Vol. 166, No. 3, 1990 February 14, 1990
HEPATIC
BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS Pages 1308-1312
MICROSOMAL OXYGENATION OF ALDEHYDES TO CARBOXYLIC ACIDS
Kazuhito
* Department
Watanabe*, and
of Hygienic
Department
of Hygienic
December
Ikuo
920-11,
Chemistry,
University,
Yamamoto*n
of Pharmaceutical
Kanazawa
and Forensic Kyushu
18,
Faculty
University,
Sciences, Received
Narimatsu*, Yoshimura**
Chemistry,
Hokuriku **
Shizuo Hidetoshi
Japan
Faculty
Fukuoka
Sciences,
812,
of Pharmaceutical Japan
1989
SUMMARY: Hepatic microsomal oxygenation of aldehydes to carboxylic acids was investigated. Aldehydes (veratrum aldehyde, cinnamic aldehyde, myrtenal, cuminaldehyde, 3-phenylpropionaldehyde, perillaldehyde and 9-anthraldehyde) were incubated with hepatic microsomes of mice in the presence of an NADPH-generating system under I802 (97 atom%). The incorporation of oxygen-18 into carboxylic acids formed was determined by gas chromatography-mass spectrometry. Oxygen-18 was incorporated into the carboxylic acids formed from all aldehyde substrates examined. Hepatic microsomal formation of 3,4-dimethoxybenzoic acid and cumic acid from veratrum aldehyde and cuminaldehyde, respectively, was inhibited by CO and SKF 525-A. These results indicate that the oxygenation of aldehydes which may be catalyzed by cytochrome P450 is a common reaction in the biotransformation of xenobiotic aldehydes. 0 1990 Academic Pre.5~. Inc.
In
general,
aldehyde
are
dehydrogenase
mitochondria two
aldehydes
or
steps
cytosol.
(1,2),
the
biotransformed
(EC
1.2.1.3)
which
This
enzymatic
reaction
initial
complex
Therefore,
the
oxygen
incorporated
originated
from
water.
In
of
subsequent
marihuana
a
by
microsomal
1[ To whom all 0006-291X/90 Copyright All rights
aldehyde correspondence
into
(3,4),
we
mainly
of
of
of
for
acid
(carboxylic (MALDO)(S).
oxygenase should
be addressed.
1308
in
principally to
form
the complex. acid
formed
studies the
on
first
time
is
active that
oxygenated
was acid)
by
located
aldehyde
metabolic
found
acids
of
carboxylic
(aldehyde)
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
is
consists
hydrolysis
series
ll-oxo-a'-tetrahydrocannabinol a8-tetrahydrocannabinol-11-oic
carboxylic
dehydrogenation
acyl-enzyme
constituents
and the
to
which The
to
was catalyzed present
study
Vol.
166, No. 3, 1990
demonstrates which
BIOCHEMICAL
that
this
novel
may be catalyzed
reaction
in the
AND BIOPHYSICAL
oxygenation
by cytochrome
biological
P450
RESEARCH COMMUNICATIONS
of
aldehyde
is
not
oxidation
of xenobiotic
MATERIALS
AND METHODS
to
carboxylic
an unusual
but
acid a common
aldehydes.
