![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 29, Issue 12, 1567-1577, December 2001
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
(R)-(+)-Pulegone, a monoterpene ketone, is a major component of pennyroyal oil. Ingestion of high doses of pennyroyal oil has caused severe toxicity and occasionally death. Studies have shown that metabolites of pulegone were responsible for the toxicity. Previous metabolism studies have used high, near lethal doses and isolation and analysis techniques that may cause degradation of some metabolites. To clarify these issues and further explore the metabolic pathways, a study of 14C-labeled pulegone in F344 rats at doses from 0.8 to 80 mg/kg has been conducted. High-pressure liquid chromatography (HPLC) analysis of the collected urine showed the metabolism of pulegone to be extensive and complex. Fourteen metabolites were isolated by HPLC and characterized by NMR, UV, and mass spectroscopy. The results demonstrated that pulegone was metabolized by three major pathways: 1) hydroxylation to give monohydroxylated pulegones, followed by glucuronidation or further metabolism; 2) reduction of the carbon-carbon double bond to give diastereomeric menthone/isomenthone, followed by hydroxylation and glucuronidation; and 3) Michael addition of glutathione to pulegone, followed by further metabolism to give diastereomeric 8-(N-acetylcystein-S-yl)menthone/isomenthone. This 1,4-addition not only took place in vivo but also in vitro under catalysis of glutathione S-transferase or mild base. Several hydroxylated products of the two mercapturic acids were also observed. Contrary to the previous study, all but one of the major metabolites characterized in the present study are phase II metabolites, and most of the metabolites in free forms are structurally different from those previously identified phase I metabolites.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Pulegone is a monoterpene ketone present in
essential oils from many mint species (Grundschober, 1979
). Two mints,
Hedeoma pulegoides and Mentha pulegium, both
commonly called pennyroyal, contain essential oils, which are chiefly
pulegone (Budavari, 1996). Pennyroyal oil has been used as a flavoring
agent in foods and beverages, as well as a component in fragrance
products and flea repellents (Hall and Oser, 1965; Tyler, 1993).
Pennyroyal herb has also been used for the purpose of inducing
menstruation and abortion (Tyler, 1993). However, high doses of
pennyroyal oil have sometimes been taken in attempted abortion and have
resulted in central nervous system toxicity, gastritis, hepatic
and renal failure, pulmonary toxicity, and death (Anderson et al.,
1996). Pulegone was found to constitute greater than 80% of the
terpenes in pennyroyal oils that were obtained from health food stores and was found to be both hepatotoxic and pneumotoxic in mice (Gordon et
al., 1982).
Gordon et al. (1987) have shown that metabolites of pulegone were
responsible for its toxicity and have implicated menthofuran as a
proximate toxin. Metabolism studies in rats treated with relatively
high doses of pulegone have demonstrated complex metabolic pathways.
About 14 phase I pulegone metabolites were fully characterized after
acid treatment and ether extraction of urine samples from rats dosed
orally with four daily 250- or 400-mg/kg doses (Moorthy et al., 1989;
Madyastha and Raj, 1993). No quantitative data were provided in these
studies. Ten phase II biliary metabolites of pulegone were partially
characterized by tandem mass spectrometry after the rats were treated
with a single i.p. dose of 250 mg/kg (Thomassen et al., 1991). These
metabolites accounted for only 3% of total radioactivity excreted in
bile, and their structures could not be established solely from mass
spectral analysis. The dose (250 mg/kg) used in these metabolism
studies has been shown to result in centrilobular hepatic necrosis and
widespread alkylation of tissue proteins (McClanahan et al., 1989).
Pulegone has been nominated to the National Toxicology Program (NTP) for toxicity and carcinogenicity studies based on the potential for human exposure and the absence of carcinogenicity data. We were concerned that the high doses used in previous studies might alter the metabolite profile, and we wanted to characterize the expected phase II metabolites excreted in urine. To explore the metabolic pathway in detail and to quantitate the metabolites, we performed metabolism studies at lower doses and minimized chemical treatment to harvest the metabolites. A metabolism study of 14C-labeled pulegone in F344 rats at single oral (0.8, 8, and 80 mg/kg) or i.v. (0.8 mg/kg) doses or four daily oral doses (80 mg/kg/day) has been conducted. Fourteen major urinary metabolites have been characterized.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Chemicals. Methylethylidene-14C-(R)-(+)-pulegone (specific activity, 60.6 mCi/mmol; radiochemical purity, 97.4%) was obtained from Wizard Laboratories, Inc. (West Sacramento, CA). Unlabeled pulegone (98% pure), trifluoroacetic acid (TFA1), and tetrabutylammonium bromide (99% pure) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Menthofuran (95% pure) was obtained from Acro Organics (Pittsburgh, PA). Piperitone (92% pure) was obtained from Pfaltz & Bauer, Inc. (Waterbury, CT). Reduced glutathione (GSH) (97% pure) was purchased from Fluka BioChemika (Milwaukee, WI). Glutathione S-transferase (GST) (75% pure) from rat liver, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, and NADP+ were purchased from Sigma Chemical Co. (St. Louis, MO). 2'-Hydroxy-4'-methylacetophenone (approximately 75% pure) was purchased from Indofine Chemical Co., Inc. (Somerville, NJ).
Spectra. 1H NMR spectra were acquired on a Nicolet NT-360 NB (Thermo Nicolet, Madison, WI) or a Varian 300 MHz NMR spectrometer (Varian, Palo Alto, CA). The chemical shifts are reported in parts per million relative to solvents. Electrospray ionization (ESI) mass spectra were obtained on a Finnigan/ThermoQuest LCQ DUO ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). Tandem mass spectra (MS/MS) were produced by collision-induced dissociation of the selected parent ions with the He gas present in the mass analyzer. Most samples were dissolved in MeOH-H2O (1:1) for direct infusion analysis (2.5 µl/min) unless otherwise indicated. The heated capillary was maintained at 200°C and the source voltage at 4.5 kV. The GC/MS instrument used for analyzing 10-hydroxypulegone was a Finnigan/ThermoQuest TraceMS, equipped with a Trace 2000 GC. Injections were made using a J&W Scientific (Folsom, CA) cold on-column injector. Samples were injected with an SGE 10-µl syringe (SGE, Inc., Austin, TX) fitted with a fused silica needle. GC conditions were: carrier gas, He; oven temperature program, held for 10 min at 100°C, then increased at 10°C/min to 300°C, and held at 300°C for 15 min. The retention time of 10-hydroxypulegone was 14.4 min.
HPLC.
HPLC analyses were carried out with one of two systems. System A
consisted of two Waters model 510 pumps (Milford, MA), an automated
gradient controller, and a model 481 UV detector. System B consisted of
a Beckman System Gold model 126 solvent module pump and a model 168 photodiode array detector, controlled by Nouveau software (Beckman
Coulter, Inc., Fullerton, CA). System B was connected to an
IN\US (Tampa, FL) 
-Ram flow detector equipped with a liquid cell
(500 µl) for radiochemical detection. Liquid scintillation fluid
Ultima-Flo M (Packard Instrument Company, Meriden, CT) was delivered in
3:1 scintillation/elute ratio. A Metachem (Torrance, CA)
Inertsil C18 5-µm column (4.6 × 250 mm) was used for all studies unless otherwise indicated.
Animal Dosing and Sample Collection. Male and female F344 rats were obtained from Taconic Farms, Inc. (Germantown, NY). Female rats were 12 to 13 weeks old and weighed 160 to 195 g. Male rats were 11 weeks old and weighed 225 to 333 g.
Single and multiple doses of pulegone were administered by gavage or i.v. to rats (n = 4-8/treatment group), as described in Table 1. Oral doses were administered at 40 µCi/kg in a dose volume of 4 ml/kg in corn oil. The i.v. dose (0.8 mg/kg) was administered at 40 µCi/kg in a dose volume of 1 ml/kg in water (80%), Emulphor (10%), and ethanol (10%). Animals were housed individually in plastic metabolism cages and provided with food (NIH no. 31) and distilled water for ad libitum consumption. Urine was collected at room temperature 4, 8, 12, 24, 48, and 72 h after dosing. Urine samples were stored at
20°C and
centrifuged at low gravity before analysis by HPLC. The Institutional
Animal Care and Use Committee approved all animal procedures.
|
Syntheses.
Pulegol
Pulegol was synthesized according to a known method (Moorthy et al.,
1989). Its NMR data are consistent with the literature report.
Microsomal incubation of pulegone to prepare 10-hydroxypulegone.
