Detection of a "nonaromatic" NIH shift during in vivo metabolism of the monoterpene carvone in humans.

Article abstract of DOI:10.1021/jf011199h

High-resolution gas chromatography in combination with mass spectrometry and high-resolution mass spectrometry was used to determine the positions and extent of labeling in the metabolites of carvone, namely in alpha,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), alpha-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone), after human ingestion of 9,9-dideutero- and 9-(13)C-carvone. Carvonic acid was formed by oxidation at the methyl carbon of the isopropenyl group of carvone, whereas dihydrocarvonic acid was formed by oxidation at the methylene position, most probably via carvone epoxide. A "nonaromatic" NIH shift must occur during the subsequent reactions yielding dihydrocarvonic acid. Additionally, dehydrogenation of dihydrocarvonic acid and hydrogenation of carvonic acid were observed, resulting in minor amounts of both acids owning a carboxy group of opposite origin. Uroterpenolone was found to be exclusively formed by oxidation at the methylene carbon of the isopropenyl group of carvone, and thus, most probably by hydrolysis of carvone epoxide.

Full text of DOI:10.1021/jf011199h

1686 J. Agric. Food Chem. 2002, 50, 1686−1694
Detection of a “Nonaromatic” NIH Shift during In Vivo
Metabolism of the Monoterpene Carvone in Humans
WOLFGANG ENGEL*
Deutsche Forschungsanstalt fu¨r Lebensmittelchemie, Lichtenbergstrasse 4,
D-85748 Garching, Germany
High-resolution gas chromatography in combination with mass spectrometry and high-resolution mass
spectrometry was used to determine the positions and extent of labeling in the metabolites of carvone,
namely in R,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), R-methylene-4-
methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-
methyl-2-cyclohexen-1-one (uroterpenolone), after human ingestion of 9,9-dideutero- and 9-13C-
carvone. Carvonic acid was formed by oxidation at the methyl carbon of the isopropenyl group of
carvone, whereas dihydrocarvonic acid was formed by oxidation at the methylene position, most
probably via carvone epoxide. A “nonaromatic” NIH shift must occur during the subsequent reactions
yielding dihydrocarvonic acid. Additionally, dehydrogenation of dihydrocarvonic acid and hydrogenation
of carvonic acid were observed, resulting in minor amounts of both acids owning a carboxy group of
opposite origin. Uroterpenolone was found to be exclusively formed by oxidation at the methylene
carbon of the isopropenyl group of carvone, and thus, most probably by hydrolysis of carvone epoxide.
KEYWORDS: MICA; urinary metabolites; r,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid; r-methylene-
4-methyl-5-oxo-3-cyclohexene-1-acetic acid; 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one;
carvone; 9,9-dideutero-carvone; 9-(13C)-carvone; nonaromatic NIH shift
INTRODUCTION
prerequisite of a MICA experiment, contamination with other
terpenes from the diet or cosmetics is difficult to rule out.
Application of labeled carvone could conclusively prove the
origin of the proposed metabolites by the presence of the
incorporated label.
The aim of this study was, therefore, first to develop a
synthetic method that would provide carvone labeled with stable
isotopes, namely with deuterium and carbon 13, at relevant and
distinct positions; and second, to deduce the metabolic pathways
of carvone metabolism in MICA experiments in humans from
the distribution and the position of the labels found in the known
metabolites.
In a previous study (1) the following compounds were
identified as the major oxygenated metabolites of carvone in
humans using the metabolism of ingestion-correlated amounts
(MICA) approach: R,4-dimethyl-5-oxo-3-cyclohexene-l-acetic
acid (dihydrocarvonic acid, M1), R-methylene-4-methyl-5-oxo-
3-cyclohexene-1-acetic acid (carvonic acid, M2), and 5-(1,2-
dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uro-
terpenolone, M3). However, the structures of the metabolites
provided little information about the mechanisms by which they
were generated, especially if the methyl or the methylene carbon
of the isopropenyl group of carvone was oxidized. To our
knowledge there are no studies reported in the literature in which
labeling with stable or radioactive isotopes was performed to
clarify the oxidation mechanism of the isopropenyl group of
terpenes which occurs frequently, for example, in carvone,
limonene, carveol, and many others.
MATERIALS AND METHODS
Chemicals. Sodium metaperiodate was from Merck (Darmstadt,
Germany), triphenylphosphine, D3-iodomethane, 13C-iodomethane,
sodium hydride, and butyl lithium (2.5 mmol/mL in hexane) were from
Aldrich (Steinheim, Germany).
MICA Experiment. Human experimentation was performed as
previously described (1) except that only the author participated. The
amount of terpene applied was 15 mg (0.2 mmol; 200 µg/kg body mass)
of either 9,9-dideutero-carvone or 9-13C-carvone.
Isolation of Metabolites from Urine and Derivatization Proce-
dures. Isolation was performed as previously described (1). Briefly,
after enzymatic hydrolysis at pH 5.0 with glucuronidase and sulfatase
from Helix pomatia and acidification to pH 2.0 with concentrated HCl,
the metabolites were extracted with diethyl ether. The metabolites in
the acidic fraction were trimethylsilylated with BSTFA or ethylated
with iodoethane (1).
