Diastereospecific kinetics of nicotine N'-oxidation in rat liver microsomes

Article abstract of DOI:10.1080/004982598239632

1. In kinetic studies, both Eadie-Hofstee plots for cis- and trans-nicotine-1'-N-oxide formation from nicotine in rat liver microsomes were linear. For the formation of cis- and trans-nicotine-1'-N-oxide, the apparent k(m) were 0.240 ± 0.069 and 1.524 ± 0.951 mM respectively. Corresponding V(max) were 1.52 ± 0.48 and 1.19 ± 0.74 nmol/mg/min respectively. 2. The formation of cis-nicotine-1'-N-oxide was greater than the formation of trans-nicotine-1'-N-oxide in rat liver microsomes and the intrinsic clearance of cis-nicotine-1'-N-oxide formation was 8.1-fold greater than that of trans-nicotine-1'-N-oxide formation. 3. The formation of both cis- and trans-nicotine-1'-N-oxide in rat liver microsomes was inhibited by the addition of 1-(1-naphthyl)-2-thiourea or by heat-treatment of microsomes. 2-Diethylaminoethyl-2, 2-diphenylvalerate (SKF525A) and carbon monoxide did not affect these activities even at high concentrations. 4. Formations of cis- and trans-nicotine-1'-N-oxide correlated significantly with each other (r = 0.862, p < 0.01). These results suggested that the same flavin-containing monooxygenase (FMO) isoform is responsible for the formation of cis- and trans-nicotine-1'-N-oxide in rat liver.

Full text of DOI:10.1080/004982598239632

xenobiotica , 1998, vol. 28, no. 2, 127±135
Diastereospeci®c kinetics of nicotine N«-oxidation in
rat liver microsom es
M. NAKAJIMA‹*, K. IWATA‹, T. YOSHIDAŒ,
T. YAMAMOTO‹ and Y. KUROIWA‹
Department of ‹ Clinical Pharmacy and Œ Biochemical Toxicology, School of
Pharmaceutical Sciences, Showa University, Tokyo 142, Japan
Received 3 June 1997
1
. In kinetic studies, both Eadie±Hofstee plots for cis- and trans-nicotine-1 «-N-oxide
formation from nicotine in rat liver microsomes were linear. For the formation of cis- and
trans-nicotine-1 «-N-oxide, the apparent K were 0±240³0±069 and 1±524³0±951 mm
m
respectively. Corresponding V
ively.
were 1±52³0±48 and 1±19³0±74 nmol}mg}min respect-
max
2
. The formation of cis-nicotine-1 «-N-oxide was greater than the formation of trans-
nicotine-1«-N-oxide in rat liver microsomes and the intrinsic clearance of cis-nicotine-1 «-
N-oxide formation was 8±1-fold greater than that of trans-nicotine-1 «-N-oxide formation.
3
. The formation of both cis- and trans-nicotine-1 «-N-oxide in rat liver microsomes
was inhibited by the addition of 1-(1-naphthyl)-2-thiourea or by heat-treatment of
microsomes. 2-Diethylaminoethyl-2, 2-diphenylvalerate (SKF525A) and carbon mon-
oxide did not aåect these activities even at high concentrations.
4
. Formations of cis- and trans-nicotine-1 «-N-oxide correlated signi®cantly with each
other (r ¯ 0±862, p 0 01). These results suggested that the same ¯avin-containing
monooxygenase (FMO) isoform is responsible for the formation of cis- and trans-nicotine-
!
±
1
«-N-oxide in rat liver.
Introduction
In most mammalian species, nicotine is rapidly and extensively metabolized
primarily in the liver (Gorrod and Jenner 1975, Nakayama 1988). Although C-
oxidation (i.e. cotinine formation) is the major pathway of nicotine metabolism, the
formation of N-oxide derivatives of nicotine constitutes an important route of
nicotine biotransformation (Sepkovic et al. 1986). Nicotine-1«-N-oxide, the main
N-oxide metabolite of nicotine, was ®rst isolated and identi®ed from the reaction
mixture after incubation of a 9000 g supernatant of rabbit liver with nicotine
(
Papadopoulos and Kintzios 1963, Papadopoulos 1964). Nicotine-1«-N-oxide has
been identi®ed in humans and experimental animals in vivo (Beckett et al. 1971,
Thompson et al. 1985), as well as in vitro hepatic preparations from several
mammalian species (Kyerematen et al. 1990). The N-oxidation of nicotine has
received speci®c attention because of the interesting stereoselectivity in the
diastereoisomeric product formation (Jenner et al. 1973a, b). Beckett et al. (1973)
assigned the cis- and trans-nicotine-1«-N-oxide structure and showed the NMR
spectra. The separation of cis- and trans-nicotine-1«-N-oxide has been ®rstly
reported by Booth and Boyland (1970) using a paper chromatographic method.
It was reported that ¯avin-containing monooxygenase (FMO) appears to be the
major enzyme responsible for nicotine-1«-N-oxide formation in mammals (Damani
* Author for correspondence at Division of Drug Metabolism, Faculty of Pharmaceutical Sciences,
Kanazawa University, 13-1 Takara-machi, Kanazawa 920, Japan.
0
049±8254}98 $12±00 ’ 1998 Taylor & Francis Ltd