9-Anthraldehyde, (-)-myrtenal, (S)-(-)-perillaldehyde and 9-anthracene carboxylic acid were purchased from Aldrich Chem. Co. Inc.; cinnamic cumic acid, 3-phenylpropionaldehyde, aldehyde, cuminaldehyde, 3-phenylpropionic acid, veratrum aldehyde and 3,4-dimethoxybenzoic acid were from Wako Pure Chem. Co. NADP; glucose-6-phosphate were from Boehringer Mannheim GmbH.; and glucose-6-phosphate dehydrogenase (type V) was from Sigma Chem. Co.; Oxygen-18 gas (97 atom%) was from Amersham International plc. Other chemicals used were of analytical reagent grade. Hepatic microsomes from male ddN mice (25-359) were prepared by the previous method (6). A typical incubation mixture consisted of washed liver microsomes equivalent), 0.5 mM NADP, 10 mM to.29 glucose-6-phosphate, 10 mM unit glucose-6-phosphate W12, 1 dehydrogenase, 200 ug of aldehyde substrate and 100 mM potassium phosphate buffer (pH 7.4) to make a final volume of 1 ml. Incubation tube (10 ml) was covered with silicon cap. After evacuation and introduction of oxygen-18 gas, the reaction was started by the injection of substrate into the reaction mixture. The reaction mixture was incubated at 37" C for 30 min and then extracted with 5 ml of ethyl acetate after acidification with 0.5 ml of 0.2 M HCl-KCl. After evaporation of the organic solvent, carboxylic acid formed was methylated with diazomethane. Gas chromatography-mass spectrometry (GC-MS) was conducted with a JEOL-GCG-06 gas chromatograph coupled with a JEOL JMS-DX-300 mass spectrometer and a JEOL-JMA-DA 5000 mass data system. The conditions were as follows: column, 5% SE-30 on Chromosorb W (60-80 mesh, 3 mm x 2 m); column temperature, 130 "C except for 9-anthraldehyde as substrate (200 "C); carrier gas, He 40 ml/min; ionization energy 70 eV; ionizing current 300 uA. Under the above conditions, the retention times of methyl ester of carboxylic acids were 3.1 min (myrtenal), (perillaldehyde), 3.7 min (3-phenylpropionaldehyde), 55i2 minm'(ncuminaldehyde), 8.4 min (cinnamic aldehyde), 17.8 min (veratrum aldehyde) and 8.gatnri (9-anthraldehyde). oxidation of veratrum aldehyde cuminaldehyde to Microsomal 3,4-dimethoxybenzoic acid and cumic acid, respectively, was determined by a gas chromatographic method. The incubation system and derivatization were the same as described above. Gas chromatography was performed with a Shimadzu GC-6A gas chromatograph equipped with a hydrogen flame ionization detector. The conditions were as follows: column 5% SE-30 on Chromosorb W (60-80 mesh, 2 mm x 1.5 m); column temperature 120 "C; carrier gas N2 40 ml/min. RESULTS AND DISCUSSION Mouse veratrum
hepatic aldehyde
spectra
of
to
methyl
under
air
or
shown
for
methyl
derivative
microsomes
of
ester the
NADPH-dependent
3,4-dimethoxybenzoic
ester
oxygen-18
catalyzed
of
the
carboxylic
atmosphere. of
carboxylic
the
acid. acid
A single
carboxylic acid
acid formed
1309
oxidation
Figure formed
from
molecular
ion
formed
under
under
1
oxygen-18
shows the
at air,
of mass
aldehyde
m/z 196 was while
the
showed
two
Vol.
BIOCHEMICALANDBIOPHYSICALRESEARCH
166. No. 3, 1990 Under
COMMUNICATIONS
Air
m/z Under
1802
(97 atom%)
167
!i0
‘i
71
79 05
34
121
183
137 I
I, I
m/z Fig.
molecular
1 Mass Spectra of Methyl Ester of 3,4-Dimethoxybenzoic Acid Formed from Veratrum Aldehyde under Air or Oxygen-18 Atmosphere.
ions
at
that
oxygen-18
under
oxygen-18.
198
was
9
was
incorporated
100.
The
Other
cuminaldehyde,
myrtenal,
also
in
examined
corresponding
the ions, to
gas
acids
oxygenation
that acids could
ions
that
carboxylic
manner
198 indicates
acid
veratrum
and were
formed
m/z
196 and
aldehyde
by oxygenation
(9-anthraldehyde, and
at
acid
with
cinnamic
to
mouse
aldehyde,
3-phenylpropionaldehyde) found
was
were
be converted
to the
: M' t 2) formed
under
acids.
(97 atom%)
indicating carboxylic
a similar
at m/z
molecular
indicate
aldehydes
intensities
carboxylic
the
perillaldehyde
carboxylic
The relative
the
ion
3,4-dimethoxybenzoic
of
results to
The molecular into
abundance
converted
microsomes.
oxygen-18
196 and 198.
