Biosci. Biotechnol. Biochem., 70 (5), 1259–1261, 2006
Note
Biotransformation of (ꢀ)-Menthone by Human Liver Microsomes
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Mitsuo MIYAZAWA and Kyousuke NAKANISHI
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University,
Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
Received October 5, 2005; Accepted January 12, 2006
The aim of the current study was to investigate the
metabolism of (ꢀ)-menthone by liver microsomes of
humans. (ꢀ)-Menthone (1) was metabolized to (þ)-
neomenthol (2) (3-reduction) and 7-hydroxymenthone
(3) by human liver microsomes. The metabolites formed
were analyzed on GC and GC–MS. Kinetic analysis
showed that Km and Vmax values for the metabolized
(ꢀ)-menthone to respective (þ)-neomenthol and 7-
hydroxymenthone by liver microsomes of human sam-
ple HG70 were 0.37 mM and 4.91 nmol/min/mg protein
and 0.07 mM and 0.71 nmol/min/mg protein.
(ꢀ)-Menthone and (þ)-neomenthol were purchased
from Tokyo Chemical Industry. (TCI, Tokyo). Piper-
itone was purchased from Taiyo Kouryou ( ). NADPþ,
glucose-6-phosphate, and glucose-6-phosphate dehydro-
genase were purchased from Sigma-Aldrich (St. Louis,
MO).
A Hewlett-Packard model 5890A gas chromatograph
(Hewlett-Pacard, Atlanta, GA) equipped with a split
injector was combined with a direct coupling to a
Hewlett-Packard 5972 mass spectrometer. The metabo-
lites were separated by a DB-5 silica capillary column
(30 m length, 0.25 mm d. i.) using helium (at 0.5 ml/
min) as the carrier gas. The column temperature was
programmed from 60 ꢁC to 240 ꢁC at a rate of 4 ꢁC/min,
and then held at 240 ꢁC. Ionization voltage was 70 eV.
The EI mode was used. A GC–MS system equipped
with Wiley 138 K Mass Spectral Database software
(John Wiley and Sons, New York, NY) was used for
identification of products.
Key words: microsomes; (ꢀ)-menthone; kinetic analysis
The metabolism of absorptive xenobiotics, such as
aroma compounds and drug and natural compounds, is
performed by various enzymes, but among these
enzymes, cytochrome P450 play important roles in the
metabolism of xenobiotics. CYP enzymes are respon-
sible for oxidation and reductive metabolic transforma-
tion of xenobiotics. Our previous studies demonstrated
that several terpenes, 1,4-cineole, 1,8-cineole, (þ)- and
(ꢀ)-limonene, and (ꢀ)-verbenone are catalyzed by
cytochrome P450 enzymes to their respective oxidation
products in liver microsomes.1–4) 1,4-Cineole and 1,8-
cineole have determined to be oxidized at the 2-position
of products both by rat and human CYP3A enzymes.1,2)
Limonene enantiomers are metabolized to their respec-
tive carveols to and perillyl alcohols in liver microsomes
of mice, rats, guinea pigs, rabbits, dogs, monkeys, and
humans.3,4) (ꢀ)-Verbenone has been determined to be
oxidized at the 10-position of the compound by rat
CYP2B1 and human CYP2A6 and CYP2B6.5)
The monocyclic monoterpene (ꢀ)-menthone has been
shown to be present in peppermint and other plants, and
is used as a fragrance in household products, compo-
nents of artificial essential oils, and tooth-brushing
powder.6) Biotransformation of (ꢀ)-menthone has been
reported by yeasts and yeast-like fungi,6,7) but there has
been no report of the metabolism of (ꢀ)-menthone in
mammal. In this study, we examined the metabolism of
(ꢀ)-menthone by human liver microsomes.
Human liver samples (HG70) were obtained from
Gentest. (Woburn, MA)
Oxidation and reduction of (ꢀ)-menthone by liver
microsomes were determined as follows: The standard
reaction mixture contained liver microsomes (0.1 mg of
proteins/ml) and 200 mM (ꢀ)-menthone in a final
volume 0.50 ml of 100 mM potassium phosphate buffer
(pH 7.4) containing an NADPH-generating system (0.5
mM NADPþ, 5 mM glucose 6-phosphate, and 0.5 units of
glucose 6-phosphate dehydrogenase/ml). The reaction
mixture was incubated at 37 ꢁC for 60 min, terminated
by centrifugation at 3,000 rpm for 5 min, and used for
analysis with GC–MS for identification of the metabo-
lites. A reaction system without microsomes was also
prepared for a blank experiment. No metabolites were
detected.
Kinetic parameters for (ꢀ)-menthone metabolites by
microsomes were estimated using a computer program
(KaleidaGraph; Synergy Software,Version 1.5.3J, Read-
ing, PA) designed for nonlinear regression analysis.
A nutrient broth was prepared by dissolving sac-
charose (15%), glucose (15%), polypeptone (5%), KCl
.
(0.5%), MgSO4 7H2O (0.5%), K2HPO4 (1%) and
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To whom correspondence should be addressed. Tel: +81-6-6730-5880; Fax: +81-6-6727-4301; E-mail: miyazawa@apch.kindai.ac.jp
Abbreviation: P450, cytochrome P450