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Epoxidation of cholesterol by hepatic microsomal lipid hydroperoxides
Vol. 108, No. 2, 1982
6lOCHEMlCAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
30, 1982
September
EPOXIDATION
Pages 724-730
OF CHOLESTEROL BY HEPATIC MICROSOMAL LIPID
Tadashi Laboratory
Watabe,
Masakazu
of Drug Metabolism
Tokyo College Received
13,
and Atsushi
& Toxicology,
of Pharmacy,
August
Isobe,
Horinouchi,
HYDROPEROXIDES
Tsubaki
Department
of Hygienic
Hachioji-shi,
Chemistry
Tokyo 192-03,
Japan
1982
SUMMARY: [1,2-3HlCholesterol was epoxidized to radioactive cholesterol a- and a-epoxides (5,6a-epoxy-5aand 5,68-epoxy-5a-cholestan-3a-01s) in the ratio 1:4 by hepatic microsomal lipid hydroperoxides (MsOOH, 1 mM as active oxygen) in the presence of ferrous ion. MsOOH could be replaced by methyl linoleate hydroperoxides (MOOH) under the same conditions although the latter was less effective than the former. None of cumene hydroperoxide, t-butyl hydroperoxide, and hydrogen peroxide was an effective oxidant even at 10 mM. Neither ADP nor EDTA had an effect on the epoxidation of cholesterol by MsOOH as well as by MOOH. Ferrous ion could not be replaced by ferric ion in the hydroperoxide-mediated epoxidation. Cyanide anion potentially inhibited the reaction.
INTRODUCTION Cholesterol
a-epoxide
has been suggested mice irradiated
to be closely
related
by ultraviolet
hyperpressure cancer
(5,6a-epoxy-5a-cholestan-3s-011,
(2)
ray
and Wolman's
have been demonstrated
to skin
(11 as well
disease
tumorigenesis
larger
5a-cholestane-38,5,6a-triol,
in their
feces
not only
covalently
DNA (51,
but also
aberrations
to isolated
!G) and malignant
Cholesterol
a-epoxide
transformations is
found
(5,6s-epoxy-5a-cholestan-38-01) in various
tissues
have demonstrated
hepatic
microsomes
by the
addition
increasing chelating monoxide lipid
that
91,
in the presence
of a ferrous
microsomal (121,
strongly
hydroperoxides
suggesting in viva
as well
0
I982
by Academic Press, Inc. in any form reserved.
of reproduction
The steroid
of cells the
complex
colon epoxide
chromosome
in culture.
isomeric
human (3).
with
with
hydrolytic
induces
s-epoxide constituents
The previous
of cholesterol
is mediated system,
concomitant
by
accelerated
formation
of
and potentially inhibited by the metalformation of the dialdehyde (10-12).
of cholesterol
is
that
by reaction
it
occurs
as in vitro.
0006-291X/82/180724-07$01.00/0 Copyright All rjghts
(4).
of an NADPH-generating
ion-ADP
epoxidation
of its
1 to 2-4 as normal
including
epoxidation
amounts of malondialdehyde, agent, EDTA, with suppressed
The hepatic
with
in the ratio
of mammals (8,
workers
(7)
together
in hairless
patients
amounts
product,
binds
steroid,
as to hypercholesterolemic In addition,
(3).
to excrete
a toxic
724
not
Actually,
inhibited with
by carbon microsomal
the ratio
of
Vol. 108, No. 2, 1982 cholesterol
a- and s-epoxides
be very close sterol
BIOCHEMICAL
to that
in vitro
(13).
has been available as true
oxidants
investigation
for
present
observed
provides with
requirement
of ferrous
the first
microsomal ion
hepatic
it
should
participation
in the microsomal
cholesterol
in rat
with
However, the
AND BIOPHYSICAL
lipid
liver
RESEARCH COMMUNICATIONS has been demonstrated
microsomal be noticed
epoxidation that
of microsomal
cholesterol direct
evidence
hydroperoxides
as an essential
for
the
of chole-
no direct
lipid
epoxidation.
to
evidence
hydroperoxides
The present epoxidation of
(MsOOH) and also
for
the
factor.