Therefore, a MICA experiment using site-specifically labeled
carvone should provide further valuable information about the
in vivo metabolic pathways of carvone in humans and more
generally of the isopropenyl group of terpenes. Additionally,
in the previous study (1) the metabolites of carvone were
identified only by comparison of control and test urine. Because
of the very low amount of carvone applied, which is a
* Author may be contacted by telephone 0049 (0)89 28914375, fax 0049
(0)89 28914183, or e-mail wolfy@dfa.leb.chemie.tu-muenchen.de.
10.1021/jf011199h CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/14/2002

Carvone Metabolism Mechanisms
J. Agric. Food Chem., Vol. 50, No. 6, 2002 1687
Purification of the Acidic Metabolites by High-Pressure Liquid
Chromatography (HPLC). The ethylated mixture of the acidic
metabolites was concentrated to about 50 µL and diluted with pentane
to a total volume of 100 µL. HPLC separation was performed in a
single run on a polar diol phase column (250 mm × 4.6 mm i. d.;
Lichrospher 100 Diol, 5 µm, Merck, Darmstadt, Germany) using a
pentane-diethyl ether gradient at a flow of 1 mL/min. The gradient
started with pentane/diethyl ether 92:8, v/v, and was raised within 40
min to a final concentration of pentane/diethyl ether 12:88, v/v.
Fractions were collected at 1-min. intervals, concentrated, and checked
by HRGC-MS for the presence of the ethyl esters of M1 (M1-ET) or
M2 (M2-ET). Both acids were localized in the fraction eluting between
12 and 13 min.
High-Resolution Gas Chromatography (HRGC)-Mass Spec-
trometry (MS). High-resolution gas chromatography was performed
using a nonpolar capillary column (30 m × 0.25 mm i. d.; DB-5,
J + W Scientific, Folsom, CA) in a model 5890 gas chromatograph
(Hewlett Packard, Heilbronn, Germany). Sample injection was per-
formed using the cold on-column technique. The volume injected ranged
from 0.2 to 0.5 µL. The temperature during HRGC was held at 35 °C
for 1 min and raised to 230 °C with a rate of 6 °C/min. Mass spectra
were recorded on a Finnigan MAT 95S (Finnigan, Bremen, Germany)
mass spectrometer in the electron ionization (EI) mode at 70 eV, in
the chemical ionization (CI) mode at 115 eV with isobutane as reagent
gas, and in high-resolution (HR) mode using perfluorokerosene as
internal standard.
Synthesis of 9,9-Dideutero-Carvone and 9-13C-Carvone. 5-(1,2-
dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one. The synthesis
was performed as described earlier (1). Briefly, carvone was epoxy-
genated, and the epoxide was cleaved by H2SO4 in aqueous THF.
5-Acetyl-2-methyl-2-cyclohexen-1-one. To a stirred solution of 5-(1,2-
dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (6.5 mmol, 1.2
g) in methanol (10 mL) and water (10 mL), a solution of sodium
metaperiodate (6.5 mmol, 1.4 g) in water (20 mL) was added dropwise
within 20 min. The mixture was stirred for further 40 minutes during
which a white precipitate appeared. The methanol was evaporated under
vacuum using a rotary evaporator, and the target compound was
extracted from the aqueous phase with diethyl ether (2 × 200 mL).
The extract was dried over Na2SO4 and evaporated to dryness. Yield
of 5-acetyl-2-methyl-2-cyclohexen-1-one was 82% (810 mg, 5.33
mmol); elemental composition (HRMS): C9H12O2, found (152.0845),
calculated (152.0834). MS (EI): m/z (relative intensity) 109 (100), 43
RESULTS AND DISCUSSION
Possible Mechanisms of Oxidative In Vivo Metabolism.
It is generally accepted that metabolism of xenobiotics starts
with either hydroxylation or epoxidation and, therefore, the
following mechanisms seem possible to explain the formation
of dihydrocarvonic acid (M1) and carvonic acid (M2) during
oxidative in vivo metabolism of carvone. In pathway 1 (Figure
1) carvone epoxide would be the first intermediate, which after
isomerization to the aldehyde and subsequent further oxidation
would lead to M1. Consequently, M2 would be a dehydroge-
nation product of M1. In pathway 1 the carboxy carbon atom
of both the acids is derived from the methylene carbon atom of
carvone. Pathway 2 (Figure 1) starts with hydroxylation at the
methyl group of the isopropenyl side chain of carvone yielding
an alcohol (3) which after further oxidation leads to M2.
Hydrogenation of the latter will result in formation of M1. The
carboxy carbon of the acids is now derived from the methyl
carbon of the isopropenyl side chain of carvone. Finally, in
pathway 3, a stabilized radical could be formed by hydrogen
abstraction prior to oxidation leading to equivalency of the
methyl and the methylene position yielding the acids M1 and
M2 with a 50% scrambling of methyl- and methylene-derived
carboxy groups.
