BIOCHEMICAL
Vol. 74, No. 3, 1977
PHOTOSENSITIZATION RADICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
BY HEMATOPORPHYRIN: ESR EVIDEYCE
FOR FREE
INDUCTION IN UNSATURATED FATTY ACIDS AND FOR SINGLET OXYGEN PRODUCTION
Salvatore Department
of
Cannistraro
Atomic
B-4000 Received
and Albert
and Molecular SART-TILMAN
Van de Worst
Physics, par Lisge
Universitv 1
of LiPge
9elaium
December 20,1976
SUMMARY Hematoporphyrin ability to photoreact by type I and tyne II mechanisms was investigated in some model systems. At room temperature, visible irradiation of hematonorphvrin-unsaturated fatty acids and hematoporphyrin-cholesterol systems resulted in the Electron Spin Resonance (ESR) spectrum of the hematoporphyrin free radical. Triplet state hematoporphyrin is shown to be involved in the electron transfer from the lipid moiety . Moreover an ESR method to monitor the singlet oxvaen production by hematoporphyrin was used. B-carotene effect on both mechanisms (type I and type II) was tested. INTRODUCTION It tes
is
associated
membranes, the teins
generally
accepted
with
the oorphyrias
following
photoexcitation
presence and/or
of
oxygen
(l-6).
photoperoxidation
of membranalipids
are believed
damage (3-11).
Very
chemical
leading
events
photodynamic In the
Abbreviations: Fatty Acids; Copyright AU rights
that
process
little
of
from damage to cells
of porphvrin
molecules
in
of membrane nro-
of unsaturated
fatty
to be responsible is known about
the
to the photosensitization, some kind
is usually
1177
acids for
(UFA)
such a
primary
nhoto-
even if postulated.
protoporphvria,
ESR, Electron Spin HP, Hematoporphyrin; Press, Inc. form reserved.
results
of erythrocv-
Photooxidation
case of erythropoietic
0 1977 by Academic of reuroduction in any
photohemolvsis
Goldstein
Resonance; UFA, Unsaturated IO2 , singlet oxyuen.
a
Vol. 74, No. 3, 1977
and Harber rin
BIOCHEMICAL
(8) have postulated
results
porphyrins
semm to be able of
pletely
suitable
lipid
(102)
(15),
interact
tion
UFA (4,7),
of
action
In a recent
on the
by hematoporphyrin
mechanisms,
HP is
adsorbed
be favoured molecules takinq
type
can assume that HP-UFA would direct
its
a suitable durinq
com-
--in vitro
(4,
6-l-2
that
membrane
in the oxidaamino acids(4,7)
of erythrocytes al.(ZO)
are probably hence type
lipid
environment
almost
to cell
et --
interaction
relative
oxy-
by pornhyrins.
Nilsson II,
other
sinfflet
and certain
surface,
occurring
suqqest involved.
I reaction
between Alona
of
lipid:
model to
thus
study
would
sensitizer
this
solubility is
that
line
and
HP, one mixtures
an eventual
photosensitization
of
red
by HP. We report
HP-cholesterol
here
In all
observed.
Moreover
in
detectinq
the
systems,
perature.
nism
leadins
on the membrane.
local
constitute
interaction
cells
the
In the
suqqested
photohemolysis
(HP),
to red cells
account
I mechanism
effective
red cells
by the more direct
into
aqent
I and type
and acceptors
Indeed,
of porphyrins
(19)
of
both
to double
(6,12-14).
stronqly
cholesterol
in the photosensitizatiqon
induced
by a type
1 O2 has been postulated
of
study
transfer
has been shown to prevent,
16) and in vivo (17,18). This -could be the primary oxidation Intermediacy
of protoporchy-
peroxidation.
substrates
the photosensitizing
damage.
excitation
known as an extremely
B-carotene,
gen quencher
to
RESEARCH COMMUNICATIONS
electron
membrane producinq
the nresence
side,
that
in an oxygen-dependent
bond of cells
in
AND BIOPHYSICAL
various lO2 .