MATERIALS AND METHODS Materials [1,2-3HlCholesterol (43 Ci/mmol) was purchased from Radiochemical Centre, Amersham, diluted with unlabeled cholesterol, and purified before use by HPLC on a Lichrosorb RP-18 ODS column in methanol. Adenosine 5'-diphosphate (ADP), EDTA, and methylene blue were obtained from Wako Pure Chemical Industries, Ltd., Osaka, and cumene hydroperoxide (98% pure as active oxygen) from Nakarai Chemical Co., Kyoto. Cholesterol a-epoxide (141, B-epoxide (151, t-butyl hydroperoxide (99% pure as active oxygen) (161, and methyl linoleate hydroperoxides (MDOH, 95% pure as active oxygen) (171 were synthesized by the previous methods. Microsomal lipid hydroperoxides (MsOOH) were prepared as follows: a suspension of microsomes (protein content: 540 mg by the Lowry method (181) from male young adult Wistar rat liver (30 g) in 0.1 M phosphate buffer, pH 7.4 (90 ml), was shaken with a mixture of chloroform and methanol (2:l v/v, 500 ml 1. The residue obtained on the evaporation of the solvent from the organic phase separated by centrifugation was suspended in ethanol (200 ml). To the ethanolic solution was added methylene blue (0.11 mM) after the removal of undissolved materials by centrifugation. The mixture was vigorously aerated at 15" for 6 hr in light of a tungsten lamp (300 W) and then condensed in mew at 15" to 50 ml. The condensate was shaken with ether and water (100 ml each). The organic layer separated was washed twice with water (0.5 vol. each), dried over anhydrous sodium sulfate, and condensed to 10 ml. The final condensate had 35 vmol/ml of active oxygen and was able to be stored without any appreciable loss of active oxygen at -20" in dark at least for 2 months. Active oxygen of the hydroperoxides was determined by iodometry as previously reported (19). Incubation of Radioactive Cholesterol A typical incubation system was made as follows: to a solution of 13H1cholesterol (1 PCi, 0.05 pmol) and a hydroperoxide (1 pmol as active oxygen) in methanol (0.1 ml) containing 0.5% Tween 80 was added a solution of ADP (1 pmol) in 0.1 M phosphate buffer, pH 7.4 (0.5 ml). The mixture was preincubated for 5 min at 37", and the reaction was started by the addition of an aqueous solution (0.4 ml) of ferrous sulfate (1 pmol). Other ingredients when used were dissolved in water and added to the incubation mixture. Incubations were carried out at 37" for 60 min. High Pressure Liquid Chromatography (HPLC) HPLC was carried out for the separation of cholesterol epoxides on a LDC model Constametric II high pressure liquid chromatograph equipped with a RI monitor and a Lichrosorb RP-18 ODS column (Merck Co., Darmstadt, 5 p in particle size, 4 x 150 mm). A silica 4 x 300 mm) was used for the column (Lichrosorb SI-60, 5 P in particle size, determination of radiochemical purity of the epoxides isolated on the ODS The ODS and silica columns were eluted at 20" with 1 and 2 ml/min column. developing solvents, respectively. Determination of Epoxides Radioactive cholesterol Q- and s-epoxides formed from tritirated cholesterol by reactions with hydroperoxides were separated by HPLC and determined by the reverse isotope dilution method, including the scintillation counting of their radioactivities and the 725
Vol. 108, No. 2, 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
determination of carrier steroids by GLC as follows: from the reaction mixture were extracted radioactive epoxides into peroxide-free ether (2 ml) containing (I- and a-epoxides (0.5 mg each) in the presence of a unlabeled cholesterol saturating amount of sodium chloride. The solvent was evaporated in a nitrogen stream from the organic phase separated. The residue obtained was dissolved in a mixture of methanol and ethanol (10:3, 0.2 ml) and immediately subjected to reverse partition HPLC carried out in the same solvent mixture to collect the epoxides appearing at 4.7-5.6 min; under these conditions, cholesterol was eluted at 9.8 min. The eluate corresponding to the epoxide fraction was redissolved in methanol (0.2 ml) after the evaporation of the solvent in nitrogen. The methanolic solution was subjected to HPLC carried out on the same column in methanol; under these conditions cholesterol a- and B-epoxides were eluted at 8.0 and 6.4 min, respectively. The epoxide fractions were separately collected, and their radioactivity to carrier ratios were measured by the previously reported method (12). RESULTS AND DISCUSSION Epoxidation
of
[3H]Cholesterol
[1,2-3H]Cholesterol ferrous
ion
was incubated
and ADP in an aqueous
cholesterol
epoxides
formed
a- and a-epoxides epoxides
by Hepatic
were
with
medium,
were
as carriers
Microsomal rat
for
the
reverse
separated
from the
an ODS column
in methanol
containing
ethanol
4.7-5.6
The
min.
homogeneity retention
of the separated times
combination
to carrier
of the
and after Per cent
epoxides ion
epoxide
in the ratio
hepatic
microsomal linoleate
epoxidation was very t-butyl
ion
of
very
separated
with
methanol.
The
by HPLC on
fraction
eluted
at 8.0
and 6.4 min,
by HPLC on a silica
11.3
and 13.5 min,
with
with
at
Radiochemical column;
respectively.
determined
GLC (121,
were
the specimens
similar
to the radioactive
by a
unchanged
separated
was 1.6% in the
was yielded
to that (1.3%)
hydroperoxides
presence
preferentially
of the
previously
of cholesterol
(0.34
(MOOH) were one half The a- to B-ratio
of the
of ferrous to the a-
demonstrated PM) in the presence system
as effective epoxides
(12).
as MsOOH for formed
by MOOH
to the case of MsOOH. oxygen
and hydrogen under
the
However, none of cumene hydroperoxide, peroxide was an effective oxidant even at
same incubation
conditions.