1
28
M. Nakajima et al.
et al. 1988, Nakayama 1988). In addition, some isoforms of cytochrome P450 are
also capable of the formation of nicotine-1«-N-oxide (Park et al. 1993). However, no
kinetic studies have yet been carried out with hepatic microsomes in any species. In
the current study the stereoselective formation of nicotine-1«-N-oxide diastereo-
isomers and its kinetic analyses in rat liver microsomes have been investigated.
M aterials and m ethods
Chemicals
(
S)-nicotine was purchased from Sigma Chemical Co. (St Louis, MO, USA). NADP+, glucose 6-
phosphate and glucose 6-phosphate dehydrogenase were from Oriental Yeast (Tokyo, Japan). Other
chemicals were of the highest grade commercially available. Analytical and preparative TLC were done
with 10¬20-cm FUNACEL SF cellulose thin layer plate (Funakoshi, Tokyo, Japan) and with 20¬
2
0-cm, 1000-lm cellulose microcrystalline PK2F (Whatman, Clifton, NJ, USA) respectively.
Synthesis of nicotine-1«-N-oxide
Nicotine-1«-N-oxide was synthesized by a method previously described (Taylor and Boyer 1959).
The diastereomers of nicotine-1«-N-oxide were separated on preparative TLC with 1-butanol}2-
propanol}ammonium hydroxide (2 : 1: 1, v}v}v) as described previously (Cashman et al. 1992). The
purities of each diastereomer have been con®rmed by HPLC. The structure of the each puri®ed synthetic
diastereomer was con®rmed by "H-NMR. "H-NMR spectra were recorded on a FT NMR SYSTEM,
JNM-LA500 (JEOL, Tokyo, Japan). These spectra (data not shown) were almost identical with a
previous report (Cashman et al. 1992). Thus, the synthesized nicotine-1«-N-oxide diastereomers were
used for authentic standards.
Preparation of rat liver microsomes
Male Sprague-Dawley rats (6 weeks old, 200±250 g) were supplied from Saitama Experimental
Animals Supply Co. Ltd (Saitama, Japan). Liver microsomes were prepared from the untreated rat as
described previously (Kamataki and Kitagawa 1974) and were stored at ®80 °C until use. Protein
concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as a
standard.
In vitro formation of nicotine-1«-N-oxide diastereomers
A typical incubation mixture (®nal volume, 0±5 ml) consisted of 50 mm potassium phosphate buåer
(
pH 8±4), NADPH-generating system (0±5 mm NADP+, 5 mm glucose 6-phosphate, 5 mm MgCl ,
#
1
U}ml glucose 6-phosphate dehydrogenase), 0±5 mg}ml microsomal protein. We selected the incubation
condition at pH 8±4 because it has been reported to be the optimum pH for FMO (Gold and Ziegler
973). The reaction was initiated by the addition of nicotine following a 2-min preincubation at 37 °C.
1
The reaction was terminated with 0±5 ml ice-cold acetone followed by addition of caåeine (50 ng) as an
internal standard. After removal of protein by centrifugation, the reaction mixtures were extracted with
4
ml 2-propanol}CH Cl (1 : 2, v}v) after saturation with solid Na CO and centrifuged at 1000 g for
# # # $
0 min to separate the aqueous and organic fractions. The organic fraction was evaporated under a gentle
1
stream of nitrogen at 40 °C and the residue redissolved in 50 ll of the mobile phase and a 20 ll portion
of the samples was subjected to HPLC.
HPLC analyses were performed using a DG-980-50 degasser, PU-980 intelligent pump (both Jasco,
Tokyo, Japan), AS-8010 autosampler (Tosoh, Tokyo, Japan), 807-IT integrator (Jasco) equipped with
a Capcell Pak C18 UG120 column (4±6¬250 mm, 5 lm ; Shiseido, Tokyo, Japan). The eluent was
monitored at 260 nm using a UV-970 intelligent UV}VIS detector (Jasco). The mobile phase was 7±5 %
acetonitrile containing 0±01% acetic acid and 1 mm sodium heptane sulphonate. The ¯ow rate was
1
±0 ml}min and the column temperature was 35 °C. The quantitation of the metabolite was performed by
comparing HPLC peak heights to those of authentic standards with reference to an internal standard
caåeine).
(
Kinetic analysis
The kinetic studies were performed using liver microsomes prepared from four diåerent rats with a
nicotine concentration ranging from 0±05 to 1 mm. Eadie±Hofstee plots were constructed for
determination of the presence of a mono- or a biphasic model. The maximum velocity (Vmax ) and
Michaelis±Menten constant (Km) were evaluated by non-linear regression analyses of untransformed
kinetic data.