1
(24), 81 (18), 79 (16), 95 (14), 108 (12), 53 (12), 82 (11). H NMR
(CDCl3): CH3-Cd (1.79, s, 3H), CH3-CdO (2.21, s, 3H), -CH2-
CH-CH2- (2.47-2.73, m, 4H), CH-CdO (3.07-3.17, m, 1H), CHd
C (6.71, m, 1H). 13C NMR (CDC3): CH3-Cd (15.6, DEPT +), CH2-
CHd (27.4, DEPT -), CH3-CdO (27.7, DEPT +), CH2-CdO (39.3,
DEPT -), CH-CdO (47.0, DEPT +), CH3-Cd (135.7, DEPT 0),
CH2-CHd (142.5, DEPT +), CH2-CdO (197.4, DEPT 0), CH3-
CdO (207.7, DEPT 0).
5-(2,2-dideutero-1-methylethenyl)-2-methyl-2-cyclohexen-1-one (9,
9-dideutero-carVone) and 5-(1-methyl-2-(13C)-ethenyl)-2-methyl-2-cy-
clohexen-1-one. A suspension of either 13C-methyl- or D3-methyl-
triphenyl-phosphonium bromide (2) (2.5 mmol, 1.0 g) in dry diethyl
ether (100 mL, dried over NaH) was stirred under a nitrogen
atmosphere, and a solution of butyllithium in hexane (1 mL, 2.5 mmol/
mL) was added dropwise at room temperature. The deep orange mixture
was stirred for an additional 10 min, and a solution of 5-acetyl-2-methyl-
2-cyclohexen-1-one (2.5 mmol, 380 mg) in dry diethyl ether (10 mL)
was then added dropwise. Stirring was continued for further 60 min,
after which the reaction mixture was washed with a solution of
KH2PO4 (3 mmol, 408 mg) in water (50 mL). The diethyl ether phase
was dried over Na2SO4 and evaporated to dryness by means of a rotary
evaporator at a water bath temperature of 37 °C. By HRGC the purity
of the crude product was in the range of 50-70% with a major impurity
of unreacted 5-acetyl-2-methyl-2-cyclohexen-1-one of approximately
20-40%. The crude reaction mixture was purified by column chro-
matography on a diol phase (Bakerbond Diol, 40 µm, pentane/diethyl
ether gradient). Yield of purified 5-(2,2-dideutero-1-methylethenyl)-
2-methyl-2-cyclohexen-1-one (9,9-dideutero-carvone) was 16% (62.5
mg, 0.41 mmol); purity 95% determined by HRGC; isotopic purity
determined by MS(CI) 94% D2, 5% 6 D1, 1% D0; elemental composition
(HRMS): C10H12D2O, found (152.1159), calculated (152.1165). MS
(EI), m/z (relative intensity) 82 (100), 110 (50), 95 (40), 109 (36), 54
(35), 39 (32), 107 (28), 41 (20), 152 (18). Yield of purified 5-(1-methyl-
2(13C)-ethenyl)-2-methyl-2-cyclohexen-1-one (9-13C-carvone) was 11%
(42.3 mg, 0.28 mmol); purity 97% determined by HRGC; isotopic purity
determined by MS(CI) 99%; elemental composition (HRMS): C913CH14O,
found (151.1059), calculated (151.1075). MS (EI), m/z (relative
intensity) 82(100), 109 (53), 94 (51), 54 (49), 108 (30), 107 (28), 39
(18), 41 (15), 151 (14).
For uroterpenolone (M3) also three possible pathways of
formation can be postulated (Figure 1). The primary alcohol
function of M3 might stem from either the methylene group
(pathway 1) or the methyl group (pathway 2) of the isopropenyl
side chain of carvone. Additionally, there might also be a
pathway operative in which scrambling occurs (pathway 3). In
pathway 1 the hypothetical intermediate carvone epoxide is
hydrated, whereas in pathways 2 and 3 hydration occurs at the
double bond of 10-hydroxy-carvone, a compound that has been
identified as a carvone metabolite in rabbits (3).
Synthesis of 9,9-Dideutero-carvone or 9-13C-Carvone. On
the basis of the postulated oxidation mechanisms (Figure 1)
only the positions 9 and 10 of carvone are suitable for labeling.
The following straightforward approach was developed to
synthesize carvone labeled at the methylene position at carbon
9. The synthesis started from unlabeled carvone which was
epoxygenated to yield 8,9-epoxy-carvone. The epoxide was
hydrolyzed with diluted sulfuric acid in water/THF producing
uroterpenolone which was cleaved with sodium metaperiodate
yielding 5-acetyl-2-methyl-2-cyclohexen-1-one. From reaction
of the latter with dideutero-methylene-triphenylphosphorane or
with 13C-methylene-triphenylphosphorane in a Wittig Reaction
either 9,9-dideutero-carvone or 9-13C-carvone was obtained
(Figure 2).
Deuterium Distribution in M1 and M2 from 9,9-Dideu-
tero-carvone. The deuterium-labeled carvone was metabolized
in a MICA experiment, and the metabolites were obtained as
NMR Spectroscopy. NMR spectra were recorded on a Bruker AM
360 (Bruker, Karlsruhe, Germany) in CDCl3 with TMS as internal
standard (δ ) 0 ppm).