Factors Influencing Cholesterol Epoxidation by Lipid Hydroperoxides Lipid hydroperoxidation in hepatic microsomes has been well recognized to be remarkably
by
a- and a-
mM), ADP (1 mM), and an NADPH-generating
hydroperoxide,
10 mM as active
oxygen)
The 6-epoxide
of cholesterol. similar
column
[3H]cholesterol
epoxidation
(0.2
method.
HPLC.
conversion
I).
well
method
cholesterol
substrate
a single
of
Radioactive
unlabeled
of the a- and 6-epoxides, counting
by MsOOH (1 mM as active
of ferrous
were
system).
dilution
was confirmed
HPLC on the adsorbent
and ADP (Table
Methyl
ratios
scintillation
the 2nd reverse partition
isotope
were then
epoxides
with
into
(MsOOH)
MsOOH in the presence
radioactive
by the elution
of the a- and a-epoxides
The radioactivity before
and s-epoxides
a-
on an ODS column
Hydroperoxides
(complete
together
imnediately
respectively,
liver
pH 7.4
extracted
Lipid
accelerated
either
by ferric
126
ion
or by ferrous
ion
in the
Vol. 108. No. 2, 1982 Table
BIOCHEMICAL
I.
Epoxidation
of
AND BIOPHYSICAL
Cholesterol
Concentration active oxygen (mM)
Hydroperoxide
Microsomal lipid hydroperoxide
(MsOOH)
Methyl linoleate hydroperoxide
(MOOH)
RESEARCH COMMUNICATIONS
by Various
of
Hydroperoxides
Radioactive products formed (%l a-Epoxide s-Epoxide
s- /a-Epoxides
1
0.31
1.33
4.3
1
0.19
0.76
4.0
Cumene hydroperoxide
1 10
0.00 0.03
0.03 0.16
t-Duty1 hydroperoxide
1 10
0.01 0.02
0.04 0.08
Hydrogen
1 10
0.00 0.02
0.01 0.02
peroxide
The reaction mixture contained ADP (1 mM), FeS04 (1 mM), [1,2-'HIcholesterol (1 PCi, 50 PM), Tween 80 (0.05%), and the hydroperoxide (1 mM as active in 0.05 M phosphate buffer, pH 7.4.
presence
of NADPH and ADP and potentially
concentrations
of EDTA (201.
been
to
suggested
activation
be
ferrous up
complete not
was
10 mM to
system
occur
inhibited
little the
oxygen
Incubation
by
by
ADP
omitting mixture.
ferrous
ion
extent
(Table
at a seven times
Epoxidation
of
a possibly
cholesterol
incubation
to any appreciable
II.
ion,
system,
Complete'
effective
Cholesterol
MsOOH or
in by
the the
Omitting
ion,
II).
Potassium
higher
by Microsomal
presence
cyanide
Lipid
Hydroperoxides
B-/a-Epoxides
0.31
1.47
4.7
+ EDTA (1 mM) (10 mM)
0.31 0.33
1.20 1.33
3.9 4.0
- Fe2+
0.03
0.14
0.02
0.10
0.00
0.00
a The reaction mixture as active oxygen) and
the
contained microsomal lipid hydroperoxides other ingredients as described in Table
727
(1 mM I.
EDTA
from
the
did
completely
than
- ADP
+ KCN (7 mM1
for
of ion
concentration
4.3
mM)
has
the epoxidation
1.33
+ Fe3+(1
ion
of
addition
0.31
- Fe2+
form
ferrous
by ferric
Radioactive products formed (%) cY-Epoxide p-Epoxide
system
ferric
(21).
influenced
or replacing
by 0.5 mM or higher
hydroperoxidation
ferrous
of
complete
the epoxidation
Table
this
to
epoxidation
ion
to
reduced
of molecular
Surprisingly,
In
inhibited
oxygen)
that
of
Vol. 108, No. 2, 1982 Table
III.
BIOCHEMICAL Epoxidation
Incubation
AND BIOPHYSICAL
of Cholesterol
RESEARCH COMMUNICATIONS
by Methyl Linoleate
Radioactive products formed (%) a-Epoxide 8-tpoxide
system
Completea
B- /a-Epoxides
0.19
0.76
4.0
- ADP
0.18
0.68
3.8
+ EDTA (1 mM) (10 mM)
0.17 0.18
0.60 0.77
3.5 4.3
- Fe*+
0.00
0.01
- Fe*+ + Fe3+ (1 mM)
0.00
0.02
+ KCN (7 mM)
0.00
0.00
a The reaction mixture contained methyl linoleate active oxygen) and other ingredients as described ferrous very
ion.
