Stereoselective metabolism of the monoterpene carvone by rat and human liver microsomes

Article abstract of DOI:10.1211/0022357001773841

The large amounts of carvone enantiomers consumed as food additives and in dental formulations justifies the evaluation of their biotransformation pathway. The in-vitro metabolism of R-(-)- and S-(+)-carvone was studied in rat and human liver microsomes using chiral gas chromatography. Stereoselective biotransformation was observed when each enantiomer was incubated separately with liver microsomes. 4R, 6S-(-)-Carveol was NADPH-dependently formed from R-(-)-carvone, whereas 4S, 6S-(+)-carveol was produced from S-(+)-carvone. Metabolite formation followed Michaelis-Menten kinetics exhibiting a significant lower apparent K(m) (Michaelis-Menten Constant) for 4R, 6S-(-)-carveol compared with 4S, 6S-(+)-carveol in rat and human liver microsomes (28.4±10.6 μM and 69.4±10.3 μM vs 33.6±8.5 μM and 98.3±22.4 μM). The maximal formation rate (V(max)) determined in the same microsomal preparations yielded 30.2±5.0 and 32.3±3.9 pmol (mg protein)^-1 min^-1 in rat liver and 55.3±5.7 and 65.2±4.3 pmol (mg protein)^-1 min^-1 in human liver microsomes. Phase II conjugation of the carveol isomers by rat and human liver microsomes in the presence of UDPGA (uridine S'-diphosphogluaronic acid) only revealed glucuronidation of 4R, 6S-(-)-carveol. V(max) for glucuronide formation was more than 4-fold higher in the rat liver compared with human liver preparations (185.9±34.5 and 42.6±7.1 pmol (mg protein)^-1 min^-1, respectively). K(m) values, however, showed no species-related difference (13.9±4.1 μM and 10.2±2.2 μM). This study demonstrated stereoselectivity in phase-I and phase-II metabolism for R-(-)- and S-(+)-carvone and might be predictive for carvone biotransformation in man.

Full text of DOI:10.1211/0022357001773841

J. Pharm. Pharmacol. 2000, 52: 191±197
Received August 12, 1999
Accepted October 8, 1999
# 2000 J. Pharm. Pharmacol.
Stereoselective Metabolism of the Monoterpene Carvone by Rat
and Human Liver Microsomes
È
WALTER JAGER, MARION MAYER, PETER PLATZER, GOTTFRIED REZNICEK*,
HERMANN DIETRICH{ AND GERHARD BUCHBAUER
Institute of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, A-1090 Vienna, *Institute of
Pharmacognosy, University of Vienna and {Central Laboratory Animal Facilities, University of Innsbruck,
Medical School, Innsbruck, Austria
Abstract
The large amounts of carvone enantiomers consumed as food additives and in dental
formulations justi®es the evaluation of their biotransformation pathway. The in-vitro
metabolism of R-(� )- and S-( ‡ )-carvone was studied in rat and human liver microsomes
using chiral gas chromatography.
Stereoselective biotransformation was observed when each enantiomer was incubated
separately with liver microsomes. 4R, 6S-(� )-Carveol was NADPH-dependently formed
from R-(� )-carvone, whereas 4S, 6S-( ‡ )-carveol was produced from S-( ‡ )-carvone.
Metabolite formation followed Michaelis±Menten kinetics exhibiting a signi®cant lower
apparent Km (Michaelis-Menten Constant) for 4R, 6S-(� )-carveol compared with 4S,
6
S-( ‡ )-carveol in rat and human liver microsomes (28Á4Æ 10Á6 mM and 69Á4Æ 10Á3 mM
vs 33Á6Æ 8Á5 mM and 98Á3Æ 22Á4 mM). The maximal formation rate (V ) determined
max
in the same microsomal preparations yielded 30Á2Æ 5Á0 and 32Á3Æ 3Á9 pmol
�
1
� 1
� 1
� 1
(
mg protein) min in rat liver and 55Á3Æ 5Á7 and 65Á2Æ 4Á3 pmol (mg protein) min
in human liver microsomes. Phase II conjugation of the carveol isomers by rat and human
liver microsomes in the presence of UDPGA (uridine S -diphosphogluaronic acid) only
0
for glucuronide formation was more
revealed glucuronidation of 4R, 6S-(� )-carveol. V
max
than 4-fold higher in the rat liver compared with human liver preparations (185Á9Æ 34Á5
�
1
� 1
and 42Á6Æ 7Á1 pmol (mg protein) min , respectively). K values, however, showed no
m
species-related difference (13Á9Æ 4Á1 mM and 10Á2Æ 2Á2 mM).