Kinetics of nicotine N«-oxidation in rat liver
129
Figure 1. Representative HPLC chromatograms of formation of cis- and trans-nicotine-1 «-N-oxide
from nicotine in rat liver microsomes. The standard mixture spiked with cis- and trans-nicotine-
1
«-N-oxide, and caåeine as an internal standard extracted under the same conditions as described
for samples (A); the incubation mixture of rat liver microsomes without (B) and with (C) nicotine.
Peak : 1, caåeine ; 2, cis-nicotine-1 «-N-oxide ; 3, trans-nicotine-1 «-N-oxide; 4, nicotine.
Inhibition analysis
The eåects of the cytochrome P450-speci®c inhibitor, 2-diethylaminoethyl-2, 2-diphenylvalerate
hydrochloride (SKF525A) or a substrate of FMO, 1-(1-naphthyl)-2-thiourea, on cis- and trans-nicotine-
«-N-oxide formation were determined at a 0±1 or 1 mm nicotine concentrations respectively. The
1
incubation mixture including the chemical inhibitor (1 mm) was pre-incubated for 2 min before the
reaction was initiated by addition of the substrate. Separately, microsomes were bubbled with carbon
monoxide for 2 min or heat-treated at 45 °C for 5 min.
Statistical analyses
Correlation between cis- and trans-nicotine-1 «-N-oxide formation in liver microsomes from ten
diåerent rats at 0±1 mm nicotine concentration was determined by Pearson’s product moment method
(
performed by Student’s t-test for paired samples. Diåerences were considered signi®cant when p 0 05.
Pearson 1895). Values were expressed as mean³SD throughout the text. Statistical analysis was
!
±
Results
Representative HPLC chromatograms of formation of cis- and trans-nicotine-1«-
N-oxide from nicotine in rat liver microsomes are shown in ®gure 1. Retention times
for caåeine, cis-nicotine-1«-N-oxide, nicotine and trans-nicotine-1«-N-oxide were
1
6±0, 20±5, 23±0 and 28±0 min respectively. Cis- and trans-nicotine-1«-N-oxide
formation at a 0±1 mm nicotine concentration increased linearly with an incubation
time up to 30 min in the presence of 0±5 mg}ml microsomal protein (®gure 2A) and
with 1±0 mg}ml microsomal protein with 20-min incubation (®gure 2B). Unless
speci®ed, an incubation time of 20 min and 0±5 mg}ml microsomal protein were
employed to ensure initial rate conditions for the formation of nicotine-1«-N-oxide.
The calibration graphs were obtained with standards extracted under the same
conditions as described for the samples (®gure 1A). The stability of cis- and trans-
nicotine-1«-N-oxide during incubation was determined. As shown in ®gure 3, cis-

