.A. Davies, Ed.) Pergamon Press, Oxford, in press. Berliner, L.J. (1976) Spin Labeling. Theory and Applications. Academic Press, New York. Takeshita, K., Utsumi, H., and Hamada, A.(1987) Biophys. J. 52, 187-197. Morse II, P.D., and Swartz, H.M. (1985) Magn. Reson. Med. 2, 114-127. Chen, K., Glockner, J.F., Morse II, P.D., and Swartz, H.M. (1986) Biochemistry 28, 2496-2501. Utsumi, H., Shimakura, A., Kashiwagi, M., and Hamada, A. (1989) J. Biochem. 105, 239-244. Miura, Y., Utsumi, H., Kashiwagi, M., and Hamada, A. (1990) J. Biochem. 108. 516-518. Fujii, H. and Kakinuma, K.(1990)J. Biochem. 108, 295-296. Masumizu, T., Tatebe, T., Masuda, S., Muto, E., Utsumi, H., and Hamada, A. (1991) Jap. Mag. Reson. Med. (in Japanese)2, in press. Couet, W.R., Eriksson, U.G., Tozer, T.N., Tuck, L.D., Wesbey, G.E., Nitecki, D., and Brasch, R. C (1984) Pharm. Res. 1, 203-209. Couet, W.R., Brasch, R.C., Sosnovsky, G. and Tozer, T.N. (1985) Magn. Reson. Imaging 3, 83-88. Eriksson, U.G., Brasch, R.C., and Tozer, T.N. (1987) Drug Metab. Dispos. 15, 155-160. Swartz, H.M., Sentjurc, M., and Morse II, P.D. (1986) Biochim. Biophys. Acta 888, 82-90. Chen, K.and Swartz, H.M. (1988)B' iochim. Biophys. Acta 970, 270-277. Kveder,M., Sentjurc,M., and Schara,M. (1988) Magn.Reson.Med. 8, 241-247.
880
177,
June
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages 874-880
14, 1991
ESR
MEASUREMENT
Keizo
OF RADICAL
Takeshita,
Department of Health Showa University, Received
May 6,
CLEARANCE
Hideo
IN
Utsumi't
Chemistry, Hatanodai,
LUNG
OF
and Akira
MOUSE
WHOLE
Hamada
School of Pharmaceutical Shinagawa-ku, Tokyo 142,
Sciences, JAPAN
1991
hydroxy-TEMPO and carboxySUIWARY: Clearance of the nitroxide radicals, PROXYL, in whole-mouse lung was directly measured by --in vivo ESR. After injecting a nitroxide radical, distribution of the nitroxide radical all over the The ESR signal of hydroxy-TEMPO was reduced lung was confirmed by ESR imaging. in the lung and the clearance obeyed first-order kinetics, whereas the signal Comparison of the clearance rates of live of carboxy-PROXYL remained constant. and dead mice indicated the presence of 2 different clearance systems in the lung: loss of its paramagnetism in the lung, and transfer from alveolar to the 0 1991 AcademicPress, Inc. blood circulation system.
Hyperbaric cause
oxygen
lung
injuries
including sion
behavior Some
with enabled
in of
are
oxidants
great
usefulness
active
oxygens
ESR spectrometer
by the
and active
dioxide
suggested
that
lung
injury
using
lipids
after
gas inhalation
radicals,
oxygens
Recent
of --in
vivo
difficult (10).
in
nitrogen
--in
vivo
free
(1,2).
and tissue
and
ozone,
radicals,
This
conclu-
homogenates,
(1,3). experiments
and
To understand on radical
necessary.
measurement
is
have
may be involved
injury
an ESR technique. --in vivo
Reports
per-oxidation
lung
lung
gases,
experiments
of lipid of
pollutant
as edema.
on -~ in vitro
mechanism
the
oxygens,
on measurement the
such
active
is based
and
Thus,
are
development
radicals of
an L-band
of radicals
in whole
ESR, direct
detection
because nitroxide
of
insufficient radicals
*To whom correspondence should be addressed. Abbreviations used: hydroxy-TEMPO, 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl; PROXYL, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl. 0006-291X/91 $1.50 Copyight 0 1991 by Academic Press. Inc. All rights of reproduction in cur?: jbrm reserved.