F344 rat liver microsomal fractions were prepared by the method of
Guengerich (1982). A large scale (50 ml) incubation of [14C]pulegone (1 mM; specific activity, 0.02 µCi/µmol) with rat liver microsomes (2 mg of protein/ml) was
conducted in 0.1 M potassium phosphate buffer, pH 7.4, in the presence
of 25 mM glucose 6-phosphate, glucose-6-phosphate dehydrogenase (2 units/ml), 4 mM NADP+, 3 mM
MgCl2, and 1 mM EDTA. Pulegone was added as a
dimethyl sulfoxide solution (0.5 ml). After a 30-min incubation at
37°C in capped vials, reactions were terminated by addition of 0.3 N
Ba(OH)2 (5 ml) and 0.3 N
ZnSO4 (5 ml). Following centrifugation, the
supernatant was filtered through an Acrodisc (Gelman, Ann Arbor, MI;
0.45 µm, 13 mm), and the filtrate was extracted with ether (2 × 50 ml). The combined ether layers were evaporated, and the residue was
redissolved in H2O (5 ml) for HPLC separation (method 16, system A, 250 nm). Spectral properties of the major radiolabeled metabolite (10.9 min): 1H NMR (300 MHz, D2O): 
4.17 (AB quartet,
J = 12.6 Hz, 2H, 10-CH2), 2.82 (dt, J = 15.1, 4.5 Hz, 1H,
3-CHeq), 2.56 (ddd, J = 14.8, 4.4, 1.9 Hz, 1H, 6-CHeq), 2.29 (br. t,
J = 12.6 Hz, 1H, 3-CHax), 2.22 (dd, J = 14.6, 10.7 Hz, 1H,
6-CHax), 2.13 to 2.01 (m, 1H, 5-CHax), 1.97 to 1.89 (m, 1H,
4-CHeq), 1.86 (d, J = 1.1 Hz, 3H, 9-CH3), 1.40 (qd, J = 11.7, 4.4 Hz, 1H, 4-CHax), 1.00 (d, J = 6.3 Hz, 3H, 5-CH3). Positive ion ESI-MS/MS [in
MeOH-H2O (1:1) + 2% acetic acid]:
m/z 169 (M + H+), 151 (M + H+ H2O). GC/EI-MS (in
acetone): m/z 168 (M·+), 150 (M H2O), 139 (M HCO), 108 (M H2O C3H6); UV:
max 248 nm. The GC/MS data of the major
product are consistent with those of 10-hydroxypulegone, as reported in
the literature (McClanahan et al., 1988).
7a-Hydroxy-3,6-dimethyl-5,6,7,7a-tetrahydro-2(4H)-benzofuranone.
This compound was prepared by a known method (Thomassen et al., 1992).
1H NMR (CDCl3, 360 MHz):
2.96 (s, 1H, -OH), 2.68 (dt, J = 13.8, 2.1 Hz, 1H,
4-CHeq), 2.41 to 2.32 (m, 2H), 2.06 to 1.93 (m,
2H), 1.81 (s, 3H, 3-CH3), 1.27 (t,
J = 12.7 Hz, 1H, 6-CHax), 1.02 (qd, J = 13.2, 4.1 Hz, 1H,
5-CHax; overlapping with
6-CH3), 0.98 (d, J = 6.2 Hz, 3H,
6-CH3); UV: max 220 nm.
2-(2-Hydroxy-4-methylphenyl)propionic acid.
The synthesis was carried out following modification of a literature
method for the desmethyl analog (Rewcastle et al., 1991). 2'-Hydroxy-4'-methylacetophenone (1.98 g, 0.013 mol) was stirred with
benzyl chloride (2.3 ml), aqueous NaOH (0.67 g in 10 ml of H2O), and tetrabutylammonium bromide (0.45 g,
0.001 mol) in CH2Cl2 (10 ml) at room temperature. After 24 h, the organic layer was separated, washed three times with H2O, and dried
with anhydrous CaCl2. After the solvent was
removed, the product was purified by flash column chromatography
(ether/hexane, 1:4). Thin layer chromatography (silica gel 60 plate,
ether/hexane, 1: 4); Rf = 0.22; yield,
2.07 g (81%). 1H NMR
(CDCl3, 360 MHz): 7.69 (d, J = 8.1 Hz, 1 H, 6-H), 7.46 to 7.38 (m, 5 H, benzyl-Ph), 6.84 (s, 1H,
3-H), 6.83 (d, J = 7.7 Hz, 1 H, 5-H), 5.15 (s, 2H,
benzyl-CH2), 2.57 (s, 3H,
4-CH3), 2.38 (s, 3H,
COCH3).
7.44 to 7.35 (m, 5H, benzyl-Ph), 7.25 (d, J = 8.8 Hz, 1H, 6-H), 6.80 (d, J = 8.8 Hz, 1H, 5-H), 6.79 (s,
1H, 3-H), 5.16 (q, J = 7.0 Hz, 1H, CHOH),
5.12 (s, 2H, benzyl-CH2), 2.58 (br. s, 1H, OH),
2.34 (s, 3H, 4-CH3), 1.51 (d, J = 7.0 Hz, 3H, CHCH3).
The alcohol (1.72 g, 0.007 mol) in dioxane (10 ml) was added to a
solution of anhydrous CaCl2 (1.69 g, 0.015 mol)
in concentrated HCl (3.4 ml). The viscous mixture was stirred at room
temperature for 1 h and then diluted with a mixture of EtOAc and
ice. The organic layer was washed with H2O and 2 N NaOH (20 ml), dried with anhydrous
Na2SO4, and the solvent was
removed (yield, 1.45 g; 80%). 1H NMR
(CDCl3, 360 MHz): 7.47 to 7.32 (m, 5H,
benzyl-Ph), 7.35 (d, J = 8.8 Hz, 1H, 6-H), 6.82 (d,
J = 8.1 Hz, 1H, 5-H), 6.77 (s, 1H, 3-H), 5.64 (q,
J = 6.6 Hz, 1H, CHCl), 5.11 (ABq,
J = 11.7 Hz, 2H, benzyl-CH2),
2.34 (s, 3H, 4-CH3), 1.82 (d, J = 7.0 Hz, 3H, CHCH3).
The chloride from the above reaction (1.45 g, 0.006 mol) was dissolved
in dimethyl sulfoxide (10 ml). NaCN (0.49 g, 0.009 mol) was added, and
the mixture was refluxed for 1 h. After cooling and dilution with
ice, the mixture was extracted twice with EtOAc, dried with anhydrous
Na2SO4, and the solvent was
removed (yield, 2.28 g; 150%). 1H NMR
(CDCl3, 360 MHz): 7.32 to 7.23 (m, 5H,
benzyl-Ph), 7.24 (d, J = 10.0 Hz, 1H, 6-H), 6.78 (d,
J = 9.2 Hz, 1H, 5-H), 6.77 (s, 1H, 3-H), 5.18 to 5.06 (m, 3H, benzyl-CH2 + CHCN), 2.33 (s, 3 H, 4-CH3), 1.49 (d, J = 6.6 Hz,
3H, CHCH3).
The crude nitrile from the above reaction (2.28 g, 0.009 mol) was
dissolved in a solution of EtOH (15 ml) and 2 N NaOH (15 ml) and
refluxed for 24 h. After EtOH was removed, the pH of the remaining
mixture was adjusted to 9 by addition of 2 N HCl. The mixture was
extracted with EtOAc, and the aqueous layer was acidified to pH 2 with
2 N HCl, then extracted again with EtOAc. The second EtOAc extraction
layer was dried with anhydrous
Na2SO4, and the solvent was
removed. 1H NMR of the residue showed a mixture
of two products, which were separated by HPLC (method 7, system A, 225 nm). 1H NMR of the product with retention time
10.3 min (CDCl3, 300 MHz): 7.05 (d,
J = 7.8 Hz, 1H, 6-H), 6.74 (d, J = 8.4 Hz, 1H, 5-H), 6.71 (s, 1H, 3-H), 3.93 (q, J = 7.5 Hz,
1H, CHCOOH), 2.29 (s, 3H, 4-CH3),
1.56 (d, J = 7.5 Hz, 3H,
CHCH3); UV:
max 204, 217, 275 nm; negative ion ESI-MS/MS:
m/z 179 (M H+), 135 (M COOH+). The spectral results are
consistent with formation of 2-(2'-hydroxy-4'-methylphenyl)propionic acid.
2-(N-Acetylcystein-S-yl)menthofuran.
The synthesis was carried out following modification of a literature
method for 2-(glutathion-S-yl)menthofuran (Thomassen et al.,
1991). To CH3CN/H2O (3:1)
(10 ml) was added 
,'-dimethoxydihydromenthofuran (100 mg,
0.47 mmol), which was synthesized by a method described by McClanahan
et al. (1989), and N-acetylcysteine (776 mg, 4.75 mmol). The
mixture was stirred at room temperature for 1 h.
2-(N-Acetylcystein-S-yl)menthofuran was separated
by HPLC using a Rainin (Varian) Microsorb C18
column (5 µm, 4.6 × 250 mm) (method 3, system A, 250 nm, 24.5 min). 1H NMR
(CD2Cl2, 300 MHz): 6.61 (br. s, 1H, CONH), 4.55 (q, J = 5.8 Hz, 1H, Cys
-CH), 3.14 to 3.02 (m, 2H, Cys -CH2), 2.63 (dd, J = 16.2, 5.2 Hz, 1H), 2.35 to 2.25 (m, 2H), 2.17 to 1.79 (m, 3H), 2.03 (s, 3H, COCH3), 1.94 (s,
3H, 3-CH3), 1.38 to 1.24 (m, 1H), 1.05 (d,
J = 6.6 Hz, 3H, 6-CH3).
1H NMR (D2O, 300 MHz): 4.27 (dd, J = 8.4, 2.9 Hz, 1H, Cys -CH), 3.24 (dd,
J = 14.0, 3.3 Hz, 1H, Cys
-CHa), 2.95 (dd, J = 14.0, 8.8 Hz, 1H, Cys -CHb), 2.66 (dd, J = 16.5, 4.7 Hz, 1H), 2.40 to 2.29 (m, 2H), 2.21 to 1.81 (m, 3H), 2.07 (s, 3H, COCH3), 1.96 (s, 3H,
3-CH3), 1.40 to 1.27 (m, 1H), 1.06 (d,
J = 6.6 Hz, 6-CH3); negative ion
ESI-MS/MS: m/z 310 (M H+), 181 (2-thiomenthofuran anion); UV:
max 202, 221, 256 nm.
Reactions of Pulegone with GSH.