Relative
:
predominantly hepatic
m/z
of molecular are
formed
summarized from
all
aldehydes
with
mouse
be determined
ions in Table
seven
aldehydes
examined hepatic
were
microsomes.
by comparing 1310
(Mt
the
I.
Under
the
showed
two
predominantly
conditions, molecular oxygenated
Contribution relative
intensities
of the of
Vol.
BIOCHEMICAL
166, No. 3, 1990
Table
I Incorporation Mouse Hepatic Aldehydes
AND BIOPHYSICAL
of Molecular Oxygen during Microsomal Oxidation of to Carboxylic Acids
Substrates
32 : 100
Cinnamic
35 : 100
aldehyde
Cuminaldehyde
69 : 100
Myrtenal
17 : 100
Perillaldehyde
15 : 100
aldehyde
9 : 100
3-Phenylpropionaldehyde
Mt + 2
ions
oxygen-U.
Veratrum
exclusively
oxygenated
abundance under
of
in that
M' t
oxygen-18
oxygenation
Relative intensities of M” : M’+2 ions
Structures
9-Anthraldehyde
Veratrum
M' and
RESEARCH COMMUNICATIONS
of
’
methyl
(Mt
:
mechanism
in
carboxylic and
corresponding the
M' +
mediated
of
myrtenal
to the
=
69
about
:
acids
perillaldehyde
carboxylic
carboxylic 2
56: 100
Cl+-CH2-CHO
ester
aldehyde,
2 ion
’
o-
acid
formed
100)
was
60% of the
were
Control
from
cuminaldehyde
lowest
carboxylic
Cuminaldehyde
5.1a) (100)
co
1.4
(CO/02=4)
(27)
(59)
SKF 525-A
1.2
1.6
(1 mM1
(24)
(36)
2.6
a) nmoles carboxylic acid formed/min/mg protein Numbers in parentheses represent percent of the control activity. The results were expressed as the mean of two experiments.
1311
almost
The relative
of CO and SKF 525-A on Carboxylic with Hepatic Microsomes of Mice Veratrum aldehyde
under
acids.
case.
Table II Effects Acid Formation
formed
even acid
though
formation
Vol.
166, No. 3, 1990 Table
of
which
cytochrome veratrum
II
BIOCHEMICAL
summarizes
are
known
to
aldehyde
to
cytochrome
the
oxygenation lipophilic
of
aldehyde
aldehyde
, on the
the
of
oxidation
oxidation
to
carboxylic
acids.
including
that is
microsomal
a
and
CO and SKF 525-A
aldehydes,
oxidation
demonstrates acid
hepatic
both
of cuminaldehyde
of both
the microsomal study
substrates
of CO and SKF 525-A,
carboxylic
present
RESEARCH COMMUNICATIONS
inhibitors
corresponding
P450 catalyzed the
effects
typical
reaction
inhibited
In conclusion,
inhibiotry
be the
P450-dependent
(1 mM) considerably that
the
AND BIOPHYSICAL
of the
suggesting aldehydes.
hepatic common
microsomal reaction
to
11-oxo-A8-tetrahydrocannabinol.
ACKNOWLEDGMENT We thank University for
Miss. carrying
of H. Shimomura out GC-MS analysis.
Analytical
Center
of
Hokuriku
REFERENCES 1. Feldman, R.I. and Weiner, H. (1972) J. Biol. Chem., 247, 267-272. 2. Weiner, H., Hu, J.H.J. and Sanny, C.G. (1976) J. Biol. Chem., 251, 3853-3855. 3. Watanabe, K., Yamamoto, I., Oguri, K. and Yoshimura, H. (1979) Biochem. Biophys. Res. Commun., 88, 178-182. 4. Watanabe, K., Yamamoto, I., Oguri, K. and Yoshimura, H. (1981) Drug Metab. Dispos., 9, 261-264. 5. Yamamoto, I., Watanabe, K., Narimatsu, S. and Yoshimura, H. (1988) Biochem. Biophys. Res. Commun., 153, 779-782. 6. Watanabe, K., Kita, M., Yamamoto, I., Oguri, K. and Yoshimura, H. (1983) J. Pharmacobio-Dyn., 6, 581-587.