This study demonstrated stereoselectivity in phase-I and phase-II metabolism for
R-(� )- and S-( ‡ )-carvone and might be predictive for carvone biotransformation in man.
The R-(� )- and S-( ‡ )-enantiomers of the mono-
terpene ketone carvone are found in various plants.
While S-( ‡ )-carvone is the main constituent of the
essential oil of caraway, the oil of spearmint leaves
contains about 50% of R-(� )-carvone besides other
terpenes. Both enantiomers differ in odour and
taste; S-( ‡ )-carvone has an aromatic ¯avour while
R-(� )-carvone has a characteristic minty ¯avour.
They also have different uses in the food, fragrance
and pharmaceutical industry (Ziegler 1982; Wichtl
formulations, whereas R-(� )-carvone is frequently
added to toothpastes and chewing gum. Though the
acute toxicity of both enantiomers is of low order,
�
1
with reported LD50 values of 1640 mg kg in rats
and 766 mg kg
�
1
in guinea-pigs (Opdyke 1973,
1978), the large amounts consumed as food addi-
tives and in dental formulations should justify the
evaluation of their biotransformation pathway.
So far, only one metabolite has been detected in
urine samples after oral administration of rac-car-
vone to rabbits (Ishida et al 1989). Structural elu-
cidation of this biotransformation product revealed
a hydroxylation of the isopropenyl group. How-
ever, neither reduction of the carbonyl group to the
corresponding alcohol, as reported for the terpe-
noids pulegone and 2,5-bornanedione (Leibmann &
Ortiz 1973; Moorthy et al 1989 ), nor subsequent
1
997). S-( ‡ )-Carvone is widely used as a taste
enhancer in the food industry and, in smaller
amounts, as a carminative in many pharmaceutical
Correspondence: W. J aÈ ger, Institute of Pharmaceutical
Chemistry, University of Vienna, Althanstrasse 14, A-1090
Vienna, Austria.
E-Mail: wjaeger@speedy.pch.univie.ac.at

È
WALTER JAGER ET AL
1
92
glucuronidation of the formed hydroxyl group has
been demonstrated, as has been found for menthol
and other hydroxylated terpenes (Yamaguchi et al
(T : 95 min and 103 min, respectively), a Perkin
R
Elmer Auto System XL Gas Chromatograph (Per-
kin Elmer, Norwalk, USA) was used. The GC
column was a 12 ftÂ4 mm (i.d.) glass packed col-
umn (Chromasorb W-AW 80=100, 10Á0% OV-1;
Perkin Elmer, Norwalk, USA). The nitrogen carrier-
1
994; Green et al 1995; Green & Tephly 1996).
The aim of the present study was to investigate
the implication of stereochemistry on metabolite
formation of R-(� )- and S-( ‡ )-carvone in rat and
human liver microsomes using a sensitive chiral
gas chromatography method. Furthermore these
biotransformation products should be identi®ed by
enzymatic hydrolysis, by synthesized standards and
by mass spectroscopy.
�
1
gas ¯ow rate was 30 mL min . The injection port
and the detector were set at 260 C and the oven
ꢀ
temperature was held isothermally at 100 C.
ꢀ
Samples (10 mL; 3 mg) were introduced onto the
column by split injection (split ratio ˆ 1 : 3) and the
corresponding peaks were collected on dry ice
(purity of the single isomers > 95%, checked by
chiral GC).