Factors
similar
influencing
to those
inhibitory
by hydroperoxides chelating
agent
in hepatic
would for
aldehyde
formation of ferrous
ion
(12)
to catalyze acyl
which
likely
catalyzes
to block
the
which
illustrated
in Fig.
involves
ration
of hydrogen
by OH radical
of the
That
active
mechanism
factor
to be an and cholesterol
suggests
that
the
of an iron
complex
polyunsaturated
fatty
oxygen from MsOOH and ferrous Potassium cyanide is
oxygen-iron for
in the
an essential (20)
from
may
malondi-
of cholesterol
the formation
of active
MsOOH as a direct
of the epoxides
with oxidizing the a-epoxide peroxide
when cholesterol
and t-butyl
for
the
complex
microsomal
oxidant
to catalyze
epoxidation
may be depicted
of as
1.
The a- to B-ratio double bond varies times higher than
strongly
of cholesterol.
a proposed
that
hydroperoxide
necessarily
hydroperoxides
epoxidation
Thus,
lipid
epoxidation.
has been demonstrated
This agent
of lipid
the the
epoxidation
it
of cholesterol
suggests
of malondialdehyde
the formation
the formation
cholesterol
product
whereas
epoxidation
EOTA of the microsomal
ADP was not
as a chelating
but not for
the epoxidation.
with
microsomes.
by MOOH were
strongly
influence
as of microsomal
the formation
lipids,
ion step for
inhibition
the formation
in hepatic
may serve
initial
and NADPH.
for
of EDTA on the
but not
epoxidation
factor
nucleotide
ion
as well
the cholesterol
epoxides
the
the potent
presence accelerating
inhibit
(1 mM as
of cholesterol
of ferrous
microsomes,
hydroperoxides in Table I.
III).
effect
in the presence
formation
be the reason
epoxidation
by MsOOH (Table
No appreciable
for
Hydroperoxides
by oxidation
agents, e. g., by air oxidation
(23). is treated
in an aqueous hydroperoxide
formed
solution
A ferric
have been shown to convert 72%
cholesterol
the 6-epoxide is yielded 8-10 (22) or by a high concent-
On the contrary, with peracids (25).
of the
the a-epoxide is in organic solvents ion-acetylacetone cholesterol
a major (24) or complex
in chloroform
Vol. 108, No. 2, 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
HO Active
0' a-Epoxide
oxygen-
(Non-enzymatic)
Cholesterol
HO a-Epoxide
Fig.
to the
1
A proposed mechanism for microsomal epoxidation of cholesterol. LH and LOOH represent polyunsaturated fatty acid residues of microsomal lipids and their hydroperoxides, respectively. Hydroperoxidation of LH with molecular oxygen is mediated by NADPHcytochrome c reductase and accelerated by an ADP-Fe2+ complex as has previously been demonstrated (21).
a- and s-epoxides
epoxidation
(26,
27).
in the However,
ratio
very
the
similar
present
to the microsomal
investigation
demonstrated
that
neither ferric ion nor t-butyl hydroperoxide was an effective agent for the epoxidation of cholesterol in an aqueous medium. A study on the participation of hydroperoxides sterol
of other
epoxidation
is
microsomal
polyunsaturated
now in progress
in our
fatty
acids
in chole-
laboratory.
REFERENCES
:: 3. 4. 5. 6. L: 1:: 11. 12. 13.
Black, H. S., and Douglas, D. R. (1972) Cancer Res. 32, 2630-2632. Gray, M. F., Lawrie, T. D. V., and Brooks, C. J. W. (1971) Lipids 6, 836-843 Assmann, G., Fredrickson, D. S., Sloan, H. R., Falesa, H. M., and Highet, R. J. (1975) J. Lipid Res. 16, 28-38. Reddy, B. S., and Wynder, E. L. (1977) Cancer 39, 2533-2539. Blackburn, G. M., Rashid, A., and Thompson, M. H. (1977) J. Chem. Sot., Chem. Commun. 420-421. Parsons, P. G., and Patricia, G. (1978) Aus. J. Exp. Biol. Med. Sci. 56, 287-296. Morris, I. K., and Roman, J. P. (1979) Cancer Lett. 6, 143-149. Sevanian, A., Mead, J. F., and Stein, R. A. (1979) Lipids 14, 634-643 Watabe, T., Isobe, M., and Kanai, M. (1980) J. Pharm. Dyn. 3, 553-556. Aringer, L., and Eneroth, P. (1974) J. Lipid Res. 15, 389-398. Watabe, T., Kanai, M., Isobe, M., and Orawa, N. (1980) Biochem. Biophys. Res. Commun. 92, 977-985. Chem. 256, Watabe, T., Kanai, M., Isobe, M., and Ozawa, N. (1981) J. Biol. 2900-2907. Isobe, M., and Watabe, T. Abstracts of Papers Presented at the 54th General Meeting
14. 15. 16. 17.
of
Fieser, L. John Wiley Davis, M., Milas, N. Terao, J.,
the
Japanese
Biochemical
Society,
Tokyo,
1981,
p.