1
30
M. Nakajima et al.
Figure 2. Formation of cis- and trans-nicotine-1 «-N-oxide from nicotine as a function of incubation
time or the amount of microsomal protein. Incubations were carried out for varying times (A) or
with varying amounts of rat liver microsomes (B). D , cis-nicotine-1 «-N-oxide formation ; E ,
trans-nicotine-1 «-N-oxide formation. Data are the mean of duplicate determination.
Figure 3. Eåects of incubation time on the stability of cis- and trans-nicotine-1 «-N-oxide. Each value
represents the HPLC peak±height ratio to an internal standard. D , cis-nicotine-1 «-N-oxide; E ,
trans-nicotine-1 «-N-oxide. Data are the mean of duplicate determination.
Figure 4. Typical Eadie±Hofstee plots for cis- and trans-nicotine-1 «-N-oxide formation in rat liver
microsomes. D , cis-nicotine-1 «-N-oxide formation ; E , trans-nicotine-1 «-N-oxide formation.
Data are the mean of duplicate determination.

Kinetics of nicotine N«-oxidation in rat liver
131
Table 1. Kinetic parameters for nicotine-1«-N-oxide formation in rat liver microsomes.
K
mm)
Vmax
V
}K
m
max m
(
(nmol}mg}min) (ll}mg}min)
cis-nicotine-1 «-N«oxide
formation
trans-nicotine-1 «-N«oxide
formation
0±240³0±069
1±524³0±951
1±52³0±48
1±19³0±74
6±41³1±27
0±79³0±03
Kinetic analyses were determined by nicotine concentrations between 0±05 and 1 mm. All values
represent mean³SD (n ¯ 4).
Figure 5. Eåects of FMO or cytochrome P450 inhibitors, carbon monoxide or heat-treatment of
microsomes on the formation of cis- and trans-nicotine-1 «-N-oxide by rat liver microsomes. The
formation of cis- and trans-nicotine-1 «-N-oxide by rat liver microsomes were determined at
0
1
±1 mm (A) or 1 mm (B) nicotine concentration. The concentration of the chemical inhibitor was
mm. Microsomes were bubbled with carbon monoxide for 2 min or treated at 45 °C for 5 min.
Each column represents the percentage of the control (mean³SD, n ¯ 4). * , cis-nicotine-1 «-N-
oxide formation ; 7, trans-nicotine-1 «-N-oxide formation. ND, not detected. * p 0 05 and
!
±
!
±
* p 0 01 compared with control.
*
and trans-nicotine-1«-N-oxide in the incubation mixture were stable up to 90 min.
In addition, when one diastereomer was incubated, the diastereomer was stable and
the other diastereomer was not detected. Furthermore, no reduction to nicotine was
observed (data not shown).
The kinetic studies for cis- and trans-nicotine-1«-N-oxide formation in rat liver
microsomes were determined with 0±05±1 mm nicotine concentrations. Figure 4
shows typical Eadie±Hofstee plots and both plots for cis- and trans-nicotine-1«-N-
oxide formation in rat liver microsomes were linear. The apparent Km for cis- and
trans-nicotine-1«-N-oxide formation from the four samples were 0±240³0±069 and
1
±524³0±951 mm respectively. The Vmax were 1±52³0±48 and 1±19³0±74 nmol}
mg}min respectively (table 1). The intrinsic clearance (V }K ) of cis-nicotine-1«-
max
m
N-oxide formation was 8±1-fold greater than that of trans-nicotine-1«-N-oxide
formation. In rat liver microsomes, cis-nicotine-1«-N-oxide formation was greater
than trans-nicotine-1«-N-oxide formation.
The eåects of FMO or cytochrome P450 inhibitors, treatment of microsomes
with carbon monoxide or heat-treatment on the formation of cis- and trans-nicotine-
1
«-N-oxide were investigated at low (0±1 mm ) and high (1 mm) substrate concen-
trations. The formations of cis- and trans-nicotine-1«-N-oxide in rat liver micro-