In basic medium
The reaction was carried out to produce authentic standards for
comparison with the GST incubation products. GSH (200 mg, 0.65 mmol)
and NaHCO3 (167 mg, 2 mmol) were dissolved in
H2O (1 ml). 14C-Labeled
pulegone (107 µl, 100 mg, 0.65 mmol, 0.0108 µCi/µmol) was added.
The mixture was stirred as tetrahydrofuran was added dropwise until 1 ml had been added. The head-space was flushed with
N2, capped, and then stirred at room temperature
for 72 h. The unreacted pulegone was extracted with ether. The two
radiolabeled products were isolated from aqueous phase by two
successive HPLC systems (retention times 12.5 and 15.2 min, method 15, system A, 220 nm; 14.2 and 18.5 min, method 5, system A, 220 nm). The peak at 12.5 min (method 15): 1H NMR
(D2O, 360 MHz): 4.54 (dd, J = 8.8, 5.5 Hz, 1H, Cys -H), 3.96 (s, 2H, Gly
-CH2), 3.81 (t, J = 5.7 Hz,
1H, Glu -CH), 3.10 (dd, J = 13.2, 5.5 Hz, 1H, Cys
-CHa), 2.89 (dd, J = 13.2, 8.8 Hz, 1H, Cys -CHb), 2.76 to 2.67 (m, 2H), 2.58 to 2.47 (m, 2H, Glu 
-CH2), 2.43 to 2.38 (m,
1H), 2.24 to 2.20 (m, 1H), 2.16 (q, J = 7.1 Hz, 2H, Glu
-CH2), 2.08 (dd, J = 12.5, 2.2 Hz, 1H), 1.95 to 1.87 (m, 2H), 1.68 to 1.64 (m, 1H), 1.43 (s, 3H,
9-CH3), 1.38 (s, 3H,
10-CH3), 0.90 (d, J = 7.0 Hz, 3H,
5-CH3); negative ion ESI-MS/MS:
m/z 458 (M H+), 306 (glutathione anion). This molecule was assigned as
8-(glutathion-S-yl)isomenthone. The peak at 15.2 min (method
15): 1H NMR (D2O, 300 MHz):
4.57 (dd, J = 8.3, 5.5 Hz, 1H, Cys -CH), 3.99 (s, 2H, Gly -CH2), 3.85 (t, J = 6.6 Hz, 1H, Glu -CH), 3.11 (dd, J = 13.8, 5.4 Hz,
1H, Cys -CHa), 2.91 (dd, J = 13.5, 8.7 Hz, 1H, Cys -CHb), 2.76 (dd,
J = 12.9, 5.4 Hz, 1H), 2.54 (td, J = 7.5, 2.9 Hz, 2H, Glu -CH2), 2.45 to 2.36 (m,
1H), 2.29 to 2.22 (m, 2H), 2.18 (q, J = 6.9 Hz, 2H, Glu
-CH2), 1.97 to 1.88 (m, 2H), 1.61 (q,
J = 12.6 Hz, 1H), 1.51 to 1.36 (m, 1H), 1.45 (s, 3H,
9-CH3), 1.38 (s, 3H,
10-CH3), 1.02 (d, J = 6.0 Hz, 3H, 5-CH3); negative ion ESI-MS/MS:
m/z 458 (M H+), 306 (glutathione anion). This molecule was assigned as
8-(glutathion-S-yl)menthone.
Under catalysis of GST.
A similar method as in the literature was used (Thomassen et al.,
1990). Initial conditions were 1.0 mM GSH, 1.0 mM
14C-labeled pulegone (1 µCi/µmol), 0.2 mg of
GST in 0.1 M potassium phosphate buffer, pH 7.7. Pulegone was added as
a CH3CN solution (5 µl). Controls lacking GST
or GSH were included. The final volume was 0.5 ml. Three replicates of
each set were carried out. The components were combined in an ice-cold
3-ml vial that was subsequently capped, vortexed, and incubated at
37°C for 15 min. The reactions were stopped by placing these vials on
ice. The products were analyzed by HPLC (method 14, system B) for
comparison with the standards prepared above.
Enzyme Hydrolysis of 24 h Urine and Metabolites D2, E2, and
9-Hydroxypulegone Glucuronide.
Samples of male and female rat urine (single doses, 80 mg/kg, 24 h) were incubated with glucuronidase (5150 units) or sulfatase (33 units) in a total volume of 0.4 ml of sodium acetate buffer (0.05 M, pH
5.0), as described previously (Burka et al., 1996). All reaction
mixtures, including enzyme-free controls, were incubated at 37°C for
17 h. Samples of each incubated mixture were analyzed by HPLC
(method 1 and 2, system B) to determine whether any of the peaks were
affected by enzymatic hydrolysis.
20°C to
separate the CH3CN and H2O
layers. Both layers were analyzed by HPLC (method 2, system B).
Standards of 10-hydroxypulegone,
7a-Hydroxy-3,6-dimethyl-5,6,7,7a-tetrahydro-2(4H)-benzofuranone,
pulegone, menthofuran,
2-(N-acetylcystein-S-yl)menthofuran, piperitone,
and pulegol were subjected to the same HPLC analyses for comparison.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Identification and Quantitation of Urinary Metabolites. At 24 h postdosing, 44 to 71% of pulegone-derived radioactivity was present in urine (Table 1). An additional 14 and 5% of radioactivity was excreted in the urine of male and female rats dosed with 8 mg/kg pulegone from 24 to 72 h, respectively. Urine samples (24 h) from rats treated with various doses were analyzed by HPLC (method 1, system B) to reveal several major radiolabeled peaks (A-M) (Fig. 1). The percentage of each major peak (A-M) in urine with each dosing method was calculated based on the 14C count in each peak compared with the total amount of 14C excreted in each urine sample (Table 1). HPLC peaks A through M accounted for approximately 60% of the radioactivity that was excreted in urine.
|
|
4.43 (br. s, 1H), 3.04 (s,
3H), 2.74 to 2.67 (m, 1H), 2.05 (t, J = 15.4 Hz, 4H),
1.76 to 1.66 (m, 2H), 1.51 (s, 3H), 1.45 (s, 3H); UV:
max 219 nm. However, all attempts to obtain a
molecular weight of this metabolite failed. It is also not clear
whether all the NMR peaks belong to the metabolite. We cannot deduce
the structure of metabolite A at this point.
Peak B (RT = 16.7 min, method 1) was further separated by HPLC
(method 5, 225 nm) to give two main metabolites [RT = 6.6 min (B1) and 7.0 min (B2)]. The more abundant
metabolite (B1) had the following spectral properties:
1H NMR (D2O, 360 MHz): 4.53 (dd, J = 7.7, 4.8 Hz, 1H, Cys -CH), 3.11 (dd,
J = 12.8, 4.8 Hz, 1H, Cys
-CHa), 2.95 (dd, J = 12.8, 7.7 Hz, 1H, Cys -CHb), 2.77 (dd, J = 12.8, 4.4 Hz, 1H, 2-CH), 2.71 (d, J = 12.8 Hz, 1H,
6-CHa), 2.37 to 2.31 (m, 1H), 2.28 (dd, J = 13.6, 1.1 Hz, 1H, 6-CHb),
2.05 (s, 3H, COCH3), 2.00 to 1.84 (m, 2H), 1.46 (s, 3H, 9-CH3), 1.37 (s, 3H,
10-CH3), 1.33 (s, 3H, 5-CH3); negative ion ESI-MS/MS:
m/z 330 (M H+), 162 (N-Ac-Cys anion); UV: max 202 nm. MS suggested
that this metabolite was a hydroxylated product of metabolite
K or L
(8-(N-acetylcystein-S-yl)menthone/isomenthone).
The NMR spectrum showed the presence of 5-CH3
group at 1.33 ppm as a singlet, indicating the ---OH substitution to be
at the C-5 position. The analogous CH3 group in
pulegone is a doublet at 1.0 ppm. B1 was more abundant than
its diastereomer C3, and its 5-CH3 group is 0.15 ppm more downfield (see below); therefore, it was identified as
8-(N-acetylcystein-S-yl)-5-hydroxymenthone
for the same assignment for K and L.
Nevertheless, the assignment of the stereochemistry at the C-2 position
was not definitive.
The less abundant metabolite (B2) had the following spectral
properties: 1H NMR (D2O,
360 MHz): 2.05 (s, 3H, COCH3), 1.91 (s, 3H,
9-CH3), 1.90 (s, 3H,
10-CH3), 1.04 (d, J = 7.7 Hz, 3H,
5-CH3); the other signals were not well resolved;
negative ion ESI-MS/MS: m/z 328 (M H+), 162 (N-Ac-Cys anion); UV:
max 195, 248 nm. MS analysis showed that the
mercapturic acid metabolite had a molecular weight 2 Da less than
B1, indicating the possibility of one carbon-carbon double
bond in the structure. The metabolite had a UV maximum at 248 nm, and
its NMR spectrum showed the presence of two allylic CH3 groups at 1.91 and 1.90 ppm. The spectral
results indicated that the cyclic-isopropylidene ketone structure of
the parent pulegone (max 255 nm) remained
intact in the metabolite. The 5-CH3 group
remained a doublet at 1.04 ppm, so B2 appeared to be a
pulegone with one hydroxyl and one
N-acetylcystein-S-yl substitutions at two of the
C-3, C-4 and C-6 positions.