874
and should
animals
ESR technique
has
(4-9).
Despite
the
radicals
or
of gas-phase sensitivity
are
commonly
be detectable
of the used
L-band
as probes
carboxy-
in
Vol.
in
177,
No.
vivo
ESR measurements.
radicals
2, 1991
are
suitable
and
macromolecules
the
biological In the
whole
BIOCHEMICAL
probes (11,12),
redox present
mouse
Recent
using
only
but
also
(14-16)
oxygen
and generation --in
vivo
of
in
live
nitroxide lipids
concentration active
compounds,
the
of membrane
(5,13),
oxygens
ESR measurement
in
(17).
the
lung
of
4-hydroxy-2,2,6,6and
(carboxy-PROXYL),
system
COMMUNICATIONS
that
dynamics
(hydroxy-TEMPO)
tetramethylpyrrolidine-1-oxyl clearance
the
monitor
nitroxide
tetramethylpiperidine-l-oxyl
radical
to
RESEARCH
demonstrated
to study
we performed the
BIOPHYSICAL
investigation
not
systems
study,
AND
3-carboxy-2,2,5,5-
as probes,
and
found
the
lungs.
MATERIALSANDMETHODS Reagents. Hydroxy-TEMPO and carboxy-PROXYL were purchased from Aldrich Chemical Co., Inc.. The radicals were dissolved in isotonic buffer (50 mM sodium phosphate buffer, pH 7.4, containing 0.6% sodium chloride) in 5 mM concentration. Other reagents were reagent grade. Animals. Female, 4 week old, ddY strain mice (15-18 g) were purchased from Nippon Bio-Supp. Center, Co., Ltd., Tokyo and used throughout these experiments. Injection radical mouse lung. Mice were anesthetized by -of nitroxide --into intramuscular injection of 40 ~1 Nembutal (Abbott Laboratories) or killed by administration of 100 pl Nembutal into the peritoneal cavity, and placed ventral side up on a Teflon plate (40 mm x 150 mm x 1 mm). After cervical incision, the chest was pressed gently to squeeze the remaining air from the a polyethylene tube (0.8 mm i.d., 1.4 mm o.d.) was inserted into a lung, trachea, and 0.9 ml of radical solution was then injected into the lung. The trachea was then closed tightly so the solution would not leak. Measurement with a JEOL JES-RE-3X spectrometer --of ESR. L-band ESR was measured equipped with a JEOL L-band ESR unit (ES-LBlA) and a loop-gap resonator (33 mm i.d. x 24 mm long; JEOL L-Horizontal cavity). ESR spectra were recorded at 1.3 GHz, 1 mW, 100 kHz field modulation, 0.2 mT modulation width. ESR imaging. The magnetic field gradient was 1.6 mT/cm. For each image, 18 spectra at angular increments of 10' of the gradient direction were obtained (1024 data points/spectrum, 0.013 mT/point). Reconstruction of the image was performed by the Filtered Back-Projection method. This system has been previously described in detail (18).
RESULTSANDDISCUSSION An aqueous and
the
spectra
L-band of
due
Fig.
1 shows
The
hyperfine
signal
of nitroxide
ESR spectrum
nitroxide
level
tumbling
solution
motion
was observed,
the
injected but
spectra
constants of
at
radical
to respiration, typical
compounds
was injected
breast to
distinct
was
nitroxide indicating
mT)
and
molecules
mouse lung
immediately.