1
32
M. Nakajima et al.
Figure 6. Correlation between cis- and trans-nicotine-1 «-N-oxide formation in microsomes from ten rat
livers at a 0±1 mm nicotine concentration.
somes were inhibited by 1-(1-naphthyl)-2-thiourea or heat treatment of microsomes.
The addition of SKF525A (2-diethylaminoethyl-2, 2-diphenylvalerate) to the
incubation mixture nor carbon monoxide aåect the formation of both cis- and trans-
nicotine-1«-N-oxide in rat liver microsomes even at a high substrate concentrations
(
®gure 5).
In the correlation analysis, the formations of cis- and trans-nicotine-1«-N-oxide
in ten rat liver microsomes signi®cantly correlated each other at a 0±1 mm
!
concentration, as shown in ®gure 6 (r ¯ 0±862, p
0±01).
Discussion
The HPLC method achieved a good separation of cis- and trans-nicotine-1«-N-
oxide as shown in ®gure 1. Compared with the incubation mixture of rat liver
microsomes without nicotine, all chromatograms were free from endogenous
compound interference. Nicotine eluted later than cis-nicotine-1«-N-oxide and
earlier than trans-nicotine-1«-N-oxide. However, since almost all nicotine volatilized
under the nitrogen stream, nicotine did not interfere with both cis- and trans-
nicotine-1«-N-oxide, as shown in ®gure 1C. In contrast with nicotine, it was
con®rmed (data not shown) that nicotine-1«-N-oxide was not volatile under the
nitrogen stream. It has been reported that the reduction of nicotine-1«-N-oxide to
nicotine could occur in vitro under anaerobic conditions (Dajani et al. 1972) and in
vivo in the gastrointestinal tract (Jenner et al. 1973c). However, the reduction of
neither cis- nor trans-nicotine-1«-N-oxide was observed during the incubation and
extraction procedures under aerobic conditions in this study. In addition, no
detectable diastereomeric conversion of either cis- and trans-nicotine-1«-N-oxide
was observed on incubation. This result is in agreement with the report of Cashman
et al. (1992). Therefore, the precise activities for nicotine-1«-N-oxide formation in
rat liver microsomes were determined reproducibly in the current study.
The formation ratios of cis-}trans-nicotine-1«-N-oxide in rat liver microsomes
were previously reported to be about 1±1 at a 5 mm nicotine concentration (Jenner et
al. 1973). In the current study, the formation of cis-nicotine-1«-N-oxide was greater
than the formation of trans-nicotine-1«-N-oxide in rat liver microsomes, over the