ESR results under
cases,
the
visible
B-carotene
is
deronstrated
effect
tested. 1178
from HP-UFA and
irradiation
photoreduced
hematoporphyrin
solvents
obtained
at room tem-
HP free
reaction by
on both
radical
by tyne an
II
was mecha-
l?SR method
mechanisms
was
of
BIOCHEMICAL
Vol. 74, No. 3, 1977
YATERIALS ---------__-
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AND METHODS
Clleic, linoleic and linolenic acids, linoleic acid methyl ester and all trans B-carotene were Sigma oroducts. Cholestehematoporohyrin IX dihvdrorol acetate y*Jas from Calhiochem, Methanol, ethanol and chloroform, all chloride from Fluka. 2,2,6,6for spectroscopic use, were nurchased from pterck. tetramethyl-oineridin was an Aldrich Furora nroduct. The liqht source for all irradiation exneriments was a 500 watt hiqh pressure mercury lamn(Osram). Cut-off filters (Jenear Glaswork Schott) were used to select the opportune wavelengths. ESR spectra were recorded at 25'C, by a Varian specand equinped with trometer E-3 type, with 100 kHz modulation a variable temperature accessory. All q-factor measurements were performed by graphical interpolation between the two centermost lines of Mq++. To promote solubility of HP in UFA, 10 ml of hematoporphyrin 5.10-5M in chloroform were added to one ml. of UFA (all UFA studied were liquid); then purified nitroqen was bubbled through until all chloroform was evaporated. Thus, HP resulted dissolved in UFA at an approximate concentration of 5.1Qe4M. HP-cholesterol solutions were prepared in chl roform; solutes were concentrated up respectively up to 4.10 3 Y and 1 M by bubblinq with N2. To perform experiments in deareated conditions, aliquots of solutions were placed in 3 mm ID quartz tubes and sealed after five cycles of freezing-thawinq under a vacuum of 10-5 Torr. Samples were irradiated in the cavity of the spectrometer through a collimatina lens at 15 cm by the lamn. Singlet oxygen production by HP was demonstrated by the ESR method set up by Lion et HP was dissolved in (21). -- al different organic solvents at a concentration of lo-5M; solutions were then bubbled with 02 for 15 min. (to avoid evaporation, 02 was bubbled throuqh the solvent before passina it over the sample). Finally 2,2,6,6-tetramethyl-piperidin was added at a concentration of 1.15.10-3M. A volume of 1.5 ml of such a solution was irradiated in a thermostatic quartz cuvette (18 2 1'C) under one atmosphere of air. Alicuots were taken in calibrated capillaries (Drummond microcaps, 50~1) before exposure to light and after different exposure times. ESR quantitative measurements were performed by comparison with a calibrated concentration of nitroxide in solution. RESULTS AND DISCUSSION Irradiation
at room temperature
and chloroform resulted
in the
was turned failed
off
It width
ESR spectrum
a hiqher consists
slightly
deqassed HP-UFA systems
of HP-cholesterol
the signal
to obtain
amplitude. to-peak
solutions
of
shown in Fig.
decayed resolution in a sinqle
varied
with
1179
very
with
light
(X>340nm)
la.
When the
quickly
(~1 set)
by lowerinq
the
line
whose a-value
the different
light and we
modulation and peak-
systemsstudied.
Vol. 74, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b)
Fig. 1. a) ESR spectrum of deareated HP-UFA and HP-cholesterol systems: b) same as (a) but in the presence of B-carotene (10m4K). Irradiation in the cavity, with A>340 nm at 25'C. Microwave power level: 10 mW. Modulation amplitude: 10 Gauss.
Table
1.
in the rated
y-values
and line
photoreaction fatty
acids
width
of
of hematoporphyrin
acid
HP + Linoleic
ac.
HP + Cholesterol
These values, agreement authors porphyrins
with (12-14),
methyl (in
which those
different
unsatu-
y-value
acid
HP + Linolenic
obtained
and cholesterol.
acid
HP + Linoleic
ESR signal
with
System
HP + Oleic
the
ester
chloroform)
2.0020+0.0002
5.520.5
2.0022+0.0001
5.7+0.3
2.0024+0.0002
5.920.5
2.0021~0.0001
5.7+0.3
2.0026+0.0002
6.2tO.5
have been resumed in Table found
for
free
who investigated
(HP included),
AH (Gauss)
porphyrin
1, are in excellent radical
the photoreduction
in the presence 1180
of various
by many of
several
reducing
BIOCHEMICAL
Vol. 74, No. 3, 1977
agents.