1312
HEPATIC
BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS Pages 1308-1312
MICROSOMAL OXYGENATION OF ALDEHYDES TO CARBOXYLIC ACIDS
Kazuhito
* Department
Watanabe*, and
of Hygienic
Department
of Hygienic
December
Ikuo
920-11,
Chemistry,
University,
Yamamoto*n
of Pharmaceutical
Kanazawa
and Forensic Kyushu
18,
Faculty
University,
Sciences, Received
Narimatsu*, Yoshimura**
Chemistry,
Hokuriku **
Shizuo Hidetoshi
Japan
Faculty
Fukuoka
Sciences,
812,
of Pharmaceutical Japan
1989
SUMMARY: Hepatic microsomal oxygenation of aldehydes to carboxylic acids was investigated. Aldehydes (veratrum aldehyde, cinnamic aldehyde, myrtenal, cuminaldehyde, 3-phenylpropionaldehyde, perillaldehyde and 9-anthraldehyde) were incubated with hepatic microsomes of mice in the presence of an NADPH-generating system under I802 (97 atom%). The incorporation of oxygen-18 into carboxylic acids formed was determined by gas chromatography-mass spectrometry. Oxygen-18 was incorporated into the carboxylic acids formed from all aldehyde substrates examined. Hepatic microsomal formation of 3,4-dimethoxybenzoic acid and cumic acid from veratrum aldehyde and cuminaldehyde, respectively, was inhibited by CO and SKF 525-A. These results indicate that the oxygenation of aldehydes which may be catalyzed by cytochrome P450 is a common reaction in the biotransformation of xenobiotic aldehydes. 0 1990 Academic Pre.5~. Inc.
In
general,
aldehyde
are
dehydrogenase
mitochondria two
aldehydes
or
steps
cytosol.
(1,2),
the
biotransformed
(EC
1.2.1.3)
which
This
enzymatic
reaction
initial
complex
Therefore,
the
oxygen
incorporated
originated
from
water.
In
of
subsequent
marihuana
a
by
microsomal
1[ To whom all 0006-291X/90 Copyright All rights
aldehyde correspondence
into
(3,4),
we
mainly
of
of
of
for
acid
(carboxylic (MALDO)(S).
oxygenase should
be addressed.
1308
in
principally to
form
the complex. acid
formed
studies the
on
first
time
is
active that
oxygenated
was acid)
by
located
aldehyde
metabolic
found
acids
of
carboxylic
(aldehyde)
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
is
consists
hydrolysis
series
ll-oxo-a'-tetrahydrocannabinol a8-tetrahydrocannabinol-11-oic
carboxylic
dehydrogenation
acyl-enzyme
constituents
and the
to
which The
to
was catalyzed present
study
Vol.
166, No. 3, 1990
demonstrates which
BIOCHEMICAL
that
this
novel
may be catalyzed
reaction
in the
AND BIOPHYSICAL
oxygenation
by cytochrome
biological
P450
RESEARCH COMMUNICATIONS
of
aldehyde
is
not
oxidation
of xenobiotic
MATERIALS
AND METHODS
to
carboxylic
an unusual
but
acid a common
aldehydes.