Additional characterization of the puri®ed car-
veol isomers was carried out by GC=MS (gas
chromatography=mass spectrometry) using a Shi-
madzu QP-1000EX system (Shimadzu, Kyoto,
Japan). The GC column (SE 54) was a
Material and Methods
Materials
R-(� )- and S-( ‡ )-carvone ( p-mentha-6,8-dien-2-
one; optical purity > 99%), (� )-carveol ( p-men-
tha-6, 8-dien-2-ol; optical purity 97%; Z=E:
50 : 50), 2,2-dimethoxy-propane and dimethyl-
sulphoxide (DMSO) were obtained from Aldrich,
5
0 mÂ0Á25 mm (i.d.) capillary of 0Á25 mm ®lm
thickness (Machery & Nagel, Germany) operated
with a temperature program of 60 C for 2 min, and
ꢀ
then increased at 3 C min to 250 C. The helium
ꢀ
�
1
ꢀ
�
1
Munich, Germany. b-Glucuronidase type B-3 from
bovine liver, digitonin and uridine 5 -diphos-
carrier-gas ¯ow rate was 2 mL min . Samples
were introduced onto the column by split injection
0
phoglucuronic acid (UDPGA) were purchased
(
split ratio ˆ 1 : 2, opened 30 s after the injection;
from Sigma, Munich, Germany. rac-Piperitone
R=S:
ꢀ
injection port temperature 250 C). The column
ef¯uent was directly introduced into the ion source,
which was held at 180 C. Electron-impact spectra
were recorded at 50±600 amu (2 sec) , with an
ionization energy of 70 eV and with a vacuum
(
8
1-methyl-4-isopropyl-1-cylohexen-3-one;
5=15) was obtained from Roth, Karlsruhe, Ger-
ꢀ
�
1
many and methanol (Lichrosolv) was from Merck,
Darmstadt, Germany. All other chemicals and sol-
vents were of analytical grade and were used
without further puri®cation.
�
6
pressure of 8Á10 torr. Chemical ionization spectra
were recorded with ammonia as reactant gas (pre-
�
6
pressure of 1 bar, vacuum pressure of 8Á10 torr,
ionization energy 200 eV).
Synthesis of carveol isomers
Stereoselective reductions of R-(� )- and S-( ‡ )-
carvone (1Á6 mmol) were carried out at room tem-
perature with NaBH (3Á2 mmol) in an aqueous
Preparation of microsomes
4
Male Sprague±Dawley rats (180±250 g, raised at
the Institut f uÈ r Versuchstierzucht und -haltung,
University of Vienna, Himberg, Austria) were used
as liver donors. The human liver samples HL8
solution of sucrose (1 mM) according to the litera-
ture (Herv e du Penhoat et al 1991; Adams & Lerner
1
®
992; Denis et al 1996). After stirring for 12 h the
nal product was extracted with diethylether and
(
female, 57 years), HL11 (male, 57 years) and
the organic layer was evaporated under reduced
pressure. The presence of sucrose dramatically
enhanced axial entrance of the hydride since more
than 75% of 4R, 6S-(� )- and 4S, 6S-( ‡ )-carveol,
but less than 25% of the 4R, 6R-(� ) and 4S, 6R-
HL12 (male, 55 years) were obtained from patients
undergoing partial hepatectomy with their written
informed consent (II. University Clinics of Surgery,
Innsbruck, Austria). Liver samples were homo-
genized in 0Á1 M phosphate buffer (pH 7Á4) and
microsomes subsequently prepared using standard
procedures in our laboratory (J aÈ ger et al 1996).
Microsomes were stored as pellets (overlayed with
(
‡ ) stereoisomers, was formed as determined by
chiral gas chromatography (GC). The residue was
dissolved in dichloromethane and further puri®ed
by preparative GC.
ꢀ
glycerine=PBS) at � 70 C until use. Protein con-
centration (using bovine serum albumin as the
standard), total cytochrome P450 content (rat:
Puri®cation of carveol isomers by preparative GC
For separation of the isomeric mixtures 4R, 6R-(� )-
�
1
0Á92Æ 0Á21 ng (mg protein) ; human: 0Á61Æ
�
1
0
=
4R, 6S(� )- and 4S, 6S-( ‡ )-=4S, 6R-( ‡ )-carveol
0Á17 ng (mg protein) ) and Uridine 5 -diphos-

STEREOSELECTIVE METABOLISM OF CARVONE
193
ꢀ
phoglucuronosyl-transferase (UDPGT) activity
37 C in te¯on-capped glass tubes. After 2 h the
samples were centrifuged at 10 000g for 5 min and
the supernatant extracted and analysed for carvone
and itsmetabolites as describedpreviously. Incontrol
samples b-glucuronidase was replaced by buffer.
�
1
(
1
rat: 17Á38Æ 5Á78 mU (mg protein) ; human:
�
1
1Á03Æ 4Á01 mU (mg protein) ) were determined
according to the methods of Lowry et al (1951),
Omura & Sato (1964) and J aÈ ger et al (1997),
respectively.