1688 J. Agric. Food Chem., Vol. 50, No. 6, 2002
Engel
Figure 1. Possible mechanisms of oxidation of carvone during metabolism leading to dihydrocarvonic acid (M1), carvonic acid (M2), and uroterpenolone
(M3). The filled square represents the methylene carbon, and the empty square represents the methyl carbon of the isopropenyl side chain of carvone.
ion m/z in the cluster analyzed, the amount of the naturally
occurring (m + 1)/z isotopic ion caused by 13C was calculated
as (amount of C-atoms) × 0.011 × (area of m/z ion). The value
obtained was subtracted from the total area of (m + 1)/z
measured to obtain the value for the deuterium-labeled ion
(Tables 1 and 2). The same results for both compounds were
also obtained in the electron ionization (El) mode (data not
shown). In this case for D0-2-M1-ET the ions with m/z 102,
103, and 104 (Figure 3) resulting from McLafferty rearrange-
ment (4, 5) of the molecular ion and for D0-2-M2-ET the
molecular ions 208, 209, and 210 (Figure 4) were chosen for
the calculations. The area values (Tables 1 and 2) are uncor-
rected for the incomplete labeling of 9,9-dideutero-carvone
which was found to be 94% D2, 5% D1, and 1% D0 by HRGC-
MS in the CI mode, causing a negligible shift of the calculated
values.
Figure 2. Synthesis of 9,9-dideutero-carvone and 9-13C-carvone: (a)
From the results of the deuterium labeling it is obvious that
MCPBA in CH Cl2 at room temperature; (b) H SO in THF−H O (50:50,
2
2
4
2
at least two different mechanisms of oxidation must be present,
because the distribution of deuterium is not consistent with one
of the three possible pathways alone. Since about half of M1
carries only one deuterium, there must be the possibility of H-D
exchange during metabolism. The existence of nearly 20% of
mono-deuterated carvonic acid shows that the reaction between
both acids has to be reversible to some extent, as from the
possible pathways only nondeuterated or double-deuterated
carvonic acid can be generated directly. However, labeling with
13C is necessary to prove whether the undeuterated acids stem
from complete deuterium exchange or from oxidation at the
methylene position which, as a consequence, should also result
in complete loss of deuterium. Therefore, in a second experi-
ment, 9-13C-carvone was administered in a MICA experiment.
v/v) at room temperature; (c) NalO in MeOH−H 0 (50:50, v/v) at room
4
2
temperature.
described recently for unlabeled carvone (1). The acidic fraction
of an ethereal extract of the 24-h urine containing the metabolites
was derivatized with iodoethane and further purified by HPLC
on RP-18 material. The fractions containing the deuterated ethyl
esters of M1 and M2 (D0-2-M1-ET and D0-2-M2-ET) were
analyzed by high-resolution gas chromatography-mass spec-
trometry (HRGC-MS) in the chemical ionization (CI) mode
to determine the amount of deuterium labeling. Because the most
abundant isotopic ion and the mono-deuterated ion have the
same mass, the measured intensities in all cases had to be
corrected using the known natural isotopic distribution of carbon,
namely 98.9% 12C and 1.1% 13C. The labeling distribution was
calculated using the following method. Starting with the lowest
Mass Spectrometric Fragmentation of M1-ET. To ensure
proper interpretation of the 13C labeling experiment, the mass
spectrometric fragmentation of M1-ET had to be deduced by

Carvone Metabolism Mechanisms
J. Agric. Food Chem., Vol. 50, No. 6, 2002 1689
Figure 3. Mass spectra of unlabeled M1-ET and 13C-M1-ET and proposed mass spectrometric fragmentation of M1-ET. The filled square represents the
13C label of the methylene carbon, and the empty square represents the methyl carbon of the isopropenyl side chain of carvone.
comparison of high-resolution mass spectrometry (HRMS) data
of unlabeled M1, unlabeled M1-ET, D0-2-M1-ET, and 13C-M1-
ET (Figure 3, mass spectrum of unlabeled M1 and D0-2-M1-
ET not shown). The ion with m/z 165 (C10H13O2, all following
m/z values and elemental compositions refer to unlabeled M1-
ET) must be formed by R-cleavage of the molecular ion m/z
210 (C12H18O3) by loss of an ethoxy radical. The ion m/z 137
(C9H13O) is formed by further loss of carbon monoxide from
m/z 165 by charge-induced cleavage (Figure 3). The ions m/z
109 (C7H9O) and m/z 82 (C5H6O) are formed from the ring
carbonyl mother ion and upon further fragmentation give m/z
91 (C7H7) and m/z 54 (C4H6). Ion m/z 95 (C6H7O) is most
probably formed from ion m/z 137 by loss of propene. In the
case of 13C-M1-ET ion m/z 95 carries no label and therefore
its carbon atoms must stem from the ring, whereas, from the
spectrum of D0-2-M1-ET a considerable H-D exchange for
ion m/z 95 was detected giving rise to about 50% m/z 96.
Consequently, several hydrogen shifts involving the deuterium
from the methyl group and a ring contraction have to take place
during the reaction. Unfortunately, the base peak at m/z 102
formed by McLafferty rearrangement and the second most
intensive signal at m/z 74 are not suitable for the determination
of the labeling distribution. They contain both the methyl and
the carboxy carbon of M1 and thus are always totally shifted
by one unit to m/z 103 and m/z 75, respectively. In conclusion,
the ion with m/z 137, even though of minor relative intensity,
is the only one suitable for successful determination of the 13
distribution between the methyl and carboxy position.