The
mice
contained
a high
noise
spectra
were
obtained
in dead
mice.
and carboxy-PROXYL
sharp in
few molecules 875
recorded
the
living
of hydroxy-TEMPO (1.72
into
the
triplet aqueous in
lines phase.
the biological
in
dead
indicate
mice. rapid
No superimposed membrane.
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
a
to-
0
1
a
I
loo
2.6 min
l.OmT
0
5
10
15
0L--
20
10
5
Time
15
20
Cmln)
of hydroxy-TEMPO (a) and carboxy-PROXYL (b) Fin. 1. L-band ESR spectra injected into mouse lung. L-band ESR spectra were recorded at the breast after 0.9 ml of 5 mM nitroxide radicals dissolved in phosphate-buffered Time after injection is saline was injected into dead mouse lungs. indicated at the left of each spectrum. Fin. mouse
2. Clearance curve of hydroxy-TEMPO (a) and carboxy-PROXYL (b) in lung. The experimental details are described in the legend of Fig. 1.
The peak la).
height
of the
The decrease
peak-to-peak
in
line
semi-logarithmic
the
width
clearance
of the
rate
constant
calculated
was essentially ring
compounds
(19-21), the
same tendency in
the
performed
using
hydroxy-TEMPO.
Fig.
the
with
of images
the did
to and
structural system of
rat
not
carefully exactly
slope of
lung
in
tissue
of living
mouse
injection,
(because
of its
stability),
coincide 876
images to the
after with
those
was
in
5-membered
tissue
homogenate
(24).
We observed on their
(9).
after
removed
The
spectrum
compounds
radicals
(b)
the
first-
experiments
radical
of nitroxide
since in
carboxy-PROXYL
stable
the
spin,
of hydroxy-TEMPO.
the
excised
(Fig.
indicating
of three
effect
parallel
time
relation
2a),
The nitroxide
3 a and b demonstrate
ribs
(Fig. radical.
be relatively
nitroxide
(a) and
time
height
lb and 2b).
with
was a linear
nitroxide
peak
carboxy-PROXYL
perpendicular
mouse
shapes
site
There
clearance
The
(Fig.
circulation the
directions;
the
decreased
be due to decreased
against
of the
(22-24),
in the
To delineate was
height
(mean+S.D.). -
cells
should
change.
has been reported
isolated
clearance
not
from
constant
spectrum
height
did
of peak
kinetics
mine1
peak
plot
order
0.107+0.014
hydroxy-TEMPO
ESR imaging instead
of
from
two
of nitroxide body axis.
Fig.
3c shows
measurement.
Although
of the
the
lungs,
the clarity
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
a
b
Fig. 3. ESR image of nitroxide radical injected into mouse lung dissected mouse (c). ESR imaging was performed from two directions ml of 15 mM carboxy-PROXYL dissolved in phosphate-buffered injected into mouse lungs; from the ventral side (a)and from the body axis (b). After ESR measurement, the chest of the incised and the ribs were carefully removed (c).
of the
images
indicate
that
nitroxide
radical
(a, b) and after-O.9 saline was tail along mouse was
was distributed
all
over
the
lung. The
clearance
circulation from
were
the
times difference
the
To evaluate the
and the
a living
4).
Since
declined,
organ
was
heartbeat
two
phases phase
clearance
constants
was
than
2 times
min -I
The
and/or in the
radical
magnetic
field
linear,
II are
defined
to for
than
shown
in Table
as large
3 min
as that 877
the
between
dead
lungs
of living
mice,
The clearance
curve
the
point
inflection
measured
and
live
and
(Fig.
then
soon
I (up to 3 min after
injection). clearance
or dead.
was observed point
phase
after
l.The
from
6
decrease
convenience;
(later
arise
than
mouse was alive
was traced.
the
more
the
and an inflection
was vigorous
blood
(calculated
are
might
into
(I=+l)
in the
min -'
constants
whether
was injected
were
and 0.051
(9)
discrepancy
solution
not
and carboxy-PROXYL
respectively.These
difference
the
be 0.71
examined
at a low mouse
hydroxy-TEMPO
obtained.
hydroxy-TEMPO
injection)and
more
values
the
signal
in
to
in ref.8),
present in
of
reported
half-life
the
mice,
constants
The calculated constant
immediately
in after
phase death
I
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
100 -i 80 -' 60 I "m 40 B
30
z rd 9, ar
20
10
0
5
10
15 20
Time
(min)
curve of hydroxy-TEMPO injected Fig. 4. Clearance living mouse. The experimental details are described 1 except that the mouse was living.