In the
of HP results form the roform it
present
in
in the
This
the
added to Sinqlet
postulated
(12,14).
ment of
sinqlet
effect
radical.
state
of
dicates
the
effect
is
of porphyrins
responsible
for
case,
out by the
while
since
B-carotene
and of other
of
The above observations
their direct
fact
triplet
that
photoreinvolve-
no oxygen-
emission,
quenches
electron
are consistent
in aqree-
reduced
the
triplet
photosensitizers
state
lb.
have been
O2 drastically
on the photoinduced
participation
be detected.
shown in Fiq.
on the HP fluorescence
(23)
signal,
(10 -4 M) had been pre-
in the present
et al. (221, --
chlorophyll
chlo-
process
could
as it
state
ruled
In addition,
to
Since
transfer
02, no signal
states
is
moiety,
system.
solutions,
However
lipid
show any detectable
same electron
as triplet
state
inhibitory
not
case when B-carotene
deareated
Cauzzo
ESR signal.
did
the
was encountered
ment with
the
free
as the excited
duction
photoexcitation
hematoporphyrin
of air,or
as well
that the
HP-cholesterol
was also
viouly
we can affirm from
that
In the presence
RESEARCH COMMUNICATIONS
transfer
of HP alone
can be inferred
occurs
case,
an electron
photoreduced
solution
AND BIOPHYSICAL
(24), transfer
in-
of HP. with
the
followinq
the
singlet
scheme:
'HP
hv ->
3HP*
+ L
->
Where 'HP* and triplet lesterol.
intersystem crossing
'HP *
excited Radical
'HP2
+
and 3HP* indicate
LG respectively
states of HP,while . L+ escaped detection,
recombination.
1181
, 3RP X
L represents probably
IJFA or choowinq
to
fast
BIOCHEMICAL
Vol. 74, No. 3, 1977
No correlation and the
was found
between
number of unsaturated
were compared
at the
Among the susceptible result
is
replaced
qroups
with
in
to predict
rules
in UFA or in cholesterol
the
Anyway results
constitute
leadinq
to
the
lipid
methyl out
OH groups; ester,
where the
free
the most important free
initiators
of
peroxidation
of
radicals
we are
the photo-
conclusion
emer-
are photoinduced
these
the
where OH
to
radicals
systems;
but
Thus at present
correspondinqly
is that
most
the intermediacy
of HP.
sites
in HP-UFA and HP-cholesterol could
radicals
UFA theoretically are the
qroup,
induced
from these
the
acid
able
ging
of
the photoreduction
of HP.
HP free
bonds of UFA, when they
process
are not
reduction
amount of
linoleic
by the methyl
OH groups
RESEARCH COMMUNICATIONS
same molarity.
functional
obtained
the
double
to a photoreducing
the
the
AND BIOPHYSICAL
very
reactive
radicalar
snecies
chain
and consequently
reaction
to membrane da-
mage. The method used to monitor by HP is based on the
high
the production
specificity
2,2,6,6-tetramethyl-piperidin of
the
oxydation
products
dicals
easily
nation
of nitroxide
tion
under
detectable
of HP in
direct
for
the presence
Experiments
to
Prior
irradiation
This
to
spectrum
an impurity
with radicals a slight
characterizes in the
commercial
via
amine, triplet
words,
is
its
irradiabear
solvents than
a
reported
radicals
and
340 nm. in Fiq.
was already
free
determi-
after should
ra-
HP.
longer
ESR signal
1182
amine
free
different
formation
oxygen
stabilization
induced
wavelengths
product:
tertiary
In other
in three
nitroxide
sinplet
form of nitroxide
of O2 and the
1O2 produced
samples were irradiated of nitroxide
the
the
and the
concentration
were performed
The rate
'02
by ESR (21).
radical
relationship
of
of
2.
observed. present
concentration
as
does
BIOCHEMICAL
Vol. 74, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.
>-
./ ./
)-
./ /
,-
J
1 2
4
6
6
10
Irradiation
timelminJ
Fig. 2. Nitroxide concentration versus light exposure duration HP (lo-5M) dissolved in : 0 chloroform A ethanol o methanol o chloroform + B-carotene. 2,2,6,6 tetramethyl-piperidin concentration: 1.15.10V3M. Insert: ESR spectra before (....) and after ( -) illumination (A>340 nm). Microwave power level: 2 ml*'. Vodulation amplitude: 2 Gauss. Temperature:25"C.
not
exceed
rate
of
This
could
0.3
'02
%a. HP dissolved
as compared
constant Adding
for
is
effect the
@-carotene
-5 10 M in chloroform as it
to HP dissolved
be due to the higher
and to a solvent rate
in chloroform
on the reaction to the
resulted
shown in Fig.
2.
in ethanol
O2-solubility rate of
decay
samples,
should
1183
or in methanol.
in chloroform of
10~ with
in the It
shows a higher
102 and/or
the
amine
on the
(26).
at a concentration
complete be noted
quenching that
the
(25)
of of
IO2 I
$-caro-
Vol. 74, No. 3, 1977
tene
quenching
ried
out
effect
as where
is quickly
does not
with
destruction
of oxygen
dene blue
about
Anderson B-carotene generated
its
of
et --
al.
the
the
lipid singlet
of
milieu, oxygen
ACKNOWLEDGEMENT A Euratom fellowship gratefully acknowledged.
it
above
at
of HP,
460 nm.