9-Anthraldehyde, (-)-myrtenal, (S)-(-)-perillaldehyde and 9-anthracene carboxylic acid were purchased from Aldrich Chem. Co. Inc.; cinnamic cumic acid, 3-phenylpropionaldehyde, aldehyde, cuminaldehyde, 3-phenylpropionic acid, veratrum aldehyde and 3,4-dimethoxybenzoic acid were from Wako Pure Chem. Co. NADP; glucose-6-phosphate were from Boehringer Mannheim GmbH.; and glucose-6-phosphate dehydrogenase (type V) was from Sigma Chem. Co.; Oxygen-18 gas (97 atom%) was from Amersham International plc. Other chemicals used were of analytical reagent grade. Hepatic microsomes from male ddN mice (25-359) were prepared by the previous method (6). A typical incubation mixture consisted of washed liver microsomes equivalent), 0.5 mM NADP, 10 mM to.29 glucose-6-phosphate, 10 mM unit glucose-6-phosphate W12, 1 dehydrogenase, 200 ug of aldehyde substrate and 100 mM potassium phosphate buffer (pH 7.4) to make a final volume of 1 ml. Incubation tube (10 ml) was covered with silicon cap. After evacuation and introduction of oxygen-18 gas, the reaction was started by the injection of substrate into the reaction mixture. The reaction mixture was incubated at 37" C for 30 min and then extracted with 5 ml of ethyl acetate after acidification with 0.5 ml of 0.2 M HCl-KCl. After evaporation of the organic solvent, carboxylic acid formed was methylated with diazomethane. Gas chromatography-mass spectrometry (GC-MS) was conducted with a JEOL-GCG-06 gas chromatograph coupled with a JEOL JMS-DX-300 mass spectrometer and a JEOL-JMA-DA 5000 mass data system. The conditions were as follows: column, 5% SE-30 on Chromosorb W (60-80 mesh, 3 mm x 2 m); column temperature, 130 "C except for 9-anthraldehyde as substrate (200 "C); carrier gas, He 40 ml/min; ionization energy 70 eV; ionizing current 300 uA. Under the above conditions, the retention times of methyl ester of carboxylic acids were 3.1 min (myrtenal), (perillaldehyde), 3.7 min (3-phenylpropionaldehyde), 55i2 minm'(ncuminaldehyde), 8.4 min (cinnamic aldehyde), 17.8 min (veratrum aldehyde) and 8.gatnri (9-anthraldehyde). oxidation of veratrum aldehyde cuminaldehyde to Microsomal 3,4-dimethoxybenzoic acid and cumic acid, respectively, was determined by a gas chromatographic method. The incubation system and derivatization were the same as described above. Gas chromatography was performed with a Shimadzu GC-6A gas chromatograph equipped with a hydrogen flame ionization detector. The conditions were as follows: column 5% SE-30 on Chromosorb W (60-80 mesh, 2 mm x 1.5 m); column temperature 120 "C; carrier gas N2 40 ml/min. RESULTS AND DISCUSSION Mouse veratrum
hepatic aldehyde
spectra
of
to
methyl
under
air
or
shown
for
methyl
derivative
microsomes
of
ester the
NADPH-dependent
3,4-dimethoxybenzoic
ester
oxygen-18
catalyzed
of
the
carboxylic
atmosphere. of
carboxylic
the
acid. acid
A single
carboxylic acid
acid formed
1309
oxidation
Figure formed
from
molecular
ion
formed
under
under
1
oxygen-18
shows the
at air,
of mass
aldehyde
m/z 196 was while
the
showed
two
Vol.
BIOCHEMICALANDBIOPHYSICALRESEARCH
166. No. 3, 1990 Under
COMMUNICATIONS
Air
m/z Under
1802
(97 atom%)
167
!i0
‘i
71
79 05
34
121
183
137 I
I, I
m/z Fig.
molecular
1 Mass Spectra of Methyl Ester of 3,4-Dimethoxybenzoic Acid Formed from Veratrum Aldehyde under Air or Oxygen-18 Atmosphere.
ions
at
that
oxygen-18
under
oxygen-18.
198
was
9
was
incorporated
100.
The
Other
cuminaldehyde,
myrtenal,
also
in
examined
corresponding
the ions, to
gas
acids
oxygenation
that acids could
ions
that
carboxylic
manner
198 indicates
acid
veratrum
and were
formed
m/z
196 and
aldehyde
by oxygenation
(9-anthraldehyde, and
at
acid
with
cinnamic
to
mouse
aldehyde,
3-phenylpropionaldehyde) found
was
were
be converted
to the
: M' t 2) formed
under
acids.
(97 atom%)
indicating carboxylic
a similar
at m/z
molecular
indicate
aldehydes
intensities
carboxylic
the
perillaldehyde
carboxylic
The relative
the
ion
3,4-dimethoxybenzoic
of
results to
The molecular into
abundance
converted
microsomes.
oxygen-18
196 and 198.