(heartbeat-free should
result
one and/or in
the
3 min
after
amount
of
injected another
The
(i)
of
was next
radicals
into
radical
was detected
in the
no heartbeat.The
results
hydroxy-TEMPO
Table
1.
in
indicate
reported
that, into
the
Clearance
transfer
living
rapid
constants mouse
blood,
vessels
1.5
h after
Clearance Numbers in of experiments.
and
delivered
of hydroxy-TEMPO
hydroxy-TEMPO
in
0.22220.055
(3)
(Phase
II)
0.143+0.011
(3)
0.107+0.014
(3)
0.075+0.006
(3)
constants parentheses
are
presented
indicate
878
as
numbers
to mice part
I)
death
no nitroxide
mouse,
lung
death
mean+S.D.
into
eye of mice
was injected
clearance
a dead
a significant
but
(Phase
after
Immediately
the
lung,
k (min -l) Anesthetized
in
by diffusion
from
in a living
of
than
mouse
hydroxy-TEMPO
mouse
blood
living
lung
of
the
the
followed
hydroxy-TEMPO
was transferred have
We
into
after
a live
was collected
detected
blood
in
vessels
of
When blood
was
in
blood
The possibility
examined.
observed
activity
of hydroxy-TEMPO
nitroxide
domain.
clearance
clearance
system.
injection
rapid
higher
transfer
circulation vessels
with
from (ii)
blood
radical
state).
into the lungs of a in the legend of Fig.
of the to in
the
Vol.
177,
No.
mouse
2, 1991
circulation
radical
system
clearance
in
The clearance
Even
the
should
not
measured death
neglect
slowly is
in
organs.
cells
and
sliced
lung
This
the
is
redox
first
It
radicals
system is
in
phase
above.
The
measured
but
clearance
the
we rate
immediately in
II.
after
lung
itself
mouse.
et al.
(19)
homogenate
compared
the
nitroxide
was
quite
homogenate
also
reported
compared
for the
min-1
extremely
slow
to homogenates rate
compounds.
indicates
was slight.
was 0.0015+0.0024
reduction
different
strongly
that
radical
Our results
in the
lung, to
The
isolated
of
between
lung
dependence cells
importance
The present
has been what work
was observed
reflect
may offer
which know
clearance must
ESR measurement
lung.
9 experiments
in lung (24)
of lung
of
than
of
for
in
vivo
animals. time
interesting
in the
from
various
This
whole
whole
the
I and the
clearance,
activity
activity
Couet
al.
using
body. --In vivo
time.
the
the
that
clearance
reduction
on structure
with
of the
whole
the
et
tissue.
measurement
radical
mentioned than
phase
was observed
to the
was slower
radicals
Kveder
rate
between
heartbeat
possibility
homogenate.
tissues
reduction
may enhance
mouse.
Feeble
obtained
of nitroxide
other
in blood
was intermediate
that
COMMUNICATIONS
death. that
lung
RESEARCH
clearance
contribute
first
constant
(mean2S.D.) reduction
might
suggesting
noteworthy
The clearance
II
1).
death
after
BIOPHYSICAL
The rapid
of phase
the
l),
AND
of a living
(Table
1.5 h after
declines
lung
heartbeat
(Table
It
the
state poor
(9).
constant
heartbeat-free
lung
BIOCHEMICAL
aspects
in
the
of clearance
in
the
more important difficult
happens seems
directly
during
to be the
information to obtain the
first
action step
about
up to this of
free
in gaining
answer.