Kinetics of nicotine N«-oxidation in rat liver
133
range of nicotine concentrations investigated (0±05±1 mm ). However, the formation
ratio of cis-}trans-nicotine-1«-N-oxide was dependent on the nicotine concentration.
The higher the nicotine concentration was, the lower the formation ratio of cis-
trans-nicotine-1
«-N-oxide was, since the Km for trans-nicotine-1«-N-oxide form-
}
ation was higher than that for cis-nicotine-1«-N-oxide formation. Therefore, the
intrinsic clearance would be an adequate indicator of the formation ratio. As a result
of the kinetic analysis in this study, it was shown that the intrinsic clearance of cis-
nicotine-1«-N-oxide formation was 8±1-fold greater than that of trans-nicotine-1«-
N-oxide formation.
The eåects of the inhibitors of FMO or cytochrome P450 were determined for
investigation of the enzymes responsible for the formation of cis- and trans-nicotine-
1
1
«-N-oxide in rat liver microsomes. The lack of the eåects of SKF525A on nicotine-
«-N-oxide formation coincided with the previous work using guinea pig 10,000 g
hepatic preparations (Gorrod et al. 1971). The inhibition study suggested that the
contribution of cytochrome P450 to nicotine N-oxidation in the rat liver was
negligible and the enzymes responsible for these activities were FMOs. In a
preliminary experiment, the activities of cis- and trans-nicotine-1«-N-oxide form-
ation at pH 7±4, a physiological condition, were lower than that at pH 8±4. Because
the condition at pH 8±4 was reported to be optimum for FMO (Gold and Ziegler
1
973), the formation of nicotine-1«-N-oxide determined at pH 8±4 might be
maximum, as described herein.
In mammals, FMO has been reported to be responsible for the formation of
nicotine-1«-N-oxide (Damani et al. 1988, Nakayama 1988). The mammalian FMO
represents a multigene family and FMO1, FMO3, FMO4 and FMO5 have been
detected in human liver, and FMO2 is present in lung microsomes of the rabbit and
guinea pig (Hines et al. 1994). In contrast, only FMO1 have been reported in rat
liver (Itoh et al. 1993). It was reported that FMO1 and FMO3 produced nicotine-
1
«-N-oxide exclusively with stereoselectivity in the pig and human liver respectively
Park et al. 1993, Cashman et al. 1995). However, it is not clear which FMO isoform
(
is responsible for the formation of nicotine-1«-N-oxide diastereomers in rat liver.
The correlation of the formation of cis- and trans-nicotine-1«-N-oxide, as shown in
®
gure 6, suggest that the same FMO isoform might be responsible for both cis- and
trans-nicotine-1«-N-oxide formation, although this remains to be veri®ed.
Species diåerences in stereoselectivity of diastereoisomeric nicotine-1«-N-oxide
formation have also been reported. In rodents, cis-nicotine-1«-N-oxide formation
was reported to be preferred over trans-nicotine-1«-N-oxide formation and the
formation ratio of cis- and trans-nicotine-1«-N-oxide ranged from 1±1 to 10 (Jenner
et al. 1973b). On the other hand, in monkey and human liver, only trans-nicotine-1«-
N-oxide was formed. (Cashman et al. 1992, Park et al. 1993). Park et al. (1993) also
reported that the formation ratio of cis-}trans-nicotine-1«-N-oxide was 40}60 and
0
}100 in pig liver FMO1 and human liver FMO3 respectively. Therefore, it is
possible that the species diåerences in stereoselectivity of diastereomeric nicotine-
«-N-oxides formation are due to the diåerence of FMO isoform responsible for
1
metabolite formation. If the same isoform is involved in diastereomeric nicotine-1«-
N-oxide formation in several species, the species diåerences of stereoselectivity
might be due to the diåerences of the primary structure of the FMO isoform in these
species.
In summary, the kinetic properties of diastereoisomeric formation of nicotine-
«-N-oxide in rat liver microsomes have been established. We conclude that the
1

1
34
M. Nakajima et al.
same FMO form may be involved in the formation of both diastereomers of
nicotine-1«-N-oxide in the rat liver, but the speci®c FMO isoform which is
responsible remains to be established.
Acknow ledgem ent
This work was supported in part by a grant from The Smoking Research
Foundation, Japan.
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