Relative
:
predominantly hepatic
m/z
of molecular are
formed
summarized from
all
aldehydes
with
mouse
be determined
ions in Table
seven
aldehydes
examined hepatic
were
microsomes.
by comparing 1310
(Mt
the
I.
Under
the
showed
two
predominantly
conditions, molecular oxygenated
Contribution relative
intensities
of the of
Vol.
BIOCHEMICAL
166, No. 3, 1990
Table
I Incorporation Mouse Hepatic Aldehydes
AND BIOPHYSICAL
of Molecular Oxygen during Microsomal Oxidation of to Carboxylic Acids
Substrates
32 : 100
Cinnamic
35 : 100
aldehyde
Cuminaldehyde
69 : 100
Myrtenal
17 : 100
Perillaldehyde
15 : 100
aldehyde
9 : 100
3-Phenylpropionaldehyde
Mt + 2
ions
oxygen-U.
Veratrum
exclusively
oxygenated
abundance under
of
in that
M' t
oxygen-18
oxygenation
Relative intensities of M” : M’+2 ions
Structures
9-Anthraldehyde
Veratrum
M' and
RESEARCH COMMUNICATIONS
of
’
methyl
(Mt
:
mechanism
in
carboxylic and
corresponding the
M' +
mediated
of
myrtenal
to the
=
69
about
:
acids
perillaldehyde
carboxylic
carboxylic 2
56: 100
Cl+-CH2-CHO
ester
aldehyde,
2 ion
’
o-
acid
formed
100)
was
60% of the
were
Control
from
cuminaldehyde
lowest
carboxylic
Cuminaldehyde
5.1a) (100)
co
1.4
(CO/02=4)
(27)
(59)
SKF 525-A
1.2
1.6
(1 mM1
(24)
(36)
2.6
a) nmoles carboxylic acid formed/min/mg protein Numbers in parentheses represent percent of the control activity. The results were expressed as the mean of two experiments.
1311
almost
The relative
of CO and SKF 525-A on Carboxylic with Hepatic Microsomes of Mice Veratrum aldehyde
under
acids.
case.
Table II Effects Acid Formation
formed
even acid
though
formation
Vol.
166, No. 3, 1990 Table
of
which
cytochrome veratrum
II
BIOCHEMICAL
summarizes
are
known
to
aldehyde
to
cytochrome
the
oxygenation lipophilic
of
aldehyde
aldehyde
, on the
the
of
oxidation
oxidation
to
carboxylic
acids.
including
that is
microsomal
a
and
CO and SKF 525-A
aldehydes,
oxidation
demonstrates acid
hepatic
both
of cuminaldehyde
of both
the microsomal study
substrates
of CO and SKF 525-A,
carboxylic
present
RESEARCH COMMUNICATIONS
inhibitors
corresponding
P450 catalyzed the
effects
typical
reaction
inhibited
In conclusion,
inhibiotry
be the
P450-dependent
(1 mM) considerably that
the
AND BIOPHYSICAL
of the
suggesting aldehydes.
hepatic common
microsomal reaction
to
11-oxo-A8-tetrahydrocannabinol.
ACKNOWLEDGMENT We thank University for
Miss. carrying
of H. Shimomura out GC-MS analysis.
Analytical
Center
of
Hokuriku
REFERENCES 1. Feldman, R.I. and Weiner, H. (1972) J. Biol. Chem., 247, 267-272. 2. Weiner, H., Hu, J.H.J. and Sanny, C.G. (1976) J. Biol. Chem., 251, 3853-3855. 3. Watanabe, K., Yamamoto, I., Oguri, K. and Yoshimura, H. (1979) Biochem. Biophys. Res. Commun., 88, 178-182. 4. Watanabe, K., Yamamoto, I., Oguri, K. and Yoshimura, H. (1981) Drug Metab. Dispos., 9, 261-264. 5. Yamamoto, I., Watanabe, K., Narimatsu, S. and Yoshimura, H. (1988) Biochem. Biophys. Res. Commun., 153, 779-782. 6. Watanabe, K., Kita, M., Yamamoto, I., Oguri, K. and Yoshimura, H. (1983) J. Pharmacobio-Dyn., 6, 581-587.
1312