ACKNOWLEDGMENTS We thank Mr. Y. Saeda for experimental assistance and Dr. A. Simpson for editing the manuscript. This work was supported by the Scientific Research Promotion Fund from Japan Private School Promotion Foundation, a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
REFERENCES 1. Menzel, II, pp.
D.B. (1976) In Free Radicals in 181-202. Academic Press, New York
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Biology (W.A. and London.
Pryor,
Ed.),
Vol.
Vol.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
177,
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2, 1991
BIOCHEMICAL
AND
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Doelman, C.J. and Bast, A, (1990)F ree Radical Biol. Med. 9, 381-400. Plaa,G.L., and Witschi,H. (1976) Annu.Rev.Pharmacol.Toxicol. 16, 125-141. Nishikawa, H., Fujii, H., and Berliner,L.J. (1985) J. Mag. Res. 62, 79-86. Subczynski, W-K., Lukiewicz, S., and Hyde, J.S. (1986) Magn. Reson. Med. 3, 747-754. Bacic, G., Nilges, M.J., Magin, R.L., Walczak, T., and Swartz, H.M. (1989) Mag. Reson. Med. 10, 266-272. Ishida, S., Kumashiro, H., Tsuchihashi, N., Ogata, T., Ono, M., Kamada, H-, and Yoshida, E. (1989) Phys. Med. Biol. 34, 1317-1323. Ferrari, M., Colacicchi, S., Gualtieri, G., Santini, M.T., and Sotgiu, A. (1990) Biochem. Biophys. Res. Commun. 166, 168-173. Utsumi, H., Muto, E., Masuda, S., and Hamada, A. (1990) Biochem. Biophys. Res. Commun. 172, 1342-1348. Utsumi, H., Masuda, S., Muto, E., and Hamada, A. In Oxidative Damage & Repair: Clinical, Biological and Medical Aspects (K.J.A. Davies, Ed.) Pergamon Press, Oxford, in press. Berliner, L.J. (1976) Spin Labeling. Theory and Applications. Academic Press, New York. Takeshita, K., Utsumi, H., and Hamada, A.(1987) Biophys. J. 52, 187-197. Morse II, P.D., and Swartz, H.M. (1985) Magn. Reson. Med. 2, 114-127. Chen, K., Glockner, J.F., Morse II, P.D., and Swartz, H.M. (1986) Biochemistry 28, 2496-2501. Utsumi, H., Shimakura, A., Kashiwagi, M., and Hamada, A. (1989) J. Biochem. 105, 239-244. Miura, Y., Utsumi, H., Kashiwagi, M., and Hamada, A. (1990) J. Biochem. 108. 516-518. Fujii, H. and Kakinuma, K.(1990)J. Biochem. 108, 295-296. Masumizu, T., Tatebe, T., Masuda, S., Muto, E., Utsumi, H., and Hamada, A. (1991) Jap. Mag. Reson. Med. (in Japanese)2, in press. Couet, W.R., Eriksson, U.G., Tozer, T.N., Tuck, L.D., Wesbey, G.E., Nitecki, D., and Brasch, R. C (1984) Pharm. Res. 1, 203-209. Couet, W.R., Brasch, R.C., Sosnovsky, G. and Tozer, T.N. (1985) Magn. Reson. Imaging 3, 83-88. Eriksson, U.G., Brasch, R.C., and Tozer, T.N. (1987) Drug Metab. Dispos. 15, 155-160. Swartz, H.M., Sentjurc, M., and Morse II, P.D. (1986) Biochim. Biophys. Acta 888, 82-90. Chen, K.and Swartz, H.M. (1988)B' iochim. Biophys. Acta 970, 270-277. Kveder,M., Sentjurc,M., and Schara,M. (1988) Magn.Reson.Med. 8, 241-247.
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