Peak C (RT = 17.3 min, method 1) was further separated by HPLC
(method 11, 225 nm) to give the major metabolite [RT = 3.3 min
(C1)] and several minor metabolites (RT = 3.5-3.9
min). Metabolite C1 was further purified by HPLC (RT = 7.5 min, method 5, 250 nm). Its spectral properties were as follows:
1H NMR (D2O, 360 MHz): 4.56 (d, J = 8.1 Hz, 1H, Gluc 1'-CH), 4.20 (br.
s, 1H, 4-CH), 3.77 to 3.71 (m, 1H, Gluc 5'-CH), 3.56 to 3.48 (m, 2H,
Gluc 2'-, 4'-CHs), 3.33 to 3.28 (m, 1H, Gluc 3'-CH), 2.94 (dd,
J = 14.7, 3.3 Hz, 1H, 3-CHeq),
2.58 (dd, J = 15.7, 1.5 Hz, 1H), 2.51 to 2.37 (m, 2H),
2.37 to 2.30 (m, 1H), 1.95 (s, 3H, 9-CH3), 1.85 (s, 3H, 10-CH3), 1.04 (d, J = 6.2 Hz, 3H, 5-CH3); negative ion ESI-MS/MS:
m/z 343 (M H+), 325 (M H3O+), 193 (glucuronide ion), 175 (glucuronide ion H2O), 157 (glucuronide ion 2 H2O); UV: max 257 nm. MS
of C1 displayed a molecular ion peak at
m/z 343, consistent with the anionic form (M H+) of a hydroxypulegone glucuronide.
C1 had a UV maximum at 257 nm, and its NMR spectrum showed
the presence of two allylic CH3 groups at 1.95 and 1.85 ppm, indicating that the cyclic-isopropylidene ketone
structure of pulegone remained intact in the metabolite. NMR spectrum
demonstrated that there was one proton geminal to the ---OGluc
substituent present at 4.20 ppm, so the hydroxylation/glucuronidation must have taken place in one of the methylene protons; that is, C-3,
C-4, or C-6 positions. The metabolite was not 3-hydroxypulegone glucuronide, which was characterized (E1, 3-CH is at 5.29 ppm). The ---OGluc substitution was more likely to be at C-4 position because the proton at 4.20 ppm was upfield compared with what would be
expected for the one at the C-6 position. In addition, the
3-CHeq was a doublet of doublets in C1
compared with a doublet of triplets (J = 15.5, 4.0 Hz)
in the parent pulegone, an indication of a substitution at the C-4
position. Metabolite C1 was identified as 4-hydroxypulegone
glucuronide with unknown stereochemistry at the C-4 position. Small
amounts of the products from Michael addition of water to C1
and other hydroxypulegone glucuronides were generated in acidic
conditions but not in neutral conditions, so it was necessary to remove
TFA right after the HPLC isolation.
The minor metabolites were separated by HPLC [RT = 10.7 (C2) and 11.3 min (C3 and C4), method
9, 225 nm]. Metabolite C2 was further purified by HPLC
(RT = 4.6 min, method 6, 225 nm). Its spectral properties were as
follows: 1H NMR (D2O, 360 MHz): 7.19 (br. s, 1H, 3-CH), 4.35 (d, J = 7.3 Hz,
1H, Gluc 1'-CH), 1.56 (s, 3H, 9-CH3), 1.46 (s,
3H, 10-CH3), 1.00 (d, J = 4.0 Hz,
3H, 5-CH3); the other signals were not well resolved; negative ion ESI-MS: m/z 343 (M H+), 193 (glucuronide ion). Positive ion ESI-MS:
m/z 345 (M + H+), 169 (5-methyl-2-(1'-hydroxy-1'-methylethyl)-2-cyclohexene-1-one + H+), 151 (M glucuronide ion); UV:
max 233 nm. MS showed that C2 was a
glucuronide with the same molecular weight as hydroxypulegone
glucuronide metabolites, but its NMR and UV spectra indicated that the
carbon-carbon double bond in the parent pulegone has rearranged. NMR of
C2 showed a rather downfield olefinic proton at 7.19 nm and
two CH3 groups at 1.56 and 1.46 ppm. The NMR
signals are comparable with those of
5-methyl-2-(1'-hydroxy-1'-methylethyl)-2-cyclohexene-1-one, which has
an olefinic H at 6.88 ppm and two CH3 groups at
1.4 ppm (Madyastha and Thulasiram, 1999). C2 had a UV
maximum at 233 nm, in agreement with the presence of
2-cyclohexene-1-one as part of the structure as in piperitone (UV
maximum, 235 nm). Metabolite C2 was tentatively identified
as 5-methyl-2-(1'-hydroxy-1'-methylethyl)-2-cyclohexene-1-one glucuronide, an allylic alcohol isomer of metabolite E1. The
presence of a bulky glucuronide substituent probably moves the NMR
signals more downfield in C2 compared with those of its
aglycone. Positive ion ESI-MS/MS analysis gave a fragment with
m/z = 151, consistent with formation of a
stable allylic cation through removal of a glucuronic acid.
Metabolites C3 and C4 were a mixture of
mercapturic acids. Based on the ratio of these two metabolites, some of
their spectral properties were resolved as followed: C3: 1H NMR (D2O, 360 MHz): 2.05 (s, 3H, COCH3), 1.45 (s, 3H,
9-CH3), 1.36 (s, 3H,
10-CH3), 1.18 (s, 3H,
5-CH3); the other signals were not well resolved;
negative ion ESI-MS/MS: m/z 330 (M H+), 162 (N-Ac-Cys anion). These two mercapturic
acids could be separated by HPLC using method 1, system B
(C3, RT = 17.1 min; C4, RT = 16.8 min)
to obtain individual UV spectra. UV for C3 was the
following: max 202 nm. MS analysis suggested that C3 was a hydroxylated
8-(N-acetylcystein-S-yl)menthone/isomenthone and
the NMR results showed the 5-CH3 group was a
singlet at 1.18 ppm, indicating the ---OH substitution was at the C-5
position. C3 was a diastereomer of B1; their
5-CH3 groups showed significant difference in
chemical shifts as the corresponding CH3 groups
in diastereomeric metabolites K and L. Metabolite
C3 was tentatively identified as
8-(N-acetylcystein-S-yl)-5-hydroxyisomenthone.
C4: 1H NMR (D2O,
360 MHz): 2.01 (s, 3H, COCH3), 1.54 (s, 3H,
9-CH3), 1.52 (s, 3H,
10-CH3), 1.10 (d, J = 5.9 Hz, 3H, 5-CH3); the other signals were not well resolved;
negative ion ESI-MS/MS: m/z 328 (M H+), 162 (N-Ac-Cys anion); UV:
max 235 nm. C4 had the same molecular weight as B2, but the NMR chemical shifts of the
CH3 groups and the UV maximum suggested that
C4 was likely to be
5-methyl-2-(1'-(N-acetylcystein-S-yl)-1'-methylethyl)-2-cyclohexene-1-one with an ---OH substitution at an unknown position. C4 is a
mercapturic acid counterpart of metabolite C2 plus an ---OH substitution.
Peak D (RT = 17.9 min, method 1) was further separated by HPLC
(method 11, 250 nm) to give two major metabolites [RT = 3.8 (D1) and 4.0 (D2) min]. The less abundant
metabolite (D1) had the following spectral properties:
1H NMR (D2O, 360 MHz): 4.65 (d, J = 7.7 Hz, 1H, Gluc 1'-CH), 3.73 to 3.68 (m,
1H, Gluc 5'-CH), 3.52 to 3.47 (m, 2H, Gluc 2'-, 4'-CHs), 3.25 to 3.20 (m, 1H, Gluc 3'-CH), 2.77 to 2.66 (m, 1H), 2.73 (d, J = 15.8 Hz, 1H, 6-CHa), 2.61 (d, J = 16.1 Hz, 1H, 6-CHb), 2.53 (dt, J = 15.0, ~5 Hz, 1H), 2.11 to 2.02 (m, 1H), 1.98 to 1.84 (m, 1H), 1.95 (s, 3H, 9-CH3), 1.84 (s, 3H,
10-CH3), 1.34 (s, 3H,
5-CH3); negative ion ESI-MS/MS:
m/z 343 (M H+), 193 (glucuronide ion), 175 (glucuronide ion H2O); UV: max 259 nm.
The spectral results indicated that metabolite D1 was a
hydroxypulegone glucuronide. The NMR spectrum showed the presence of
the 5-CH3 at 1.34 ppm as a singlet, which
suggested that the ---OGluc substitution be at the C-5 position. This
metabolite was identified as 5-hydroxypulegone glucuronide
(D1).
The more abundant metabolite (D2) had the following spectral
properties: 1H NMR (D2O,
300 MHz): 4.40 (ABq, J = 12.0 Hz, 2H,
10-CH2OGluc), 4.39 (d,
J = 6.9 Hz, 1H, Gluc 1'-H), 3.71 to 3.65 (m, 1H, Gluc 5'-H), 3.55 to 3.46 (m, 2H, Gluc 2'-, 4'-CHs), 3.36 to 3.31 (m, 1H,
Gluc 3'-CH), 2.86 (dt, J = 14.8, 4.5 Hz, 1H,
3-CHeq), 2.58 (dd, J = 14.3, 3.2 Hz, 1H, 6-CHeq), 2.32 (br. t, J = 13.7 Hz, 1H, 3-CHax), 2.24 (dd, J = 14.3, 10.4 Hz, 1H, 6-CHax), 2.17 to 2.05 (m,
1H, 5-CHax), 1.96 to 1.82 (m, 1H,
4-CHeq), 1.86 (s, 3H, 9-CH3), 1.42 (qd, J = 12.0, 4.6 Hz, 1H, 4-CHax), 1.01 (d, J = 6.3 Hz, 3H, 5-CH3); negative ion ESI-MS/MS:
m/z 343 (M H+), 325 (M H3O+), 193 (glucuronide ion), 175 (glucuronide ion H2O), 167 (10-hydroxypulegone anion), 157 (glucuronide ion 2 H2O), 149 (10-hydroxypulegone anion H2O); UV:
max 248. The spectral results indicated that metabolite D2 was a hydroxypulegone glucuronide. NMR showed only one allylic CH3 group and two protons
at 4.38 ppm as an AB quartet due to the ---OGluc substitution at either
the C-9 or the C-10 position. Metabolite D2 was hydrolyzed
by glucuronidase to give a product that comigrated (20.7 min, method 1, system B) with 10-hydroxypulegone prepared from microsomal incubation of pulegone. Metabolite D2 was identified as
10-hydroxypulegone glucuronide.