(A> 340 nm) resulted (10 -5M).
B-carotene (27)
who found
nm)
B-carotene
presence
absorption
In
in more
This
is
in
a considerable mixture
by photosensitization
due to of
tolui-
discharge. experimental
HP to interact and they
provide
producing
ability
held
(x>340
In addition
in chloroform-ethanol either
was car-
absorbs
in the
optical
or by radiofrequency
the capacity
the
irradiation
bleaching
In conclusion, bigously
(h>550 nm).
during
RESEARCH COMMUNICATIONS
when irradiation
range where
min irradiation
50 % optical
agreement
within
was encountered
by monitoriny
conditions,15
singlet
it
destroyed
as resulted
than
AND BIOPHYSICAL
in the wavelenqths
as well
our
BIOCHEMICAL
by one of
results
prove
by a radicalar a direct
unammechanism
information
of HP.
the
authors
(S.C.)
is
REFERENCES L.C., Fleischer, A., and Baer ,R.L. (1964) J.Amer. 1. Harber, Med. Ass., 189, 191-197. A.S., Harber,L.C.,Cook, J.S., and Baer, R.L. 2. Fleischer, (1966) J. Invest. Derm.,E, 505-509. 3. Ludwiy,G.D., Bilheimer, D., and Iverson, L. (1967) Clin. 284-285. Res. Abstr.,g, 4. Schothorst,A.A., Van Steveninck, J., Went, L.N., and Suurmond, D. (1972) Clin. Chim. Acta, 28, 41-49. 5. Schothorst, A.A., Van Steveninck, J., Went, L.N., and Suurmond, D. (1971) Clin. Chim. Acta, 33, 207-213. B.D., and Harber, L.C. (1971) Photochem. 6. Hsu,J. , Goldstein, Photobiol., 13, 67-77. 7. Schothorst, A.A., Van Steveninck, J., Went, L.N., and Suurmond, D. (1972) Clin. Chim. Acta, 2, 161-169. 8. Goldstein, B.D., and Harber, L.C. (1972) J.Clin. Invest., 51, 892-902. 9. De Goeij, A.F.P.M., and Van Steveninck, J. (1976) Clin. Chim. Acta , 68, 115-122. 10. De Goeij, A.F.P.M., and Van Steveninck, J. (1976) Clin. Chim. Acta, 71, 485-494. A.W. (1976) Biochem. Biophys. Res. Comm., 72, 11. Girottc 1367-1374.
1184
Vol. 74, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
12. Mauzerall, D., and Feher, G. (1964) Biochim. Biophys. Acta, 79, 430-432. 13. Esenstein, K.K., and Wang, J.H. (1969) J.Riol. Chem., 244, 1720-1728. 14. Harel, Y., Manassen, J., and Levanon, H. (1976) Photochem. Photobiol., 23, 337-341. C.S., Chang, Y.C., and Denny, F.W. (1970) J.Amer. 15. Foote, Chem. Sot., 92, 5216-5218. 16. Swanbeck, G., and Wennersten, G. (1973) Acta Dermat.Vener. (Stockholm), 53, 283-289. Nature, 203, 1092. 17. Mathews, M.M.(1964) M.M., Pathak, M.A., Fitzpatrick, T.B., Harber, 18. Mathews-Roth, L.C., and Kass, E.H. (1970) New Eng. J.Med., 282, 1231-1234. 19. Lamola, A.A., Yamane, T., and Trozzolo, A.M. (1973) Science, 179, 1131-1133. 20. Nilsson, R., Swanbeck, G., and Wennersten, G. (1975) Photothem. Photobiol., 22, 183-186. 21. Lion, Y., Delmelle,M., and Van de Vorst,A. (1976) Nature, 263, 442-443. 22. Cauzzo, G., Gennari, G., Jori,G., and Spikes, J.D. (1976) Photochem. Photobiol., (in press). 23. Fujimori, E., and Livingston, R. (1957) Nature, =,1036-1038. 24. Mathis, P., and Kleo, J. (1973) Photochem. Photobiol.,E, 343-346. (1972) Textbook of Physical Chemistry, p.695 25. Classtone,S. MC Millan Press LTD, London. 26. Young, R.H., Vehrly, K., and Martin, R.L. (1971) J.Amer. Chem. Sot., 93, 5774-5779. 27. Anderson, S.M., Krinsky, N.I., Stone, M.J., and Claqett,D.C. (1974) Photochem. Photobiol., 2, 65-69.