Chiral-GC analysis
For chiral separation of the enantiomers of carvone
and its metabolites a Carlo-Erba HRGC 5160 Mega
Series instrument with a ¯ame ionization detector
and a 40 mÂ0Á25 mm Chiraldex b-cyclodextrin
In-vitro phase-I metabolism of R-(� )- and S-( ‡ )-
carvone
Carvone metabolites were formed in-vitro with rat
and human liver microsomes. The incubation
mixture contained 1 mg microsomal protein, 1 mM
NADPH and an NADPH-regenerating system
tri¯uoroacetyl column connected with
a
5 mÂ0Á25 mm retention gap guard column (both
�
1
(
5 mM MgCl , 5 mM isocitrate and 0Á5 U mL
Astec, Whippany, NJ) were used. The carrier gas
2
�
1
isocitrate dehydrogenase) in 0Á1 M sodium phos-
phate buffer, pH 7Á4 (J aÈ ger et al 1996). After a 5-
was helium, with a ¯ow rate of 0Á5 mL min .
Samples were injected onto the column by split
injection (split ratio ˆ 1 : 10; injection port temp-
ꢀ
min pre-incubation at 37 C, the reaction was star-
ꢀ
ted by the addition of 10±200 mM R-(� )- and S-
erature, 200 C). The oven temperature was held
ꢀ
(
yield an incubation DMSO concentration of not
‡ )-carvone (®nal volume 250 mL) in DMSO (to
isothermally at 90 C. The limit of quanti®cation for
R-(� )- and S-( ‡ )-carvone in liver micro-
�
1
greater than 0Á7% v=v) and allowed to proceed at
somal samples was 20Æ 3Á23 ng mL and 20Æ
�
1
ꢀ
3
7 C for 30 min with constant shaking. The reac-
2Á87 ng mL , respectively, by spiking drug-free
tion was stopped by addition of 750 mL of ice cold
methanol, and piperitone (100 ng mL ) was added
liver microsomes with the enantiomers to give ®nal
concentrations of 10, 15, 20, 25 and 30 ng mL
�
1
� 1
as internal standard. After centrifugation at 5000 g
ꢀ
(n ˆ 5). The injection volume was 1Á5 mL. The
analysed peaks were well separated from the peaks
of the internal standard, piperitone, with no over-
lapping matrix peaks.
for 5 min at 4 C, 1 mL of the supernatant was
injected onto the GC column. Control experiments
were performed in the absence of NADPH.
Data analysis
Estimates of apparent K and V were obtained
max
In-vitro phase-II metabolism of 4R, 6S-(� )- and 4S,
6
Microsomal glucuronidation of carvone metabolites
S-( ‡ )-carveol
m
by ®tting the data to the Michaelis±Menten equa-
tion, using the nonlinear curve-®tting package
Winzyme (PC version 2Á10, Biosoft, Cambridge,
UK). Assay conditions were such that metabolite
formation and parent consumption were linear with
respect to time of incubation and protein con-
centration. Results are expressed as the meanÆ s.d.
The signi®cance of differences (P < 0Á05) was
evaluated by the Student's t-test. The enzymatic
was studied by incubating 4R, 6R-(� )- and 4S, 6S-
(
‡ )-carveol (5±100 mM dissolved in DMSO),
respectively, with 1 mg microsomal protein in 0Á1 M
sodium phosphate buffer, pH 7Á4, 10 mM MgCl ,
2
�
1
and digitonin (1 mg (mg protein) ) in a total
volume of 250 mL. The reaction was started by the
addition of 2 mM UDPGA and allowed to proceed
ꢀ
for 60 min at 37 C. Incubations were stopped by
ef®cacy, that is de®ned as the ratio Vmax=Km,
the addition of 750 mL of ice-cold methanol and
piperitone (100 ng mL ), followed by centrifuga-
�
1
quanti®es the capacity of metabolism and corres-
ponds to the intrinsic clearance.
ꢀ
tion (5000Âg for 5 min at 4 C). The supernatant
(1 mL) was injected onto the GC column. Control
experiments in the absence of UDPGA were run
in parallel.