613.
M. (1967) Reagents for Organic Synthesis, p. 136, F., and Fieser, & Sons, New York. and Petrow, V. (1949) J. Chem. Sot. 2536-2539. A., and Surgenor, D. M. (1946) J. Am. Chem. Sot. 68, 205-208. and Matsushita, S. (1977) J. Am. Chem. Sot. 54, 234-238. 129
Vol. 108, No. 2, 1982 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Lawry, J. Biol. Wills, Wills, Svingen, J. Biol. Smith, Smith, J. Am. Gumulka, Ansari, Tohma, Kimura,
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
P. H., Rosebrough, N. J., Farr, A., and Randall, R. J. (1951) Chem. 193, 265-275. E. D. (1971) Biochem. J. 123, 983-991. E. D. (1969) Biochem. J. 113, 325-341. B. A., Buege, J. A., O'Neal, F. O., and Aust, S. D. (1979) CHem. 254, 5892-5899. L. L., and Kulig, M. J. (1975) Cancer Biochem. Biophys. 1, 79-85. L. L., Kulig, M. J., Miiller, D., and Ansari, G. A. S. (1978) Chem. Sot. 100, 6206-6211. J., Pyrek, J. S., and Smith, L. L. (1982) Lipids 17, 197-203. G. A. S., and Smith, L. L. (1979) Photochem. Photobiol. 30, 147-150. M., Tomita, T., and Kimura, M. (1973) Tetrahedron Lett. 4359-4362. M., and Muto, T. (1979) Chem. Pharm. Bull. 27, 109-112.
730
6lOCHEMlCAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
30, 1982
September
EPOXIDATION
Pages 724-730
OF CHOLESTEROL BY HEPATIC MICROSOMAL LIPID
Tadashi Laboratory
Watabe,
Masakazu
of Drug Metabolism
Tokyo College Received
13,
and Atsushi
& Toxicology,
of Pharmacy,
August
Isobe,
Horinouchi,
HYDROPEROXIDES
Tsubaki
Department
of Hygienic
Hachioji-shi,
Chemistry
Tokyo 192-03,
Japan
1982
SUMMARY: [1,2-3HlCholesterol was epoxidized to radioactive cholesterol a- and a-epoxides (5,6a-epoxy-5aand 5,68-epoxy-5a-cholestan-3a-01s) in the ratio 1:4 by hepatic microsomal lipid hydroperoxides (MsOOH, 1 mM as active oxygen) in the presence of ferrous ion. MsOOH could be replaced by methyl linoleate hydroperoxides (MOOH) under the same conditions although the latter was less effective than the former. None of cumene hydroperoxide, t-butyl hydroperoxide, and hydrogen peroxide was an effective oxidant even at 10 mM. Neither ADP nor EDTA had an effect on the epoxidation of cholesterol by MsOOH as well as by MOOH. Ferrous ion could not be replaced by ferric ion in the hydroperoxide-mediated epoxidation. Cyanide anion potentially inhibited the reaction.
INTRODUCTION Cholesterol
a-epoxide
has been suggested mice irradiated
to be closely
related
by ultraviolet
hyperpressure cancer
(5,6a-epoxy-5a-cholestan-3s-011,
(2)
ray
and Wolman's
have been demonstrated
to skin
(11 as well
disease
tumorigenesis
larger
5a-cholestane-38,5,6a-triol,
in their
feces
not only
covalently
DNA (51,
but also
aberrations
to isolated
!G) and malignant
Cholesterol
a-epoxide
transformations is
found
(5,6s-epoxy-5a-cholestan-38-01) in various
tissues
have demonstrated
hepatic
microsomes
by the
addition
increasing chelating monoxide lipid
that
91,
in the presence
of a ferrous
microsomal (121,
strongly
hydroperoxides
suggesting in viva
as well
0
I982
by Academic Press, Inc. in any form reserved.
of reproduction
The steroid
of cells the
complex
colon epoxide
chromosome
in culture.
isomeric
human (3).
with
with
hydrolytic
induces
s-epoxide constituents
The previous
of cholesterol
is mediated system,
concomitant
by
accelerated
formation
of
and potentially inhibited by the metalformation of the dialdehyde (10-12).
of cholesterol
is
that
by reaction
it
occurs
as in vitro.