1185
Vol. 74, No. 3, 1977
PHOTOSENSITIZATION RADICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
BY HEMATOPORPHYRIN: ESR EVIDEYCE
FOR FREE
INDUCTION IN UNSATURATED FATTY ACIDS AND FOR SINGLET OXYGEN PRODUCTION
Salvatore Department
of
Cannistraro
Atomic
B-4000 Received
and Albert
and Molecular SART-TILMAN
Van de Worst
Physics, par Lisge
Universitv 1
of LiPge
9elaium
December 20,1976
SUMMARY Hematoporphyrin ability to photoreact by type I and tyne II mechanisms was investigated in some model systems. At room temperature, visible irradiation of hematonorphvrin-unsaturated fatty acids and hematoporphyrin-cholesterol systems resulted in the Electron Spin Resonance (ESR) spectrum of the hematoporphyrin free radical. Triplet state hematoporphyrin is shown to be involved in the electron transfer from the lipid moiety . Moreover an ESR method to monitor the singlet oxvaen production by hematoporphyrin was used. B-carotene effect on both mechanisms (type I and type II) was tested. INTRODUCTION It tes
is
associated
membranes, the teins
generally
accepted
with
the oorphyrias
following
photoexcitation
presence and/or
of
oxygen
(l-6).
photoperoxidation
of membranalipids
are believed
damage (3-11).
Very
chemical
leading
events
photodynamic In the
Abbreviations: Fatty Acids; Copyright AU rights
that
process
little
of
from damage to cells
of porphvrin
molecules
in
of membrane nro-
of unsaturated
fatty
to be responsible is known about
the
to the photosensitization, some kind
is usually
1177
acids for
(UFA)
such a
primary
nhoto-
even if postulated.
protoporphvria,
ESR, Electron Spin HP, Hematoporphyrin; Press, Inc. form reserved.
results
of erythrocv-
Photooxidation
case of erythropoietic
0 1977 by Academic of reuroduction in any
photohemolvsis
Goldstein
Resonance; UFA, Unsaturated IO2 , singlet oxyuen.
a
Vol. 74, No. 3, 1977
and Harber rin
BIOCHEMICAL
(8) have postulated
results
porphyrins
semm to be able of
pletely
suitable
lipid
(102)
(15),
interact
tion
UFA (4,7),
of
action
In a recent
on the
by hematoporphyrin
mechanisms,
HP is
adsorbed
be favoured molecules takinq
type
can assume that HP-UFA would direct
its
a suitable durinq
com-
--in vitro
(4,
6-l-2
that
membrane
in the oxidaamino acids(4,7)
of erythrocytes al.(ZO)
are probably hence type
lipid
environment
almost
to cell
et --
interaction
relative
oxy-
by pornhyrins.
Nilsson II,
other
sinfflet
and certain
surface,
occurring
suqqest involved.
I reaction
between Alona
of
lipid:
model to
thus
study
would
sensitizer
this
solubility is
that
line
and
HP, one mixtures
an eventual
photosensitization
of
red
by HP. We report
HP-cholesterol
here
In all
observed.
Moreover
in
detectinq
the
systems,
perature.
nism
leadins
on the membrane.
local
constitute
interaction
cells
the
In the
suqqested
photohemolysis
(HP),
to red cells
account
I mechanism
effective
red cells
by the more direct
into
aqent
I and type
and acceptors
Indeed,
of porphyrins
(19)
of
both
to double
(6,12-14).
stronqly
cholesterol
in the photosensitizatiqon
induced
by a type
1 O2 has been postulated
of
study
transfer
has been shown to prevent,
16) and in vivo (17,18). This -could be the primary oxidation Intermediacy
of protoporchy-
peroxidation.
substrates
the photosensitizing
damage.
excitation
known as an extremely
B-carotene,
gen quencher
to
RESEARCH COMMUNICATIONS
electron
membrane producinq
the nresence
side,
that
in an oxygen-dependent
bond of cells
in
AND BIOPHYSICAL
various lO2 .