Metabolite D2 partially isomerized to its geometric isomer,
9-hydroxypulegone glucuronide, during isolation in acidic conditions,
possibly via acid-catalyzed addition of water followed by elimination.
9-Hydroxypulegone glucuronide (RT = 5.9 min, method 11, 250 nm)
had the following spectral properties: 1H NMR
(D2O): 4.44 (d, J = 12.4 Hz,
1H, 9-CHa), 4.31 (d, J = 7.7 Hz,
1H, Gluc 1'-CH), 4.30 (d, J = 13.0 Hz, 1H,
9-CHb), 3.61 to 3.56 (m, 1H, Gluc 5'-CH), 3.50 to
3.41 (m, 2H, Gluc 2', 4'-CHs), 3.31 to 3.26 (m, 1H, Gluc 3'-CH), 2.78 (dt, J = 15.6, 4.4 Hz, 1H,
3-CHeq), 2.53 (dd, J = 14.7, 2.4 Hz, 1H, 6-CHeq), 2.29 (br. t, J = 14 Hz, 1H, 3-CHax), 2.13 (t, J = 11.0 Hz, 1H, 6-CHax), 2.06 to 1.99 (m, 1H,
5-CHax), 1.95 to 1.87 (m, 1H,
4-CHeq), 1.83 (s, 3H,
10-CH3), 1.46 to 1.35 (m, 1H,
4-CHax), 0.98 (d, J = 6.3 Hz, 3H,
5-CH3); negative ion ESI-MS/MS:
m/z 343 (M H+), 325 (M H3O+), 193 (glucuronide ion), 175 (glucuronide ion H2O), 167 (9-hydroxypulegone anion), 157 (glucuronide ion 2 H2O), 149 (9-hydroxypulegone anion H2O); UV:
max 248 nm. 9-Hydroxypulegone glucuronide was analyzed by HPLC (RT = 18.7 min, method 1, system B), which showed that it was either not a metabolite or a very minor metabolite of
pulegone. It has been speculated that 9-hydroxypulegone, if formed,
would cyclize and dehydrate to give menthofuran (Gordon et al., 1987).
Therefore, it was of interest to investigate the products from
glucuronidase treatment of 9-hydroxypulegone glucuronide. HPLC analysis
(method 2, system B) of the reaction mixture showed total disappearance
of the glucuronide peak, but no other peaks were formed.
CH3CN extraction of the reaction mixture did not give the anticipated menthofuran upon HPLC analysis, either.
Peak E (RT = 18.5 min, method 1) was further separated by HPLC
(method 11, 250 nm) to three major metabolites [RT = 3.9 (E1), 4.3 (E2), and 4.8 (E3) min].
Metabolite E1 was further purified by two successive HPLC
systems (RT = 7.3 min, method 13, 250 nm; RT = 5.0 min,
method 6, 250 nm). Its spectral properties were as follows:
1H NMR (D2O, 360 MHz): 5.29 (dd, J = 7.0, 2.2 Hz, 1H, 3-CH), 4.17 (d,
J = 7.7 Hz, 1H, Gluc 1'-CH), 3.62 (d, J = 9.5 Hz, 1H, Gluc 5'-CH), 3.50 (t, J = 9.2 Hz, 1H,
Gluc 2'-CH), 3.41 (t, J = 9.2 Hz, Gluc 4'-CH), 3.24 (t,
J = 8.8 Hz, Gluc 3'-CH), 2.47 (dd, J = 17.6, 4.8 Hz, 1H, 6-CHeq), 2.41 to 2.35 (m, 1H),
2.29 (dd, J = 16.8, 11.7 Hz, 1H,
6-CHax), 2.06 to 1.95 (m, 1H), 2.01 (s, 3H,
9-CH3), 1.93 (s, 3H,
10-CH3), 1.63 to 1.54 (m, 1H), 1.03 (d,
J = 7.0 Hz, 3H, 5-CH3); negative
ion ESI-MS: m/z 343 (M H+), 193 (glucuronide ion); UV:
max 245 nm. The spectral data indicated that
the metabolite was a hydroxypulegone glucuronide. There was one proton
present at 5.29 ppm as a doublet of doublets, more than 1 ppm downfield
than its counterpart in metabolite C1, which suggested the
---OGluc substitution be at the allylic C-3 position. This metabolite
was identified as 3-hydroxypulegone glucuronide (E1),
although the stereochemistry at the C-3 position was not clear.
Metabolite E2 was further purified by HPLC (RT = 5.5 min, method 6, 210 nm) to give the following spectral properties: 1H NMR (D2O, 360 MHz): 4.45 (d, J = 7.7 Hz, 1H, Gluc 1'-CH), 4.01 (dd,
J = 10.3, 4.0 Hz, 1H, 2-CHa),
3.81 (d, J = 8.8 Hz, 1H, 2-CHb),
3.56 to 3.48 (m, 3H, Gluc 2', 4', and 5'-CHs), 3.32 (t, J = 8.1 Hz, 1H, Gluc 3'-CH), 2.43 to 2.33 (m, 2H), 2.27 to 2.20 (m, 2H), 2.13 to 2.03 (m, 1H), 2.02 to 1.94 (m, 1H), 1.82 to
1.72 (m, 2H), 1.60 to 1.53 (m, 1H), 0.97 (d, J = 6.6 Hz, 3H, 3-CH3), 0.90 (d, J = 6.6 Hz, 3H, 6-CH3); negative ion ESI-MS/MS:
m/z 345 (M H+), 327 (M H3O+), 193 (glucuronide ion), 175 (glucuronide ion H2O), 157 (glucuronide ion 2 H2O); UV: max <190 nm.
The molecular ion peak at m/z 345 (M H+) suggested that metabolite E2 was a
glucuronic acid conjugate of monohydroxylated, reduced pulegone. This
metabolite had almost no UV absorption; therefore, the reduced pulegone
was more likely to be menthone/isomenthone, which had almost no UV
absorption, and less likely to be pulegol, which showed a UV maximum at
201 nm. NMR of E2 showed only two CH3
groups at 0.97 and 0.90 ppm as doublets, which confirmed the reduction
of the carbon-carbon double bond in the metabolite and indicated the
---OH substitution on one of the CH3 groups. The
---OH substitution could be at the C-9 position of menthone, which
would cyclize to give a five-membered ring, or at the C-7 position of
menthone, which could not cyclize. A very similar metabolite
G1 showed two CH3 groups at 1.02 and
0.84 ppm as doublets and two protons on the
---OCH2--- at 3.79 and 3.57 ppm. Because the
CH2 in cyclopentane (1.50 ppm) is more downfield
than in n-pentane (1.25 ppm) (Pouchert and Campbell, 1974),
we assigned E2, whose
---OCH2--- appeared at 4.01 and 3.81 ppm as 7a-hydroxy-3,6-dimethyloctahydrobenzofuran glucuronide, and
G1 as 7-hydroxymenthone glucuronide. There was a minor metabolite isolated along with E2, which was probably the isomenthone diastereomer of E2.
Metabolite E3 was further purified by HPLC (RT = 5.6 min, method 6, 225 nm) to give a molecule with the following spectral properties: 1H NMR (D2O,
360 MHz): 4.41 (d, J = 7.7 Hz, 1H, Gluc 1'-CH), 3.61 (d, J = 9.2 Hz, 1H, Gluc 5'-CH), 3.49 (t,
J = 8.4 Hz, 1H, Gluc 2'-CH), 3.44 (t, J = 8.8 Hz, 1H, Gluc 4'-CH), 3.34 (t, J = 8.8 Hz, 1H,
Gluc 3'-CH), 2.83 (br. d, J = 13.6 Hz, 1H), 2.45 (br.
d, J = 9.4 Hz, 1H), 2.34 (td, J = 12.1, 5.1 Hz, 1H), 2.01 to 1.91 (m, 2H), 1.83 (s, 3H,
3-CH3), 1.32 (t, J = 13.0 Hz,
1H), 1.04 to 1.01 (m, 1H), 0.94 (d, J = 6.6 Hz, 3H,
6-CH3); negative ion ESI-MS/MS:
m/z 357 (M H+), 193 (glucuronide ion), 175 (glucuronide ion H2O); UV: max 222 nm.
NMR spectrum of E3 showed only one allylic
CH3 group at 1.83 ppm, which indicated that the
other allylic CH3 group was modified. MS analysis
gave a molecular weight 14 Da more than hydroxypulegone glucuronide
metabolites, consistent with oxidation of the other allylic
CH3 group to a carboxylic acid. E3
showed a UV maximum at 222 nm, indicating the carboxylic acid group was
syn to the ketone group, which cyclized to give
7a-hydroxy-3,6-dimethyl-5,6,7,7a-tetrahydro-2(4H)-benzofuranone (a hydroxylated ,-unsaturated--lactone). The ---OH group was conjugated with a glucuronic acid. This metabolite was partially hydrolyzed by glucuronidase to give a new product, which had a similar
HPLC retention time (23.0 min, method 1, system B) and UV maximum as
the synthetic
7a-hydroxy-3,6-dimethyl-5,6,7,7a-tetrahydro-2(4H)-benzofuranone (UV: max 220 nm). Metabolite E3 was
identified as
7a-hydroxy-3,6-dimethyl-5,6,7,7a-tetrahydro-2(4H)-benzofuranone glucuronide.