C
13C Distribution in M1 from 9-13C-carvone. From the areas
measured for the ions with m/z 137 and 138 for unlabeled M1-
ET, a H-transfer cluster can be ruled out (Table 3). The area
of ion with m/z 138 is completely generated by natural 13C

1690 J. Agric. Food Chem., Vol. 50, No. 6, 2002
Engel
Figure 4. Mass spectra of unlabeled M2-ET and 13C-M2-ET and proposed mass spectrometric fragmentation of M2-ET. The filled square represents the
13C label of the methylene carbon, and the empty square represents the methyl carbon of the isopropenyl side chain of carvone.
abundance in the remaining nine carbon atoms of ion m/z 137.
The theoretical value calculated from (intensity of m/z 137) ×
(natural abundance of 13C) × (number of carbon atoms in ion
m/z 137) ) 2.052 × 106 × 0.011 × 9 ) 0.207 × 106 is in
good agreement with the measured value of 0.195 × 106. For
13C-M1-ET derived from 9-13C-carvone this percentage of the
area had to be subtracted from the measured value for the ion
m/z 138 to obtain the amount for methyl-13C labeled ion with
also m/z 138 (Table 3). From these calculations, about 63% of
the carboxy groups contain 13C and thus stem from the
methylene carbon, whereas about 37% do not contain the label
and, therefore, must stem from the methyl carbon of the
isopropenyl group of carvone.
Mass Spectrometric Fragmentation of M2-ET. The mass
spectrometric fragmentation of M2-ET was deduced by com-
parison of high-resolution mass spectrometry (HRMS) data of
unlabeled M2, unlabeled M2-ET, D0-2-M2-ET and 13C-M2-
ET (Figure 4, mass spectrum of unlabeled M2 and D0-2-M2-
ET not shown). Even though M2 is very closely related to M1,
its double bond leads to a completely different mass spectro-
metric fragmentation. As can be seen from the comparison of
the mass spectra of M1-ET (Figure 3) and M2-ET (Figure
4), because of the double bond in conjugation to the ester
function, the McLafferty rearrangement to form a hypothetical
ion m/z 100 does not take place. Overall, the presence of the
double bond results in peaks with larger relative intensities at

Carvone Metabolism Mechanisms
J. Agric. Food Chem., Vol. 50, No. 6, 2002 1691
Table 1. Areas below Mass Traces m/z 211, 212, 213, and 214
Table 3. Areas below the Mass Traces m/z 137 and 138 Determined
for M1-ET Derived from Unlabeled Carvone and 13C-M1-ET Derived
from 9-13C-carvone and Calculated Distribution for 13C-M1-ET
Determined for D -M1-ET in the Chemical Ionization Mode (CI) after
0-2
Ingestion of 9,9-dideutero-carvone and Calculated Distribution for
D -M1-ET (HH), D -M1-ET (HD), and D -M1-ET (DD)
0
1
2
m/z a
137
C H O
2.052
138
8
elemental composition
unl.-M1-ET area × 106 b
13C-M1-ET area × 106 c
C 13CH O
m/z a
211
2.161
212
4.584
213
3.202
214
9
13
13
0.195 d
area × 106 b
0.402 g
15.462
10.470 e
HH : 13CHH c 2.161
HD : 13CHD d
:
0.285
4.299
23.7 ± 0.3% f
47.5 ± 0.4% f
carboxy-labeled M1
R-methyl-labeled M1
15.462
:
1.530 f
8.940
63.2 ± 0.2% g
36.8 ± 0.2% g
:
0.567
2.635
DD : 13CDD e
:
0.348 g 28.8 ± 0.4% f
a m/z Value of the ion. b Measured area units below the mass trace in the
electron ionization (EI) mode for unlabeled M1-ET. c Measured area units below
the mass trace in the electron ionization (EI) mode for 13C-M1-ET. d Area resulting
from natural abundance of 13C in ion m/z 137. e Area resulting from natural
a m/z Value of the ion. b Measured area units below the mass trace in the
chemical ionization (CI) mode. c Calculated amount of the [M + H]+ ion C H O
12 19
3
(HH, m/z 211) and C 13CH O (13CHH, m/z 212). d Calculated amount of the [M
11
18 3
+ H]+ ion C DH O (HD, m/z 212) and C 13CDH O (13CHD, m/z 213).
12
18
3
11
17 3
abundance of 13Cin ion m/z 137 and from singly 13C labeled ion C 13CH O where
e Calculated amount of the [M + H]+ ion C D H O (DD, m/z 213) and
8
9
12
2 17 3
all remaining carbon atoms are 12C. f Theoretical area resulting from natural
abundance of 13C in ion m/z 137. g Mean %-values − deviation n − 1 weighting
determined from 5 experiments. All area values in the table represent a single
experiment and are used only to illustrate the calculation procedure. They do not
necessarily produce the exact mean %-values.
C
11
13CD H O (13CDD, m/z 214). f Mean %-values − deviation n − 1 weighting
2 16 3
determined from 5 experiments. All area values in the table represent a single
experiment and are used only to illustrate the calculation procedure. They do not
necessarily produce the exact mean %-values. g Discrepancy results from
instrumentation error and nonobservance of area produced by other isotopes.