ESR results under
cases,
the
visible
B-carotene
is
deronstrated
effect
tested. 1178
from HP-UFA and
irradiation
photoreduced
hematoporphyrin
solvents
obtained
at room tem-
HP free
reaction by
on both
radical
by tyne an
II
was mecha-
l?SR method
mechanisms
was
of
BIOCHEMICAL
Vol. 74, No. 3, 1977
YATERIALS ---------__-
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AND METHODS
Clleic, linoleic and linolenic acids, linoleic acid methyl ester and all trans B-carotene were Sigma oroducts. Cholestehematoporohyrin IX dihvdrorol acetate y*Jas from Calhiochem, Methanol, ethanol and chloroform, all chloride from Fluka. 2,2,6,6for spectroscopic use, were nurchased from pterck. tetramethyl-oineridin was an Aldrich Furora nroduct. The liqht source for all irradiation exneriments was a 500 watt hiqh pressure mercury lamn(Osram). Cut-off filters (Jenear Glaswork Schott) were used to select the opportune wavelengths. ESR spectra were recorded at 25'C, by a Varian specand equinped with trometer E-3 type, with 100 kHz modulation a variable temperature accessory. All q-factor measurements were performed by graphical interpolation between the two centermost lines of Mq++. To promote solubility of HP in UFA, 10 ml of hematoporphyrin 5.10-5M in chloroform were added to one ml. of UFA (all UFA studied were liquid); then purified nitroqen was bubbled through until all chloroform was evaporated. Thus, HP resulted dissolved in UFA at an approximate concentration of 5.1Qe4M. HP-cholesterol solutions were prepared in chl roform; solutes were concentrated up respectively up to 4.10 3 Y and 1 M by bubblinq with N2. To perform experiments in deareated conditions, aliquots of solutions were placed in 3 mm ID quartz tubes and sealed after five cycles of freezing-thawinq under a vacuum of 10-5 Torr. Samples were irradiated in the cavity of the spectrometer through a collimatina lens at 15 cm by the lamn. Singlet oxygen production by HP was demonstrated by the ESR method set up by Lion et HP was dissolved in (21). -- al different organic solvents at a concentration of lo-5M; solutions were then bubbled with 02 for 15 min. (to avoid evaporation, 02 was bubbled throuqh the solvent before passina it over the sample). Finally 2,2,6,6-tetramethyl-piperidin was added at a concentration of 1.15.10-3M. A volume of 1.5 ml of such a solution was irradiated in a thermostatic quartz cuvette (18 2 1'C) under one atmosphere of air. Alicuots were taken in calibrated capillaries (Drummond microcaps, 50~1) before exposure to light and after different exposure times. ESR quantitative measurements were performed by comparison with a calibrated concentration of nitroxide in solution. RESULTS AND DISCUSSION Irradiation
at room temperature
and chloroform resulted
in the
was turned failed
off
It width
ESR spectrum
a hiqher consists
slightly
deqassed HP-UFA systems
of HP-cholesterol
the signal
to obtain
amplitude. to-peak
solutions
of
shown in Fig.
decayed resolution in a sinqle
varied
with
1179
very
with
light
(X>340nm)
la.
When the
quickly
(~1 set)
by lowerinq
the
line
whose a-value
the different
light and we
modulation and peak-
systemsstudied.
Vol. 74, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b)
Fig. 1. a) ESR spectrum of deareated HP-UFA and HP-cholesterol systems: b) same as (a) but in the presence of B-carotene (10m4K). Irradiation in the cavity, with A>340 nm at 25'C. Microwave power level: 10 mW. Modulation amplitude: 10 Gauss.
Table
1.
in the rated
y-values
and line
photoreaction fatty
acids
width
of
of hematoporphyrin
acid
HP + Linoleic
ac.
HP + Cholesterol
These values, agreement authors porphyrins
with (12-14),
methyl (in
which those
different
unsatu-
y-value
acid
HP + Linolenic
obtained
and cholesterol.
acid
HP + Linoleic
ESR signal
with
System
HP + Oleic
the
ester
chloroform)
2.0020+0.0002
5.520.5
2.0022+0.0001
5.7+0.3
2.0024+0.0002
5.920.5
2.0021~0.0001
5.7+0.3
2.0026+0.0002
6.2tO.5
have been resumed in Table found
for
free
who investigated
(HP included),
AH (Gauss)
porphyrin
1, are in excellent radical
the photoreduction
in the presence 1180
of various
by many of
several
reducing
BIOCHEMICAL
Vol. 74, No. 3, 1977
agents.