Peak F (RT = 19.3 min, method 1) was further separated by HPLC
(method 13, 210 nm) to give two major metabolites (RT = 8.6 min,
F1; RT = 9.7 min, F2). After further
purification by HPLC (RT = 6.5 min, method 6, 210 nm), metabolite
F1 had the following spectral characteristics:
1H NMR (D2O, 300 MHz): 4.64 (d, J = 8.0 Hz, 1H, Gluc 1'-H), 3.74 to 3.71 (m,
1H, Gluc 5'-H), 3.54 to 3.48 (m, 2H, Gluc 2'-, 4'-CHs), 3.28 to 3.22 (m, 1H, Gluc 3'-CH), 2.62 (ABq, J = 13.5 Hz, 2H, 6-CH2), 2.30 to 2.23 (m, 1H), 2.19 to 1.81 (m,
5H), 1.36 (s, 3H, 5-CH3), 0.89 (d,
J = 6.6 Hz, 3H, 9-CH3), 0.88 (d,
J = 6.9 Hz, 3H, 10-CH3); negative
ion ESI-MS/MS: m/z 345 (M H+), 193 (glucuronide ion), 175 (glucuronide
ion H2O); UV:
max <190 nm. The spectral data indicated that
the metabolite was a hydroxymenthone/isomenthone glucuronide. NMR
showed the presence of two CH3 groups at 0.89 and
0.88 ppm as doublets and one CH3 at 1.36 ppm as a
singlet, due to the ---OGluc substitution at the C-5 position. Because
metabolite F1 was slightly less abundant than metabolite
F2, we assigned this metabolite as an isomenthone. Metabolite F1 was identified as 5-hydroxyisomenthone
glucuronide, although the assignment of stereochemistry was not certain.
After purification by HPLC (6.3 min, method 6, 210 nm), metabolite
F2 had the following spectral characteristics:
1H NMR (D2O, 300 MHz): 4.66 (d, J = 7.4 Hz, 1H, Gluc 1'-H), 3.72 to 3.67 (m,
1H, Gluc 5'-H), 3.55 to 3.46 (m, 2H, Gluc 2'-, 4'-CHs), 3.27 to 3.22 (m, 1H, Gluc 3'-CH), 2.63 (s, 2H, 6-CH2), 2.18 to 2.01 (m, 3H), 1.97 (t, J = 5.7 Hz, 2H), 1.74 to 1.64 (m, 1H), 1.30 (s, 3H, 5-CH3), 0.92 (d,
J = 6.6 Hz, 3H, 9-CH3), 0.83 (d, J = 6.3 Hz, 3H, 10-CH3); negative
ion ESI-MS/MS: m/z 345 (M H+), 193 (glucuronide ion), 175 (glucuronide
ion H2O); UV:
max <190 nm. This metabolite appeared to be
the diastereomer of metabolite F1. Metabolite F2
was identified as 5-hydroxymenthone glucuronide.
There was a minor metabolite (7.0 min, method 13, 210 nm) isolated from
peak F. It was further purified by HPLC (6.3 min, method 6, 210 nm) and
was shown to have a molecular weight of 331. The metabolite was likely
to be a hydroxylated
8-(N-acetylcystein-S-yl)menthone/isomenthone, although the position of the ---OH substitution was unknown.
Peak G (RT = 19.8 min, method 1) was separated by HPLC (method 10, 210 nm) to three poorly resolved peaks containing radioactivity (6.8, 7.3, 7.8 min). The peak at 6.8 min contained several minor metabolites,
which were not isolated. The peak at 7.3 min was further purified
(RT = 7.3 min, method 5, 210 nm) to give the major metabolite
G1. 1H NMR (D2O,
300 MHz): 4.42 (d, J = 7.7 Hz, 1H, Gluc 1'-CH), 3.79 (t, J = 9.1 Hz, 1H, 7-CHa),
3.71 to 3.68 (m, 1H, Gluc 5'-CH), 3.57 (dd, J = 9.9, 4.2 Hz, 1H, 7-CHb), 3.52 to 3.49 (m, 2H, Gluc 2'-, 4'-CHs), 3.34 to 3.26 (m, 1H, Gluc 3'-CH), 2.71 to 2.63 (m, 1H),
2.36 (dd, J = 12.6, 4.6 Hz, 2H), 2.18 (t,
J = 12.1 Hz, 1H), 2.11 to 2.04 (m, 1H), 1.98 to 1.83 (m, 2H), 1.56 to 1.37 (m, 2H), 1.02 (d, J = 6.3 Hz, 3H,
9-CH3), 0.84 (d, J = 6.9 Hz, 3H,
10-CH3); negative ion ESI-MS/MS:
m/z 345 (M H+), 327 (M H3O+), 193 (glucuronide ion); UV: max <190 nm.
Metabolite G1 had very similar spectral data as metabolite
E2, and subsequently G1 was identified as
7-hydroxymenthone glucuronide as the result of comparison of NMR spectra.
The peak at 7.8 min was further purified (RT = 7.6 min, method 6, 210 nm) to give a mixture of two glucuronides (G2 and
G3). Metabolite G2 had the following spectral properties: 1H NMR (D2O,
300 MHz) showed 2 CH3 groups at 1.15 (d,
J = 7.2 Hz, 3H), 0.99 (d, J = 6.3 Hz,
3H), and the other signals were not well resolved; negative ion
ESI-MS/MS: m/z 359 (M H+), 193 (glucuronide ion), 183 (3,6-dimethyl-3,4,5,6,7,7a-hexahydro-2-benzofuranone anion), 175 (glucuronide ion H2O); UV:
max <190 nm. Metabolite G2 had a
molecular weight 2 Da higher than that of E3, as a result of
reduction of either the carbon-carbon double bond or the carbonyl group
of E3. NMR indicated that the reduction occurred at the
carbon-carbon double bond, as evidenced by the absence of allylic
CH3 groups and presence of two
CH3 groups at 1.15 and 0.99 ppm as doublets.
Metabolite G2 was tentatively identified as
7a-hydroxy-3,6-dimethyl-3,4,5,6,7,7a-hexahydro-2-benzofuranone glucuronide.
Metabolite G3 had the following spectral properties:
1H NMR (D2O, 300 MHz)
showed two CH3 groups at 0.88 (t,
J = 7.2 Hz, 6H), and the other signals were not well
resolved; negative ion ESI-MS/MS: m/z 361 (M H+), 193 (glucuronide ion), 185 (7a-dihydroxy-3,6-dimethyl-octahydrobenzofuran anion), 175 (glucuronide
ion H2O); UV:
max <190 nm. Metabolite G3 had a
molecular weight 4 Da higher than that of E3, as a result of
reduction of both the carbon-carbon double bond and the carbonyl group
of E3. Metabolite G3 was tentatively identified
as 2,7a-dihydroxy-3,6-dimethyl-octahydrobenzofuran glucuronide.
Peak H (isolated in fraction 20-21 min, method 1) and peak I (isolated
in fraction 21-22 min, method 1) contained minor metabolites, which
could not be identified. MS analysis showed formation of a hydroxylated
8-(N-acetylcystein-S-yl)menthone/isomenthone
(M = 331) contained in peak H, although the position of the ---OH
group was unknown.
Peak J (isolated in fraction 22-24 min, method 1) contained a major
metabolite J, which was further purified by HPLC (RT = 10.5 min, method 7, 225 nm). Metabolite J had the following
spectral characteristics: 1H NMR
(CDCl3, 300 MHz): 7.05 (d, J = 7.8 Hz, 1H, 6'-H), 6.74 (d, J = 7.5 Hz, 1H, 5'-H),
6.72 (s, 1H, 3'-H), 3.93 (q, J = 6.9 Hz, 1H,
CHCH3), 2.29 (s, 3H,
4'-CH3), 1.56 (d, J = 7.2 Hz, 3H, CHCH3); negative ion ESI-MS/MS:
m/z 179 (M H+), 135 (M COOH+); UV:
max 200, 221, 275 nm. The NMR spectrum showed
two doublets at 7.05 and 6.74 ppm with a coupling constant of 7.5 Hz,
consistent with ortho protons on an aromatic structure. The
singlet at 6.72 ppm indicated the presence of a proton isolated from
the other aromatic protons, and the singlet at 2.29 ppm was consistent
with an aromatic CH3 group. MS/MS gave a peak at
m/z 135 (M COOH+),
which showed evidence of a carboxylic acid fragmentation in the
metabolite. One proton at 3.93 ppm as a quartet and a
CH3 group at 1.56 ppm as a doublet agreed with a
2-phenylpropionic acid structure. This metabolite was identified as
2-(2'-hydroxy-4'methylphenyl)propionic acid.
2-(2'-Hydroxy-4'methylphenyl)propionic acid is also a metabolite of
thymol in rats; the reported GC-MS data of its methylated derivative are consistent with the MS result of metabolite J (Austgulen et al., 1987). An independent synthesis of this metabolite was carried
out starting from 2'-hydroxy-4'-methylacetophenone. The ---OH group was
first benzylated, followed by successive conversion of the ketone group
to alcohol, chloride, and then cyanide. Subsequent hydrolysis did not
only convert the cyanide group to the carboxylic acid but also
partially deprotected the ---OH group. NMR and mass spectra, as well as
the HPLC chromatograms of the metabolite and the authentic standard
were identical.