Results and Discussion
Microsomal preparations from three individual rat
and human livers were incubated with R-(� )- and
S-( ‡ )-carvone and subsequently analysed by
chiral GC. Under the analytical conditions selected,
R-(� )-carvone and S-( ‡ )-carvone, the two isomers
of the internal standard, R- and S-piperitone (R=S:
85=15), and the two metabolites were resolved
Incubation of microsomal samples with
b-glucuronidase
For detection of glucuronides, 250 mL of micro-
somal samples were incubated with 50 mL of b-
glucuronidase (50 000 U) in the presence of piper-
�
1
itone (5 mL of a stock solution of 10 mg mL ) at

È
WALTER JAGER ET AL
1
94
Figure 1. GC chromatogram of medium extracts from the
microsomal incubation of R-(� )-carvone. Human liver micro-
somes containing 1Á0 mg of microsomal protein were incubated
Figure 2. GC chromatogram of medium extracts from the
microsomal incubation of S-( ‡ )-carvone. Human liver micro-
somes containing 1Á0 mg of microsomal protein were incubated
ꢀ
ꢀ
with 200 mM of R-(� )-carvone for 30 min at 37 C without (A),
with 200 mM of S-( ‡ )-carvone for 30 min at 37 C without (A),
and in the presence of (B), NADPH. (� )-C: R-(� )-carvone;
and in the presence of (B), NADPH. ( ‡ )-C: S-( ‡ )-carvone;
(R)-IST: R-internal standard; (S)-IST: S-internal standard;
( ‡ )-M: metabolite 4S, 6S-( ‡ )-carveol.
(
R)-IST: R-internal standard; (S)-IST: S-internal standard; (� )-
M: metabolite 4R, 6S-(� )-carveol.
adequately and there was no interference from
endogenous medium constituents. Typical GC
chromatograms from the experiments using R-(� )-
carvone are shown in Figure 1 for the organic phase
after solid-phase extraction from an incubation with
human liver microsomes without the addition of
NADPH (Figure 1A), and from an incubation
containing NADPH (Figure 1B). Figure 1B iden-
ti®ed one R-(� )-carvone-derived peak with a GC
retention time of 32Á27 min. This peak was not
observed in the incubation extract shown in Figure
lites. The peaks derived from R-(� )-carvone
‡
exhibited M -ions at m=z 152 amu and main
fragment ions at m=z 109, 95, 91 and 84 amu,
respectively, and was different from the S-( ‡ )-
carvone-derived biotransformation product which
‡
showed no M -ions at m=z 152 amu but did have
more prominent fragment ions at m=z 134 and
119 amu. A mass-spectral library search (Adams
1989) con®rmed that R-(� )-carvone is exclusively
metabolized to (� )-carveol, whereas S-( ‡ )-car-
vone is biotransformed to ( ‡ )-carveol. By syn-
thesizing or isolating all four possible carveol
isomers, we were able to further identify the chiral
structure of the carveol metabolites formed. (� )-
Carveol, therefore, attributes to 4R, 6S-(� )-carveol
and ( ‡ )-carveol to 4S, 6S-( ‡ )-carveol. The for-
mation of 4R, 6R-(� )-carveol and 4S, 6R-( ‡ )-
carveol after incubation with either (� )-carvone or
( ‡ )-carvone, respectively, was below the detection
limit.
1
A. Parallel incubation with S-( ‡ )-carvone (Fig-
ure 2) again revealed the formation of only one new
peak in the chromatogram with a retention time of
4
0Á95 min (Figure 2B). These new GC peaks were
absent when heat-inactivated microsomes were
used, or when the substrates or NADPH-generating
system were omitted, indicating the involvement of
the cytochrome P450 system. Qualitatively similar
metabolic pro®les were observed in rat microsomal
preparations from three individual livers.
Formation of the two carveol isomers were linear
with time up to 40 min and with respect to micro1-
somal protein concentrations of 0Á4±1Á2 mg mL�
GC=MS studies were used in order to identify the
chemical structure of these two carvone metabo-

STEREOSELECTIVE METABOLISM OF CARVONE
195
Figure 3. Formation of 4R, 6S-(� )-carveol (A) and 4S, 6S-
Figure 4. Formation of (� )-(4R, 6S)-carveol (A) and ( ‡ )-
(4S;6S)-carveol (B) in human liver microsomes. A Michaelis±
Menten curve was generated by incubating (� )-(R)- and ( ‡ )-
(S)-carvone at varying concentrations in human liver micro-
somes in the presence of NADPH. Data represent the means
(Æ s.d.) of three determinations.