Table 4. Areas below the Mass Traces m/z 133 and 134 Determined
for M2-ET Derived from Unlabeled Carvone and 13C-M2-ET Derived
from 9-13C-carvone and Calculated Distribution for 13C-M2-ET
Table 2. Areas below Mass Traces m/z 209, 210, 211, and 212
Determined for D -M2-ET in the Chemical Ionization Mode (CI) after
0-2
Ingestion of 9,9-Dideutero-carvone, and Calculated Distribution for
D -M2-ET (HH), D -M2-ET (HD), and D -M2-ET (DD)
0
1
2
m/z a
133
134
elemental composition
unl.-M2-ET area × 106 b
13C-M2-ET area × 106 c
C H O
1.844
2.093
C H O + C 13CH O
m/z a
209
4.138
210
3.625
211
9.751
212
9
9
9
10
8
9
1.038 d
8.649 e
area × 106 b
1.363 g
HH : 13CHH c 4.138
HD : 13CHD d
:
0.546
3.079
24.9 ± 0.1% f
18.7 ± 0.1% f
carboxy-labeled M2
R-methylene-labeled M2
2.093
:
1.178 f
7.471
22.2 ± 0.4% g
77.8 ± 0.4% g
:
0.406
9.345
DD : 13CDD e
:
1.234 g 56.4 ± 0.2% f
a m/z Value of the ion. b Measured area units below the mass trace in the
electron ionization (EI) mode for unlabeled M2-ET. c Measured area units below
the mass trace in the electron ionization (EI) mode for 13C-M2-ET. d Area resulting
from natural abundance of 13C in ion m/z 133 and from ion C H O with m/z 134.
a m/z Value of the ion. b Measured area units below the mass trace in the
chemical ionization (CI) mode. c Calculated area of the [M + H]+ ion C H O
12 17
3
(HH, m/z 209) and C 13CH O (13CHH, m/z 210). d Calculated area of the [M +
11
17 3
H]+ ion C DH O (HD, m/z 210) and C 13CDH O (13CHD, m/z 211). e Calculated
9
10
12
16
3
11
16 3
e Area resulting from natural abundance of 13C in ion m/z 133, from ion C H O
area of the [M + H]+ ion C D H O (DD, m/z 211) and C 13CD H O (13CDD,
9
10
12
2
15
3
11
2 15 3
with m/z 134, and from singly 13C labeled ion C 13CH O where all remaining carbon
m/z 212). f Mean %-values − deviation n − 1 weighting determined from 5
experiments. All area values in the table represent a single experiment and are
used only to illustrate the calculation procedure. They do not necessarily produce
the exact mean %-values. g Discrepancy results from instrumentation error and
nonobservance of area produced by other isotopes.
8
9
atoms are 12C. f Theoretical area resulting from natural abundance of 13C in ion
m/z 133 and from ion C H O with m/z 134 g Mean %-values − deviation n − 1
9
10
weighting determined from 5 experiments. All area values in the table represent a
single experiment and are used only to illustrate the calculation procedure. They
do not necessarily produce the exact mean %-values.
higher m/z values. From the quite intense molecular ion m/z
208 (C12H16O3), formed by removal of an electron at the ring
carbonyl oxygen, the ion series m/z 82 (C5H6O) and 54 (C4H6)
is formed which is analogous to the fragmentation of M1-ET.
Removal of an electron at the carbonyl of the ester function
results in two different ion series. First, by removal of an ethyl
radical the ion m/z 179 (C10H11O3) is formed, resulting in an
ion m/z 135 (C9H11O) after loss of carbon dioxide. Second,
another ion series [(1) in Figure 4] is initiated by abstraction
of a hydrogen from the cyclohexenone ring, resulting in the
base peak m/z 162 (C10H10O2) after loss of ethanol. Ion m/z
162 has a remarkable stability due to delocalization of both the
radical and the positive charge. It decomposes further, losing
carbon monoxide (m/z 134, C9H10O) and hydrogen (m/z 133,
C9H9O). If ketene is lost prior to carbon monoxide an analogous
ion series with m/z 120 (C8H8O), m/z 92 (C7H8, not shown in
Figure 4), and m/z 91 (C7H7) results. The high intensity, and
thus stability, of ion m/z 133 results from its aromatic character
and the stabilization of the charge by the electron-donating +M
effect of the hydroxy and the ethenyl group. As this ion is the
one with the lowest mass in the H cluster m/z 133, 134, 135,
and it carries only the methylene position of M2, it is most
suitable for determining the distribution of 13C in 13C-M2-ET.
13C Distribution in M2 from 9-13C-carvone. From the
measured values of the cluster 133, 134 in unlabeled M2-ET
the distribution of the label in 13C-M2-ET was calculated as
follows (all area values from Table 4). For 13C-M2-ET an
amount of 2.093 × 106 area units for m/z 133 (C9H9O) would
result in an area of 2.093 × 106 × (1.038 × 106/1.844 × 106)
) 1.178 × 106 for the expected amount of m/z 134 (C9H10O
plus C813CH9O resulting from natural abundance). This amount
would leave an area of 8.649 × 106 - 1.178 × 106 ) 7.471 ×
106 for the ion m/z 134 (C813CH9O) with the 13C label at the
methylene position. Consequently, about 78% of the 13C label
in M2 are still at the methylene position, whereas the remaining
22% have to be located at the carboxy group forming unlabeled
ion m/z 133 in 13C-M2-ET.