0006-291X/82/180724-07$01.00/0 Copyright All rjghts
(4).
of an NADPH-generating
ion-ADP
epoxidation
of its
1 to 2-4 as normal
including
epoxidation
amounts of malondialdehyde, agent, EDTA, with suppressed
The hepatic
with
in the ratio
of mammals (8,
workers
(7)
together
in hairless
patients
amounts
product,
binds
steroid,
as to hypercholesterolemic In addition,
(3).
to excrete
a toxic
724
not
Actually,
inhibited with
by carbon microsomal
the ratio
of
Vol. 108, No. 2, 1982 cholesterol
a- and s-epoxides
be very close sterol
BIOCHEMICAL
to that
in vitro
(13).
has been available as true
oxidants
investigation
for
present
observed
provides with
requirement
of ferrous
the first
microsomal ion
hepatic
it
should
participation
in the microsomal
cholesterol
in rat
with
However, the
AND BIOPHYSICAL
lipid
liver
RESEARCH COMMUNICATIONS has been demonstrated
microsomal be noticed
epoxidation that
of microsomal
cholesterol direct
evidence
hydroperoxides
as an essential
for
the
of chole-
no direct
lipid
epoxidation.
to
evidence
hydroperoxides
The present epoxidation of
(MsOOH) and also
for
the
factor.
MATERIALS AND METHODS Materials [1,2-3HlCholesterol (43 Ci/mmol) was purchased from Radiochemical Centre, Amersham, diluted with unlabeled cholesterol, and purified before use by HPLC on a Lichrosorb RP-18 ODS column in methanol. Adenosine 5'-diphosphate (ADP), EDTA, and methylene blue were obtained from Wako Pure Chemical Industries, Ltd., Osaka, and cumene hydroperoxide (98% pure as active oxygen) from Nakarai Chemical Co., Kyoto. Cholesterol a-epoxide (141, B-epoxide (151, t-butyl hydroperoxide (99% pure as active oxygen) (161, and methyl linoleate hydroperoxides (MDOH, 95% pure as active oxygen) (171 were synthesized by the previous methods. Microsomal lipid hydroperoxides (MsOOH) were prepared as follows: a suspension of microsomes (protein content: 540 mg by the Lowry method (181) from male young adult Wistar rat liver (30 g) in 0.1 M phosphate buffer, pH 7.4 (90 ml), was shaken with a mixture of chloroform and methanol (2:l v/v, 500 ml 1. The residue obtained on the evaporation of the solvent from the organic phase separated by centrifugation was suspended in ethanol (200 ml). To the ethanolic solution was added methylene blue (0.11 mM) after the removal of undissolved materials by centrifugation. The mixture was vigorously aerated at 15" for 6 hr in light of a tungsten lamp (300 W) and then condensed in mew at 15" to 50 ml. The condensate was shaken with ether and water (100 ml each). The organic layer separated was washed twice with water (0.5 vol. each), dried over anhydrous sodium sulfate, and condensed to 10 ml. The final condensate had 35 vmol/ml of active oxygen and was able to be stored without any appreciable loss of active oxygen at -20" in dark at least for 2 months. Active oxygen of the hydroperoxides was determined by iodometry as previously reported (19). Incubation of Radioactive Cholesterol A typical incubation system was made as follows: to a solution of 13H1cholesterol (1 PCi, 0.05 pmol) and a hydroperoxide (1 pmol as active oxygen) in methanol (0.1 ml) containing 0.5% Tween 80 was added a solution of ADP (1 pmol) in 0.1 M phosphate buffer, pH 7.4 (0.5 ml). The mixture was preincubated for 5 min at 37", and the reaction was started by the addition of an aqueous solution (0.4 ml) of ferrous sulfate (1 pmol). Other ingredients when used were dissolved in water and added to the incubation mixture. Incubations were carried out at 37" for 60 min. High Pressure Liquid Chromatography (HPLC) HPLC was carried out for the separation of cholesterol epoxides on a LDC model Constametric II high pressure liquid chromatograph equipped with a RI monitor and a Lichrosorb RP-18 ODS column (Merck Co., Darmstadt, 5 p in particle size, 4 x 150 mm). A silica 4 x 300 mm) was used for the column (Lichrosorb SI-60, 5 P in particle size, determination of radiochemical purity of the epoxides isolated on the ODS The ODS and silica columns were eluted at 20" with 1 and 2 ml/min column. developing solvents, respectively. Determination of Epoxides Radioactive cholesterol Q- and s-epoxides formed from tritirated cholesterol by reactions with hydroperoxides were separated by HPLC and determined by the reverse isotope dilution method, including the scintillation counting of their radioactivities and the 725
Vol. 108, No. 2, 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
determination of carrier steroids by GLC as follows: from the reaction mixture were extracted radioactive epoxides into peroxide-free ether (2 ml) containing (I- and a-epoxides (0.5 mg each) in the presence of a unlabeled cholesterol saturating amount of sodium chloride. The solvent was evaporated in a nitrogen stream from the organic phase separated. The residue obtained was dissolved in a mixture of methanol and ethanol (10:3, 0.2 ml) and immediately subjected to reverse partition HPLC carried out in the same solvent mixture to collect the epoxides appearing at 4.7-5.6 min; under these conditions, cholesterol was eluted at 9.8 min. The eluate corresponding to the epoxide fraction was redissolved in methanol (0.2 ml) after the evaporation of the solvent in nitrogen. The methanolic solution was subjected to HPLC carried out on the same column in methanol; under these conditions cholesterol a- and B-epoxides were eluted at 8.0 and 6.4 min, respectively. The epoxide fractions were separately collected, and their radioactivity to carrier ratios were measured by the previously reported method (12). RESULTS AND DISCUSSION Epoxidation
of
[3H]Cholesterol
[1,2-3H]Cholesterol ferrous
ion
was incubated
and ADP in an aqueous
cholesterol
epoxides
formed
a- and a-epoxides epoxides
by Hepatic
were
with
medium,
were
as carriers
Microsomal rat
for
the
reverse
separated
from the
an ODS column
in methanol
containing
ethanol
4.7-5.6
The
min.