In the
of HP results form the roform it
present
in
in the
This
the
added to Sinqlet
postulated
(12,14).
ment of
sinqlet
effect
radical.
state
of
dicates
the
effect
is
of porphyrins
responsible
for
case,
out by the
while
since
B-carotene
and of other
of
The above observations
their direct
fact
triplet
that
photoreinvolve-
no oxygen-
emission,
quenches
electron
are consistent
in aqree-
reduced
the
triplet
photosensitizers
state
lb.
have been
O2 drastically
on the photoinduced
participation
be detected.
shown in Fiq.
on the HP fluorescence
(23)
signal,
(10 -4 M) had been pre-
in the present
et al. (221, --
chlorophyll
chlo-
process
could
as it
state
ruled
In addition,
to
Since
transfer
02, no signal
states
is
moiety,
system.
solutions,
However
lipid
show any detectable
same electron
as triplet
state
inhibitory
not
case when B-carotene
deareated
Cauzzo
ESR signal.
did
the
was encountered
ment with
the
free
as the excited
duction
photoexcitation
hematoporphyrin
of air,or
as well
that the
HP-cholesterol
was also
viouly
we can affirm from
that
In the presence
RESEARCH COMMUNICATIONS
transfer
of HP alone
can be inferred
occurs
case,
an electron
photoreduced
solution
AND BIOPHYSICAL
(24), transfer
in-
of HP. with
the
followinq
the
singlet
scheme:
'HP
hv ->
3HP*
+ L
->
Where 'HP* and triplet lesterol.
intersystem crossing
'HP *
excited Radical
'HP2
+
and 3HP* indicate
LG respectively
states of HP,while . L+ escaped detection,
recombination.
1181
, 3RP X
L represents probably
IJFA or choowinq
to
fast
BIOCHEMICAL
Vol. 74, No. 3, 1977
No correlation and the
was found
between
number of unsaturated
were compared
at the
Among the susceptible result
is
replaced
qroups
with
in
to predict
rules
in UFA or in cholesterol
the
Anyway results
constitute
leadinq
to
the
lipid
methyl out
OH groups; ester,
where the
free
the most important free
initiators
of
peroxidation
of
radicals
we are
the photo-
conclusion
emer-
are photoinduced
these
the
where OH
to
radicals
systems;
but
Thus at present
correspondinqly
is that
most
the intermediacy
of HP.
sites
in HP-UFA and HP-cholesterol could
radicals
UFA theoretically are the
qroup,
induced
from these
the
acid
able
ging
of
the photoreduction
of HP.
HP free
bonds of UFA, when they
process
are not
reduction
amount of
linoleic
by the methyl
OH groups
RESEARCH COMMUNICATIONS
same molarity.
functional
obtained
the
double
to a photoreducing
the
the
AND BIOPHYSICAL
very
reactive
radicalar
snecies
chain
and consequently
reaction
to membrane da-
mage. The method used to monitor by HP is based on the
high
the production
specificity
2,2,6,6-tetramethyl-piperidin of
the
oxydation
products
dicals
easily
nation
of nitroxide
tion
under
detectable
of HP in
direct
for
the presence
Experiments
to
Prior
irradiation
This
to
spectrum
an impurity
with radicals a slight
characterizes in the
commercial
via
amine, triplet
words,
is
its
irradiabear
solvents than
a
reported
radicals
and
340 nm. in Fiq.
was already
free
determi-
after should
ra-
HP.
longer
ESR signal
1182
amine
free
different
formation
oxygen
stabilization
induced
wavelengths
product:
tertiary
In other
in three
nitroxide
sinplet
form of nitroxide
of O2 and the
1O2 produced
samples were irradiated of nitroxide
the
the
and the
concentration
were performed
The rate
'02
by ESR (21).
radical
relationship
of
of
2.
observed. present
concentration
as
does
BIOCHEMICAL
Vol. 74, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.
>-
./ ./
)-
./ /
,-
J
1 2
4
6
6
10
Irradiation
timelminJ
Fig. 2. Nitroxide concentration versus light exposure duration HP (lo-5M) dissolved in : 0 chloroform A ethanol o methanol o chloroform + B-carotene. 2,2,6,6 tetramethyl-piperidin concentration: 1.15.10V3M. Insert: ESR spectra before (....) and after ( -) illumination (A>340 nm). Microwave power level: 2 ml*'. Vodulation amplitude: 2 Gauss. Temperature:25"C.
not
exceed
rate
of
This
could
0.3
'02
%a. HP dissolved
as compared
constant Adding
for
is
effect the
@-carotene
-5 10 M in chloroform as it
to HP dissolved
be due to the higher
and to a solvent rate
in chloroform
on the reaction to the
resulted
shown in Fig.
2.
in ethanol
O2-solubility rate of
decay
samples,
should
1183
or in methanol.
in chloroform of
10~ with
in the It
shows a higher
102 and/or
the
amine
on the
(26).
at a concentration
complete be noted
quenching that
the
(25)
of of
IO2 I
$-caro-
Vol. 74, No. 3, 1977
tene
quenching
ried
out
effect
as where
is quickly
does not
with
destruction
of oxygen
dene blue
about
Anderson B-carotene generated
its
of
et --
al.
the
the
lipid singlet
of
milieu, oxygen
ACKNOWLEDGEMENT A Euratom fellowship gratefully acknowledged.
it
above
at
of HP,
460 nm.