Mass Spectrometric Fragmentation of M3-TMS and
Deuterium Distribution in M3 from 9,9-Dideutero-carvone.
From the shift of m/z 147 in undeuterated trimethylsilylated M3
(M3-TMS) to m/z 149 in D2-M3-TMS (Figure 5) it could be
concluded that both deuterium atoms were still present in M3
and no deuterium exchange took place during metabolism. From
MS-EI the position was determined according to the mass
spectrometric fragmentation of M3-TMS (Figure 5), which was
deduced from HRMS. Ion m/z 153 (C9H13O2) being present in

1692 J. Agric. Food Chem., Vol. 50, No. 6, 2002
Engel
Figure 5. Mass spectra of unlabeled M3-TMS and D -M3-TMS and proposed mass spectrometric formation of relevant ions useful for the determination
2
of the deuterium labeling in M3-TMS.
the mass spectrum of M3-TMS and D2-M3-TMS with un-
changed intensity proves that the methyl group does not carry
deuterium and, therefore, stems from the unlabeled methyl group
of carvone. Ion m/z 147 (C6H15O2Si) in the mass spectrum of
unlabeled M3-TMS is completely shifted to m/z 149 in D2-
M3-TMS proving the presence of both deuterium labels in M3
at the HO-CH2- position. The 13C labeling experiment (data
not shown) fully confirmed the results obtained by the deuterium
labeling.
a methylene group would be oxidized to a carboxy group
without a hydride shift, more than 63% of M1 (Table 3) would
be expected not to carry any deuterium. However, only about
24% of M1 (Table 1) was found undeuterated and nearly 48%
was found mono-deuterated. This observation can be explained
only by the so-called NIH shift, which occurs in vivo during
enzymatic hydroxylation of aromatic compounds (6). In non-
enzymatic model experiments the NIH shift was observed during
acid-catalyzed rearrangements of deuterated epoxides (7) or 5,6-
dihydroxy-cyclohexa-1,3-dienes (8) synthesized from labeled
aromatic compounds. Probably the most common example of
a NIH shift is found in the biosynthesis of tyrosine from
phenylalanine (6, 9) where the 4-hydrogen of the phenyl ring
of phenylalanine is later partially found in the 3-position of
tyrosine. There is evidence for the involvement of an epoxide,
which in one case (10) was even isolated, or for a cationic (11)
or even radical intermediate (12). The mechanistic situation
becomes even more complicated because in two flavine mo-
nooxygenase model systems the NIH shift could not be detected
(13) indicating the possibility of a different mechanism.
However, to our knowledge a NIH shift has never been found
in connection with nonaromatic compounds and, generally, it
is assumed that aliphatic epoxides are either reduced to alcohols
or hydrated forming a 1,2-diol. Therefore, this work reports the
first NIH shift observed on a nonaromatic compound introducing
the term “nonaromatic” NIH shift. As it is yet unclear whether
epoxides are intermediates in all cases, the term “epoxide-
equivalent” will be used in the following text.
It is remarkable that the 13C distribution for M2, where about
22% of the label was located at the carboxy group and 78%
was located at the methylene group, is nearly the opposite of
the distribution found in M1, where about 64% of the label
was located at the carboxy group and only 36% was located at
the methyl group. Therefore, even though M1 and M2 are
closely related from a structural point of view, neither M2 is
the direct precursor for M1, nor is M1 the precursor for M2.
Consequently, both acids are primarily generated by independent
pathways.
From the results, the symmetrical-radical mechanism (path-
way 3 in Figure 1) cannot be completely ruled out, but because
of the following points it has to be considered unlikely or of
minor importance. The presence of 29% D2-M1 clearly shows
that reduction of D2-M2 without loss of deuterium is possible,
since there is no other formation pathway for D2-M1. As a
consequence, the presence of D1-M2 proves, that also the reverse
reaction, that is the dehydrogenation of D2-M1, is possible to
some extent. The same reaction will also generate D0-M2 from
both D0-M1 or D1-M1 being labeled at the carboxy-group.
Therefore, the amount of 22% M2 labeled at the carboxy group
can be explained without the symmetrical-radical mechanism
(pathway 3 in Figure 1).
It is noteworthy that the postulated product of the “nonaro-
matic” NIH shift, 8,9-dihydro-carvonaldehyde, will lose some
of the shifted hydride prior to further oxidation due to partial
enolization. This is very well reflected by the high percentage
(23% of total M1, Figure 6) of carboxy-labeled M1 not
containing any original hydrogens from carvone.
More importantly, the combined results of D2 and 13C labeling
for M1 prove a hydride shift during the process of oxidation. If

Carvone Metabolism Mechanisms
J. Agric. Food Chem., Vol. 50, No. 6, 2002 1693
Figure 6. Formation of M1, M2, and M3 during in vivo metabolism of carvone in humans. The outlined Hs represent the original hydrogen atoms of
carvone and the outlined Cs represent the methylene carbon of carvone. Percentage values for each metabolite calculated from the combined results
of the D- and 13C-labeling experiments.