homogeneity retention
of the separated times
combination
to carrier
of the
and after Per cent
epoxides ion
epoxide
in the ratio
hepatic
microsomal linoleate
epoxidation was very t-butyl
ion
of
very
separated
with
methanol.
The
by HPLC on
fraction
eluted
at 8.0
and 6.4 min,
by HPLC on a silica
11.3
and 13.5 min,
with
with
at
Radiochemical column;
respectively.
determined
GLC (121,
were
the specimens
similar
to the radioactive
by a
unchanged
separated
was 1.6% in the
was yielded
to that (1.3%)
hydroperoxides
presence
preferentially
of the
previously
of cholesterol
(0.34
(MOOH) were one half The a- to B-ratio
of the
of ferrous to the a-
demonstrated PM) in the presence system
as effective epoxides
(12).
as MsOOH for formed
by MOOH
to the case of MsOOH. oxygen
and hydrogen under
the
However, none of cumene hydroperoxide, peroxide was an effective oxidant even at
same incubation
conditions.
Factors Influencing Cholesterol Epoxidation by Lipid Hydroperoxides Lipid hydroperoxidation in hepatic microsomes has been well recognized to be remarkably
by
a- and a-
mM), ADP (1 mM), and an NADPH-generating
hydroperoxide,
10 mM as active
oxygen)
The 6-epoxide
of cholesterol. similar
column
[3H]cholesterol
epoxidation
(0.2
method.
HPLC.
conversion
I).
well
method
cholesterol
substrate
a single
of
Radioactive
unlabeled
of the a- and 6-epoxides, counting
by MsOOH (1 mM as active
of ferrous
were
system).
dilution
was confirmed
HPLC on the adsorbent
and ADP (Table
Methyl
ratios
scintillation
the 2nd reverse partition
isotope
were then
epoxides
with
into
(MsOOH)
MsOOH in the presence
radioactive
by the elution
of the a- and a-epoxides
The radioactivity before
and s-epoxides
a-
on an ODS column
Hydroperoxides
(complete
together
imnediately
respectively,
liver
pH 7.4
extracted
Lipid
accelerated
either
by ferric
126
ion
or by ferrous
ion
in the
Vol. 108. No. 2, 1982 Table
BIOCHEMICAL
I.
Epoxidation
of
AND BIOPHYSICAL
Cholesterol
Concentration active oxygen (mM)
Hydroperoxide
Microsomal lipid hydroperoxide
(MsOOH)
Methyl linoleate hydroperoxide
(MOOH)
RESEARCH COMMUNICATIONS
by Various
of
Hydroperoxides
Radioactive products formed (%l a-Epoxide s-Epoxide
s- /a-Epoxides
1
0.31
1.33
4.3
1
0.19
0.76
4.0
Cumene hydroperoxide
1 10
0.00 0.03
0.03 0.16
t-Duty1 hydroperoxide
1 10
0.01 0.02
0.04 0.08
Hydrogen
1 10
0.00 0.02
0.01 0.02
peroxide
The reaction mixture contained ADP (1 mM), FeS04 (1 mM), [1,2-'HIcholesterol (1 PCi, 50 PM), Tween 80 (0.05%), and the hydroperoxide (1 mM as active in 0.05 M phosphate buffer, pH 7.4.
presence
of NADPH and ADP and potentially
concentrations
of EDTA (201.
been
to
suggested
activation
be
ferrous up
complete not
was
10 mM to
system
occur
inhibited
little the
oxygen
Incubation
by
by
ADP
omitting mixture.
ferrous
ion
extent
(Table
at a seven times
Epoxidation
of
a possibly
cholesterol
incubation
to any appreciable
II.
ion,
system,
Complete'
effective
Cholesterol
MsOOH or
in by
the the
Omitting
ion,
II).
Potassium
higher
by Microsomal
presence
cyanide
Lipid
Hydroperoxides
B-/a-Epoxides
0.31
1.47
4.7
+ EDTA (1 mM) (10 mM)
0.31 0.33
1.20 1.33
3.9 4.0
- Fe2+
0.03
0.14
0.02
0.10
0.00
0.00
a The reaction mixture as active oxygen) and
the
contained microsomal lipid hydroperoxides other ingredients as described in Table
727
(1 mM I.