(
‡ )-carveol (B) in rat liver microsomes. A Michaelis±Menten
curve was generated by incubating R-(� )- and S-( ‡ )-carvone
at varying concentrations in rat liver microsomes in the
dp re et es er mn ci en a ot if o Nn sA. DPH. Data represent the meansÆ s.d. of three
(
data not shown). Contrary to ®ndings in a previous
carvone is a better substrate than the S-( ‡ ) enan-
tiomer. The ratio of enzymatic ef®cacies of R-(� )=
S-( ‡ ) ranges from 2Á6 to 2Á5 in rat and human liver
microsomes.
study (Ishida et al 1989) investigating the meta-
bolism of ( ‡ )-carvone in rabbits, neither R-(� )-
nor S-( ‡ )-carvone showed hydroxylation of the
isopropenyl group, indicating a different bio-
transformation depending on species.
The kinetic constants for this reaction were esti-
mated using R-(� )- and S-( ‡ )-carvone con-
centrations of up to 200 mM. Kinetic parameters Km
and Vmax were determined by direct least squares
regression of R-(� )- and S-( ‡ )-carvone con-
centrations (substrates) vs velocity data using the
Michaelis±Menten equation (Figures 3 and 4).
Biochemical evidence for glucuronic acid con-
jugates was obtained after incubation of 4R, 6S-
(� )-carveol and 4S, 6S-( ‡ )-carveol with rat and
human liver microsomes in the presence of
UDPGA. After UDPGA incubation the microsomal
samples were further treated with b-glucuronidase
(control experiments were run with buffer). GC
analysis of these samples only revealed an increase
of the 4R, 6S-(� )-carveol peak by b-glucuronidase
compared with non-treated samples, indicating
glucuronic acid conjugation of this biotransforma-
tion product. The formation rate of 4R, 6S-(� )-
carveol glucuronidation substantially differed in rat
and human liver preparations (Figure 5). While the
Using this approach, signi®cantly lower K values
m
for 4R, 6S-(� )-carveol were found in rat and
human liver microsomes (28Á4Æ 10Á6 mM and
6
(
9Á4Æ 10Á3 mM, respectively) compared with S-
‡ )-carveol (33Á6Æ 8Á5 mM and 98Á3Æ 22Á4 mM).
V
arations yielded higher values in human liver than
determined in the same microsomal prep-
apparent K
m
for 4R, 6S-(� )-carveol glucuronide
max
was similar in both species (rat: 13Á9Æ 4Á1 mM;
human: 10Á2Æ 2Á2 mM), Vmax was more than 4-fold
higher in the rat liver microsomes (rat:
in rat liver microsomes (55Á3Æ 5Á7 and
�
1
� 1
6
5Á2Æ 4Á3 pmol (mg protein) min vs 30Á2Æ 5Á0
� � 1
1
� 1
� 1
and 32Á3Æ 3Á9 pmol (mg protein) min ). In
terms of enzymatic ef®cacy (V =K ), R-(� )-
185Á9Æ 34Á5 pmol (mg protein) min ; human:
� � 1
1
(mg protein) min ).
42Á6Æ 7Á1 pmol
The
max
m

È
WALTER JAGER ET AL
1
96
( ‡ )-, 4R, 6R-(� )- and 4S, 6R-( ‡ )-carveol glu-
curonide was below the detection limit.
The stereoselective metabolism of R-(� )- and S-
(
summarized in Figure 6.
‡ )-carvone in rat and human liver microsomes is
In conclusion, our ®ndings show that carvone
metabolism is stereoselective and species depen-
dent. As stereoselectivity and stereospeci®city of
many endogenous biochemical processes, particu-
larly in phase-I and phase II-metabolism have been
characterized for many drugs (Williams & Lee
1985; Drayer 1986), it is becoming more apparent
that differences in metabolism may also result in
altered pharmacokinetics. An ongoing study shall
therefore elucidate the stereoselectivity of R-(� )-
carvone and S-( ‡ )-carvone in man.
Acknowledgements
This study was supported by grant P 11606-Med
from the Austrian Science Foundation, Vienna,
Austria. We also acknowledge Dragoco Comp.,
Vienna, Austria, for their interest in this study.
Figure 5. Glucuronidation of 4R, 6S-(� )-carveol in rat and
human liver microsomes. A Michaelis±Menten curve was
generated by incubating 4R, 6S-(� )-carveol at varying con-
centrations in rat (A) and human (B) liver microsomes in the
dp re et es er mn ci en a ot if o Un sD. PGA. Data represent the meansÆ s.d. of three
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m
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4
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