Peak K (isolated in fraction 24-25 min, method 1) contained one major
metabolite. After further purification by HPLC (RT = 12.5 min,
method 7, 225 nm), the major metabolite K had the following
spectral characteristics: 1H NMR
(D2O, 360 MHz): 4.34 (dd, J = 8.1, 4.0 Hz, 1H, Cys -CH), 3.09 (dd, J = 13.2, 4.4 Hz, 1H, Cys -CHa), 2.88 (dd, J = 13.2, 8.4 Hz, 1H, Cys -CHb), 2.72 to 2.64 (m, 1H), 2.70 (dd, J = 12.1, 5.1 Hz, 1H, 2-CH), 2.41 to
2.34 (m, 1H), 2.25 to 2.18 (m, 1H), 2.09 to 2.03 (m, 1H), 2.05 (s, 3H,
COCH3), 1.91 to 1.84 (m, 2H), 1.65 to 1.60 (m,
1H), 1.44 (s, 3H, 9-CH3), 1.37 (s, 3H,
10-CH3), 0.91 (d, J = 7.3 Hz, 3H,
5-CH3); negative ion ESI-MS:
m/z 314 (M H+), 162 (N-Ac-Cys anion); UV: max 202 nm. MS of
metabolite K was consistent with an
N-acetylcysteine conjugate derived from conjugation of
glutathione with unmodified pulegone. NMR demonstrated that the
N-acetylcysteine substituent was at the 8-position, as evidenced by the chemical shifts of 9- and 10-CH3
groups at 1.44 and 1.37 ppm as singlets. Michael addition of
glutathione to the ,-unsaturated ketone group of pulegone took
place in vivo to give the ultimate diastereomeric metabolites
K and L (see below). Because metabolite
K was less abundant than metabolite L, we
assigned this reduced pulegone as an isomenthone, although the
assignment was not unambiguous. Metabolite K was identified
as 8-(N-acetylcystein-S-yl)isomenthone.
Peak L and peak M were isolated in one fraction (RT = 25-26.5
min, method 1). When further purified by HPLC (RT = 9.1 min, method 8, 225 nm), the major metabolite L had the following spectral characteristics: 1H NMR
(D2O, 360 MHz): 4.42 (dd, J = 7.7, 4.4 Hz, 1H, Cys -CH), 3.08 (dd, J = 12.8, 4.4 Hz, 1H, Cys -CHa), 2.90 (dd, J = 12.8, 7.7 Hz, 1H, Cys -CHb), 2.75 (dd,
J = 13.0, 4.9 Hz, 1H, 2-CH), 2.43 to 2.38 (m, 1H), 2.25 to 2.22 (m, 2H), 2.03 (s, 3H, -COCH3), 1.93 to
1.86 (m, 2H), 1.58 (qd, J = 13.2, 2.2 Hz, 1H), 1.47 to 1.38 (m, 1H), 1.43 (s, 3H, 9-CH3), 1.35 (s, 3H,
10-CH3), 1.00 (d, J = 6.2 Hz, 3H,
5-CH3); negative ion ESI-MS/MS:
m/z 314 (M H+), 162 (N-Ac-Cys anion); UV: max 202 nm. This
metabolite appeared to be a diastereomer of metabolite K.
Its 5-CH3 group is 0.09 ppm more downfield than
the corresponding CH3 group of K. Metabolite L was identified as
8-(N-acetylcystein-S-yl)menthone.
The minor metabolite M (RT = 10.9 min, method 8, 225 nm) showed the following characteristics: 1H NMR
(D2O): 7.29 (d, J = 7.7 Hz,
1H, 6'-H), 7.28 (s, 1H, 3'-H), 7.14 (d, J = 7.7 Hz, 1H,
5'-H), 5.24 (s, 1H, olefinic H), 5.12 (s, 1H, olefinic H), 2.36 (s, 3H,
4'-CH3), 2.12 (s, 3H,
3-CH3); negative ion ESI-MS/MS:
m/z 227 (M H+), 147 (M SO3H+); UV:
max 205, 236 nm. MS/MS showed a peak at
m/z 147 (M SO3H+), indicating the
presence of a sulfate group in the metabolite. The NMR spectrum of
metabolite M displayed similar aromatic protons and an
aromatic methyl group as that of metabolite J. The presence
of the sulfate group on 2'-OH shifted 3'-H downfield, a shift to 7.28 ppm from 6.72 ppm in metabolite J. Singlets at 5.24 and 5.12 ppm were olefinic protons, and the singlet at 2.12 ppm was consistent
with an allylic methyl group. Metabolite M was identified as
2-(2'-hydroxy-4'-methylphenyl)propene sulfate. Its phenol precursor,
2-(2'-hydroxy-4'methylphenyl)propene is a known compound; its
1H NMR data in CDCl3 are as
follows: 7.1 to 6.7 (2 doublets, 2H, 5'-H and 6'-H), 6.75 (s, 1H,
3'-H), 5.7 (br. s, 1H, -OH), 5.4 (br. s, 1H, 1-olefinic H), 5.15 (br.
s, 1H, 1-olefinic H), 2.3 (s, 3H, 4'-CH3), 2.1 (s, 3H, 3-CH3) (Madyastha and Gaikwad, 1999).
M showed similar NMR results as its phenol precursor except
the 3'-H shift due to the effect of the sulfate group.
As shown in Table 1 and Fig. 2, peaks C-G are
mostly glucuronides, which account for 38 to 47% of the total radioactivity in urine. Mercapturic acids (peaks B,
K, and L) constitute 9 to 3% and phenols (peak
J and M) make up 3 to 7% of the total
radioactivity in urine.
Reactions of Pulegone and Glutathione in Vitro. The in vivo study showed that at least 9 to 13% of metabolites were derived from direct Michael addition of glutathione to pulegone. These results prompted us to reinvestigate the reactivity of pulegone with glutathione in vitro. Two radiolabeled products were isolated from the reaction of glutathione with [14C]pulegone catalyzed by 3 Eq of NaHCO3. They were identified by MS and NMR to be diastereomeric 8-(glutathion-S-yl)menthone/isomenthone. The chemical shifts of their 5-CH3 groups were 1.02 and 0.9 ppm, respectively. We assigned the product with the more downfield 5-CH3 as a menthone in agreement with the assignment for metabolites K and L. These two authentic standards showed similar retention times with the respective two GST incubation products (Fig. 3). These two GST incubation products were formed only when both GST and GSH were present in the incubation mixtures (Fig. 3). The results demonstrated that pulegone underwent Michael addition with glutathione to give the diastereomeric 8-(glutathion-S-yl)menthone/isomenthone under catalysis of GST in vitro.
|
Enzyme Hydrolysis of 24-h Urine and Metabolites D2, E2, and 9-Hydroxypulegone Glucuronide. Glucuronidase treatment of 24-h urine (80 mg/kg, single dose) resulted in hydrolysis of many metabolites to give aglycones with longer retention times, whereas sulfatase treatment did not change the metabolite profile. Pulegone (RT = 21.6 min, method 2, system B), menthofuran (RT = 26.4 min, method 2, system B), 2-(N-acetylcystein-S-yl)menthofuran (RT = 19.0 min, method 2, system B), piperitone (RT = 20.0 min, method 2, system B), and pulegol (RT = 20.4 min, method 2, system B) were not observed in either hydrolyzed or untreated urine (data not shown). The results of glucuronidase hydrolysis of metabolites D2, E2, and 9-hydroxypulegone glucuronide were described previously in this work.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
This study focused on 13 radioactive peaks (A-M) in the rat urinary metabolite profile of pulegone (Fig. 1). None of the peaks contained more than 15% of total radioactivity in urine, and several of the peaks were resolved into more than one component (Table 1). Approximately 40% of the eluted radioactivity was diffuse and poorly resolved and not pursued further. Unchanged pulegone was not detected. Pulegone was found to be metabolized via three pathways (Fig. 4): 1) hydroxylation followed by glucuronidation (metabolites C1, D1, D2, and E1) or further metabolism (metabolites E3, J, and M); 2) reduction to give menthone/isomenthone, followed by hydroxylation/glucuronidation (metabolites E2, F1, F2, and G1); and 3) formation of mercapturic acids (metabolites K and L) followed by hydroxylation (metabolite B1).
|
Approximately equal amounts of hydroxypulegone glucuronides and
hydroxymenthone/isomenthone glucuronides were the most abundant urinary
metabolites. This observation was similar to the study on biliary
metabolites of pulegone in which glucuronides of hydroxylated pulegone
and hydroxylated reduced pulegone were principle metabolites (Thomassen
et al., 1991). Four hydroxypulegone glucuronides, 4- (C1),
5- (D1), 10- (D2), and 3- (E1), were
characterized in urine. 9-Hydroxypulegone glucuronide produced from
acid-catalyzed isomerization of 10-hydroxypulegone glucuronide (D2) was obtained. 9-Hydroxypulegone glucuronide was not a
metabolite or, at best, a minor metabolite. Glucuronidase hydrolysis of
D2 gave 10-hydroxypulegone as the sole product. By contrast, glucuronidase treatment of 9-hydroxypulegone glucuronide did not give
the anticipated menthofuran or any other detectable products. Metabolites E1 and C2 are related as allylic
isomers. 3-Hydroxypulegone and
5-methyl-2-(1-hydroxy-1-methylethyl)-2-cyclohexen-1-one, the alcohol
precursors to E1 and C2, respectively, were
observed in a fungal incubation mixture of pulegone, and the latter was
believed to be converted from the former through an allylic alcohol
isomerization (Madyastha and Thulasiram, 1999). Alternatively, during
the cytochrome P450-catalyzed oxidation of pulegone, an early step is
removal of the allylic hydrogen atom on C-3; the allylic radical thus
formed can be oxygenated at either end to give the isomeric alcohols.