EDTA
from
the
did
completely
than
- ADP
+ KCN (7 mM1
for
of ion
concentration
4.3
mM)
has
the epoxidation
1.33
+ Fe3+(1
ion
of
addition
0.31
- Fe2+
form
ferrous
by ferric
Radioactive products formed (%) cY-Epoxide p-Epoxide
system
ferric
(21).
influenced
or replacing
by 0.5 mM or higher
hydroperoxidation
ferrous
of
complete
the epoxidation
Table
this
to
epoxidation
ion
to
reduced
of molecular
Surprisingly,
In
inhibited
oxygen)
that
of
Vol. 108, No. 2, 1982 Table
III.
BIOCHEMICAL Epoxidation
Incubation
AND BIOPHYSICAL
of Cholesterol
RESEARCH COMMUNICATIONS
by Methyl Linoleate
Radioactive products formed (%) a-Epoxide 8-tpoxide
system
Completea
B- /a-Epoxides
0.19
0.76
4.0
- ADP
0.18
0.68
3.8
+ EDTA (1 mM) (10 mM)
0.17 0.18
0.60 0.77
3.5 4.3
- Fe*+
0.00
0.01
- Fe*+ + Fe3+ (1 mM)
0.00
0.02
+ KCN (7 mM)
0.00
0.00
a The reaction mixture contained methyl linoleate active oxygen) and other ingredients as described ferrous very
ion.
Factors
similar
influencing
to those
inhibitory
by hydroperoxides chelating
agent
in hepatic
would for
aldehyde
formation of ferrous
ion
(12)
to catalyze acyl
which
likely
catalyzes
to block
the
which
illustrated
in Fig.
involves
ration
of hydrogen
by OH radical
of the
That
active
mechanism
factor
to be an and cholesterol
suggests
that
the
of an iron
complex
polyunsaturated
fatty
oxygen from MsOOH and ferrous Potassium cyanide is
oxygen-iron for
in the
an essential (20)
from
may
malondi-
of cholesterol
the formation
of active
MsOOH as a direct
of the epoxides
with oxidizing the a-epoxide peroxide
when cholesterol
and t-butyl
for
the
complex
microsomal
oxidant
to catalyze
epoxidation
may be depicted
of as
1.
The a- to B-ratio double bond varies times higher than
strongly
of cholesterol.
a proposed
that
hydroperoxide
necessarily
hydroperoxides
epoxidation
Thus,
lipid
epoxidation.
has been demonstrated
This agent
of lipid
the the
epoxidation
it
of cholesterol
suggests
of malondialdehyde
the formation
the formation
cholesterol
product
whereas
epoxidation
EOTA of the microsomal
ADP was not
as a chelating
but not for
the epoxidation.
with
microsomes.
by MOOH were
strongly
influence
as of microsomal
the formation
lipids,
ion step for
inhibition
the formation
in hepatic
may serve
initial
and NADPH.
for
of EDTA on the
but not
epoxidation
factor
nucleotide
ion
as well
the cholesterol
epoxides
the
the potent
presence accelerating
inhibit
(1 mM as
of cholesterol
of ferrous
microsomes,
hydroperoxides in Table I.
III).
effect
in the presence
formation
be the reason
epoxidation
by MsOOH (Table
No appreciable
for
Hydroperoxides
by oxidation
agents, e. g., by air oxidation
(23). is treated
in an aqueous hydroperoxide
formed
solution
A ferric
have been shown to convert 72%
cholesterol
the 6-epoxide is yielded 8-10 (22) or by a high concent-
On the contrary, with peracids (25).
of the
the a-epoxide is in organic solvents ion-acetylacetone cholesterol
a major (24) or complex
in chloroform
Vol. 108, No. 2, 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
HO Active
0' a-Epoxide
oxygen-
(Non-enzymatic)
Cholesterol
HO a-Epoxide
Fig.
to the
1
A proposed mechanism for microsomal epoxidation of cholesterol. LH and LOOH represent polyunsaturated fatty acid residues of microsomal lipids and their hydroperoxides, respectively. Hydroperoxidation of LH with molecular oxygen is mediated by NADPHcytochrome c reductase and accelerated by an ADP-Fe2+ complex as has previously been demonstrated (21).
a- and s-epoxides
epoxidation
(26,
27).
in the However,
ratio
very
the
similar
present
to the microsomal
investigation
demonstrated
that
neither ferric ion nor t-butyl hydroperoxide was an effective agent for the epoxidation of cholesterol in an aqueous medium. A study on the participation of hydroperoxides sterol
of other
epoxidation
is
microsomal
polyunsaturated
now in progress
in our
fatty
acids
in chole-
laboratory.
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