(A> 340 nm) resulted (10 -5M).
B-carotene (27)
who found
nm)
B-carotene
presence
absorption
In
in more
This
is
in
a considerable mixture
by photosensitization
due to of
tolui-
discharge. experimental
HP to interact and they
provide
producing
ability
held
(x>340
In addition
in chloroform-ethanol either
was car-
absorbs
in the
optical
or by radiofrequency
the capacity
the
irradiation
bleaching
In conclusion, bigously
(h>550 nm).
during
RESEARCH COMMUNICATIONS
when irradiation
range where
min irradiation
50 % optical
agreement
within
was encountered
by monitoriny
conditions,15
singlet
it
destroyed
as resulted
than
AND BIOPHYSICAL
in the wavelenqths
as well
our
BIOCHEMICAL
by one of
results
prove
by a radicalar a direct
unammechanism
information
of HP.
the
authors
(S.C.)
is
REFERENCES L.C., Fleischer, A., and Baer ,R.L. (1964) J.Amer. 1. Harber, Med. Ass., 189, 191-197. A.S., Harber,L.C.,Cook, J.S., and Baer, R.L. 2. Fleischer, (1966) J. Invest. Derm.,E, 505-509. 3. Ludwiy,G.D., Bilheimer, D., and Iverson, L. (1967) Clin. 284-285. Res. Abstr.,g, 4. Schothorst,A.A., Van Steveninck, J., Went, L.N., and Suurmond, D. (1972) Clin. Chim. Acta, 28, 41-49. 5. Schothorst, A.A., Van Steveninck, J., Went, L.N., and Suurmond, D. (1971) Clin. Chim. Acta, 33, 207-213. B.D., and Harber, L.C. (1971) Photochem. 6. Hsu,J. , Goldstein, Photobiol., 13, 67-77. 7. Schothorst, A.A., Van Steveninck, J., Went, L.N., and Suurmond, D. (1972) Clin. Chim. Acta, 2, 161-169. 8. Goldstein, B.D., and Harber, L.C. (1972) J.Clin. Invest., 51, 892-902. 9. De Goeij, A.F.P.M., and Van Steveninck, J. (1976) Clin. Chim. Acta , 68, 115-122. 10. De Goeij, A.F.P.M., and Van Steveninck, J. (1976) Clin. Chim. Acta, 71, 485-494. A.W. (1976) Biochem. Biophys. Res. Comm., 72, 11. Girottc 1367-1374.
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Vol. 74, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
12. Mauzerall, D., and Feher, G. (1964) Biochim. Biophys. Acta, 79, 430-432. 13. Esenstein, K.K., and Wang, J.H. (1969) J.Riol. Chem., 244, 1720-1728. 14. Harel, Y., Manassen, J., and Levanon, H. (1976) Photochem. Photobiol., 23, 337-341. C.S., Chang, Y.C., and Denny, F.W. (1970) J.Amer. 15. Foote, Chem. Sot., 92, 5216-5218. 16. Swanbeck, G., and Wennersten, G. (1973) Acta Dermat.Vener. (Stockholm), 53, 283-289. Nature, 203, 1092. 17. Mathews, M.M.(1964) M.M., Pathak, M.A., Fitzpatrick, T.B., Harber, 18. Mathews-Roth, L.C., and Kass, E.H. (1970) New Eng. J.Med., 282, 1231-1234. 19. Lamola, A.A., Yamane, T., and Trozzolo, A.M. (1973) Science, 179, 1131-1133. 20. Nilsson, R., Swanbeck, G., and Wennersten, G. (1975) Photothem. Photobiol., 22, 183-186. 21. Lion, Y., Delmelle,M., and Van de Vorst,A. (1976) Nature, 263, 442-443. 22. Cauzzo, G., Gennari, G., Jori,G., and Spikes, J.D. (1976) Photochem. Photobiol., (in press). 23. Fujimori, E., and Livingston, R. (1957) Nature, =,1036-1038. 24. Mathis, P., and Kleo, J. (1973) Photochem. Photobiol.,E, 343-346. (1972) Textbook of Physical Chemistry, p.695 25. Classtone,S. MC Millan Press LTD, London. 26. Young, R.H., Vehrly, K., and Martin, R.L. (1971) J.Amer. Chem. Sot., 93, 5774-5779. 27. Anderson, S.M., Krinsky, N.I., Stone, M.J., and Claqett,D.C. (1974) Photochem. Photobiol., 2, 65-69.
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