Isomeric allylic alcohol products have been reported for similar
substrates (Groves and Subramanian, 1984).
Four hydroxymenthone/isomenthone glucuronides with hydroxylation at the
C-5 position or the methyl groups were fully characterized (E2, F1, F2, and G1).
Menthone/isomenthone along with menthofuran and pulegone were detected
in plasma of rats after i.p. administration of pulegone (Thomassen et
al., 1990). Reduction of the carbon-carbon double bond probably took place before hydroxylation. Reduction to menthone/isomenthone is a
detoxification process. Menthone/isomenthone were shown to be neither
as hepatotoxic nor as extensively bound to tissue proteins as pulegone
(McClanahan et al., 1989). Two minor metabolites (hemiacetal, G3, and lactone, G2) corresponding to higher
oxidation products of E2 were isolated.
The metabolic pathway proposed for metabolites M and
J begins with hydroxylation of pulegone to
5-hydroxypulegone, followed by dehydration to give piperitenone (Fig.
5). Hydroxylation of piperitenone
followed by dehydration and a 1,5-H+ transfer led
to formation of 2-(2'-hydroxy-4'-methylphenyl)propene. Sulfation of
2-(2'-hydroxy-4'-methylphenyl)propene gave metabolite M.
Previous studies have shown 2-(2'-hydroxy-4'-methylphenyl)propene to be
a metabolite of piperitenone (Madyastha and Gaikwad, 1999). Hydration
and oxidation of 2-(2'-hydroxy-4'-methylphenyl)propene results in the
formation of metabolite J, a transformation similar to
metabolism of -methylstyrene to phenylpropionic acid (De Costa et
al., 2001).
|
Michael addition of glutathione to pulegone ultimately gave the major
mercapturic acid metabolites K and L. The
reaction also occurred in vitro under catalysis by GST or in basic
medium to give two glutathione conjugates (Fig. 3). These glutathione
conjugates were previously partially characterized in the bile of rats
treated with pulegone (Thomassen et al., 1991). The direct conjugation
of pulegone with glutathione might partially explain the
glutathione-depleting effect of pulegone, although the in vitro
reaction was neither rapid nor complete. Also, Thomassen et al. (1990)
showed that cytochrome P450-catalyzed bioactivation of pulegone was
necessary for the depletion of glutathione. Several mercapturic acid
metabolites with a molecular weight of 331, formally products of
hydroxylation of metabolites K and L, were
observed, but only B1 was fully characterized. Two mercapturic acids with molecular weight 329 were detected; they were
tentatively assigned as B2 and C4. Glutathione precursors to B2 and C4 have been partially
characterized in the bile of rats dosed with pulegone. It has been
speculated that allylic alcohol precursors to C2 and
E1, which if activated, might undergo nucleophilic
substitution with glutathione to give these conjugates (Thomassen et
al., 1991). Several glutathionyl glucuronide diconjugate metabolites
were observed in bile (Thomassen et al., 1991). The hydroxylated
mercapturic acids, such as B1, might undergo glucuronidation
to give "mixed" diconjugate metabolites.
Fourteen phase I urinary metabolites of pulegone including menthofuran
have been previously characterized (Moorthy et al., 1989; Madyastha and
Raj, 1993).
7a-Hydroxy-3,6-dimethyl-5,6,7,7a-tetrahydro-2(4H)-benzofuranone and 5-hydroxypulegone are free forms of E3 and
D1, respectively. We cannot exclude the possibility that
7a-hydroxy-3,6-dimethyl-5,6,7,7a-tetrahydro-2(4H)-benzofuranone itself is a urinary metabolite of pulegone; it had the same retention time as a peak appearing in the metabolite profile (Fig. 1).
Hydrolysis/dehydration of F1 or F2 and
D1 could give piperitone and piperitenone, respectively. The
rest of the phase I metabolites have very little structural similarity
to our phase II metabolites. Several standards of the phase I
metabolites were prepared or obtained from commercial sources. Pulegol,
piperitone, and menthofuran were not detected in untreated urine or in
urine treated with glucuronidase. We have observed that the major
microsomal metabolite of pulegone, 10-hydroxypulegone, when dissolved
in CDCl3 or
CD2Cl2, quickly isomerized
and dehydrated to give menthofuran. It is possible that metabolite
D2, once hydrolyzed to 10-hydroxypulegone under the
isolation conditions, could convert into menthofuran while dissolved in
CDCl3 for an NMR. p-Cresol, formed as
a result of the cleavage of the carbon-carbon double bond of pulegone, would be nonradioactive and not pursued in our study. Cleavage of the
carbon-carbon double bond of pulegone and subsequent metabolism should
lead ultimately to expiration of
14CO2, a relatively minor
excretion pathway (1.3% of total dose) (unpublished
observation). The difference in dose and isolation procedures might
contribute to the different outcome of the metabolic pathways.
In vitro studies have demonstrated sequential metabolism of pulegone to
menthofuran, then to mintlactones (Fig.
6) (Gordon et al., 1987; Thomassen et
al., 1992). A plausible metabolite that can be envisioned from this
pathway is E3. However, Madyastha and Raj (1992) did not
identify E3 or
7a-Hydroxy-3,6-dimethyl-5,6,7,7a-tetrahydro-2(4H)-benzofuranone as urinary metabolites of menthofuran. We have recently determined that
E3 is a major urinary metabolite from rats dosed with [14C]menthofuran (unpublished
observation). The other menthofuran metabolites are currently under
investigation. Menthofuran has been detected in plasma of rats after
i.p. administration of pulegone (Thomassen et al., 1988, 1990). Our
studies on urinary metabolites of pulegone and menthofuran support that
menthofuran is an in vivo metabolite of pulegone. The reactive
intermediate from metabolism of pulegone and menthofuran,
2-Z-(2'-keto-4'-methylcyclohexylidene)propanal, has been
shown to bind to liver proteins and is believed to be responsible for
the hepatotoxicity of pulegone and menthofuran (McClanahan et al.,
1989; Thomassen et al., 1992). Another oxidative metabolite of
pulegone, 10-hydroxypulegone, could be oxidized to
2-E-(2'-keto-4'-methylcyclohexylidene)propanal, which might bind to proteins (Fig. 6). MS analysis of the bile of rats dosed with
pulegone gave a metabolite that was tentatively assigned as
2-(glutathion-S-yl)menthofuran, the GSH conjugate of
2-Z-(2'-keto-4'-methylcyclohexylidene)propanal (Thomassen et
al., 1991). To determine whether the mercapturic acid derived from this
conjugate present in urine, we synthesized 2-(N-acetylcystein-S-yl)menthofuran as a
standard. 2-(N-Acetylcystein-S-yl)menthofuran was
not detected in urine of rats treated with single 80-mg/kg doses of
pulegone, but a minor peak (0.6% of total radioactivity in urine) with
the same retention time was observed in a multiple-dose study (4 daily
80 mg/kg/day; 3,4*; see Table 1) (data not shown). It is not certain at
this point if the minor peak is
2-(N-acetylcystein-S-yl)menthofuran.
|
In summary, we have characterized 14 major urinary metabolites of pulegone and have gathered information on some minor metabolites. The identification of these metabolites has elucidated three major pathways by which pulegone can be metabolized. The isolation of several mercapturic acids suggests that pulegone or activated allylic alcohols might be partially responsible for the toxicity of pulegone.
| |
Acknowledgments |
|---|
We acknowledge the assistance of Tiffany M. Heath, Carolyn A. Leverett, Freddy Nieves, Namrata Patel, Raki K. Prasad, and Peter J. Schupp for parts of the HPLC isolation, synthesis, and microsomal and GST incubations. We are grateful to Dr. Carol E. Parker and Dr. Kenneth B. Tomer for obtaining GC/MS data on 10-hydroxypulegone.
| |
Footnotes |
|---|
Received April 18, 2001; accepted September 14, 2001.
Dr. Ling-Jen Chen Ferguson, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. E-mail: ferguso2{at}niehs.nih.gov
| |
Abbreviations |
|---|
Abbreviations used are: TFA, trifluoroacetic acid; Gluc, glucuronide; GSH, reduced glutathione; GST, glutathione S-transferase; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; GC, gas chromatography; RT, retention time.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
-methylstyrene in rats.
Drug Metab Dispos
29:
166-171
analysis and separation, in
Principles and Methods of Toxicology (Hayes AW ed) pp 609-634,
Raven Press, New York.a hepatotoxin in rat: isolation and characterization of new metabolites.
Xenobiotica
23:
509-518[Medline].a hepatotoxin in rat.
Xenobiotica
19:
217-224[Medline].This article has been cited by other articles:
|
|
 |
|
  L.-J. Chen, E. H. Lebetkin, and L. T. Burka METABOLISM OF (R)-(+)-MENTHOFURAN IN FISCHER-344 RATS: IDENTIFICATION OF SULFONIC ACID METABOLITES Drug Metab. Dispos., October 1, 2003; 31(10): 1208 - 1213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-J. Chen, E. H. Lebetkin, and L. T. Burka COMPARATIVE DISPOSITION OF (R)-(+)-PULEGONE IN B6C3F1 MICE AND F344 RATS Drug Metab. Dispos., July 1, 2003; 31(7): 892 - 899. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