ACKNOWLEDGMENT
From the results obtained for M3 it is obvious that it is
generated by pathway 1 in Figure 2 because the deuterium
labels were found at the primary alcohol function. In this case
the usual fate of an aliphatic epoxide-equivalent is observed
resulting in 1,2-diol formation after hydration. To conclude, the
following pathways lead to the major oxygenated in vivo
metabolites of carvone in humans (Figure 6). Carvone is
partially transformed to a carvone epoxide-equivalent which is
converted to M1 by rearrangement followed by further oxida-
tion. In another reaction sequence the epoxide-equivalent can
also be hydrolyzed to the 1,2-diol M3. Carvone is also oxidized
at the methyl carbon of the isopropenyl side chain. Most
probably 10-hydroxy-carvone is an intermediate, being rapidly
and completely further oxidized to M2. The alcohol, 10-
hydroxy-carvone, was identified in rabbits (2) but could not be
detected in MICA experiments (1). The conversion of M1 to
M2 by dehydrogenation and vice versa of M2 to M1 by
hydrogenation is possible but obviously slower than conjugation
or excretion. If the opposite were the case, the 13C label should
be equally distributed between both positions and, additionally,
the deuterium label should be more or less completely removed;
neither was found to be the case.
I gratefully acknowledge the support of Ines Otte and Julius
Scho¨nauer for mass spectrometry.
LITERATURE CITED
(1) Engel, W. In vivo studies on the metabolism of the monoterpenes
S-(+)- and R-(-)-carvone in humans using the metabolism of
ingestion-correlated amounts (MICA) approach. J. Agric. Food
Chem. 2001, 49, 4069-4075.
(2) Gajewski, J. J.; Peterson, K. B.; Kagel, J. R.; Huang, Y. C. J.
Transition-state structure variation in the Diels-Alder reaction
from secondary deuterium kinetic isotope effects. The reaction
of nearly symmetrical dienes and dienophiles is nearly synchro-
nous. J. Am. Chem. Soc. 1989, 111, 9078-9081.
(3) Ishida, T.; Toyota, M.; Asakawa, Y. Terpenoid biotransformation
in mammals. V. Metabolism of (+)-citronellal, (()-7-hydroxy-
citronellal, citral, (-)-perillaldehyde, (-)-myrtenal, cuminalde-
hyde, thujone, and (()-carvone in rabbits. Xenobiotica 1989,
19, 843-855.
(4) McLafferty, F. W. Mass spectrometric analysis. Molecular
rearrangements. Anal. Chem. 1959, 31, 82-87.

1694 J. Agric. Food Chem., Vol. 50, No. 6, 2002
Engel
(5) Kingston, D. G. I.; Bursey, J. T.; Bursey, M. M. Intramolecular
hydrogen transfer in mass spectra. II. McLafferty rearrangement
and related reactions. Chem. ReV. 1974, 74, 215-242.
(6) Guroff, G.; Daly, J. W.; Jerina, D. M.; Renson, J.; Witkop, B.;
Udenfried, S. Hydroxylation-induced migration. NIH shift.
Science 1967, 157, 1527-1530.
(7) Jerina, D. M.; Daly, J. W.; Witkop, B. The role of arene oxide-
oxepin systems in the metabolism of aromatic substrates. II.
Synthesis of 3,4-toluene-4-d oxide and subsequent “NIH shift”
to 4-hydroxytoluene-3-d. J. Am. Chem. Soc. 1968, 90, 6523-
6525.
(8) Jerina, D. M.; Daly, J. W.; Witkop, B. Deuterium migration
during the acid-catalyzed dehydration of 6-deuterio-5,6-dihy-
droxy-3-chloro-1, 3- cyclohexadiene, a nonenzymatic model for
the NIH shift. J. Am. Chem. Soc. 1967, 89, 5488-5489.
(9) Lehmann, W. D.; Heinrich, H. C. The NIH-shift in the in vivo
hydroxylation of ring-deuterated L-phenylalanine in man. Arch.
Biochem. Biophys. 1986, 250, 180-185.
(10) Boyd, D. R.; Sharma, N. D.; Harrison J. S.; Hamilton, J. T. G.
McRoberts, W. C.; Harper, D. B. Isolation of a stable benzene
oxide from a fungal biotransformation and evidence for an “NIH
shift” of the carbomethoxy group during hydroxylation of methyl
benzoates. Chem. Commun. 2000, 16, 1481-1482.
(11) Vanelli, T.; Hooper, A. B. NIH shift in the hydroxylation of
aromatic compounds by the ammonia-oxidizing bacterium Ni-
trosomonas europaea. Evidence against an arene oxide inter-
mediate. Biochemistry 1995, 34, 11743-11749.
(12) Bhatt, M. V.; Periasamy, M. Rearrangements in the cerium(IV)
and manganese(III) oxidations of substituted naphthalenes and
the NIH shift mechanism. Tetrahedron 1994, 50, 3575-3586.
(13) Smith, J. R. L.; Jerina, D. M.; Kaufman, S.; Milstein, S. Aromatic
hydroxylation mediated by flavin autoxidation. Lack of the NIH
shift. J. Chem. Soc., Chem. Commun. 1975, 21, 881-882.
Received for review September 10, 2001. Revised manuscript received
December 14, 2001. Accepted December 18, 2001. I thank the Deutsche
Forschungsanstalt fu1r Lebensmittelchemie for financial support of this
work.
JF011199H