*Corresponding author,
e-mail : tkyaraki@gunma-u.ac.jp (T. Araki)
https://doi.org/10.24198/idjp.v2i1.25302
© 2020 Kawano et al
Vol 2, Issue 1, 2020 (7-13)
http://journal.unpad.ac.id/idjp
The reductive activity of human liver microsomes for vitamin K epoxides
Masashi Kawano
1,2
, Takuya Araki
1,2*
, Hideaki Yashima
1
, Tomonori Nakamura
3
, Koujirou
Yamamoto
1,2
1. Department of Pharmacy, Gunma University Hospital, 3-39-15 Showa-machi, Maebashi, Gunma 371-
8511, Japan
2. Department of Clinical Pharmacology and Therapeutics, Gunma University Graduate School of
Medicine, 3-39-22 Showa-machi, Maebbashi, Gunma 371-8511, Japan
3. Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
Received: 27 Dec 2019/Revised: 3 Jan 2020/Accepted: 13 Jan 2020/Published: 13 Feb 2020
ABSTRACT
Vitamin K (VK) is oxidized to vitamin K epoxide (VK-O) during the production of VK-dependent
blood clotting factors. Thereafter, VK-O is reduced to VK by vitamin K epoxide reductase (VKOR)
in the liver and reused. This reductive reaction is inhibited by warfarin, an oral anticoagulant. VK in
nature is roughly divided into two types, VK1 (phylloquinone) and VK2 (menaquinone). Although
VK1-O and MK4-O by VKOR was evaluated in an in vitro study using human liver microsomes. The
reduction was around 7 and 4 times higher for MK4-O than for VK1-O, respectively. The intrinsic
clearance of MK4-O, obtained by dividing the Vmax value by the Km value, was about 30 times
greater than that of VK1-O. According to these data, the production of VK-dependent blood coagulation
factors can be considered to be dominated mainly by MK4-O, at least under normal conditions. We
may thus have to be more careful about controlling the intake of MK4 than VK1 in patients receiving
warfarin therapy.
Keywords: vitamin K1 (phylloquinone), vitamin K2 (menaquinone), VKOR, warfarin
1. Introduction
Vitamin K (VK) is essential for the production
of VK-dependent blood clotting factors (blood
blood clotting. Subsequently, VK is oxidized
to vitamin K epoxide (VK-O). Thereafter,
VK-O is reduced to VK by vitamin K epoxide
reductase (VKOR) in the liver and reused [1].
Warfarin, one of the most widely used oral
anticoagulants, inhibits VKOR, depletes VK,
and reduces the production of coagulation factors
is used for the treatment and prevention of
thromboembolism in various pathologies associated
with increased blood coagulation ability, such
and prosthetic valve replacement [4,5]. On the
among individuals, with multiple genetic variations
genetics cannot fully explain individual variations,
one of the other major factors besides genetic
factors [8-10]. Because warfarin inhibits the reuse
by the ingestion of drugs and foods containing large
amounts of VK. These interactions are clinically
important, and many VK-containing foods such
as broccoli, chlorella, Japanese traditional foods,
nori (seaweed) and natto (fermented soybean),
and pig liver have been reported to potentially
VK is a general term for a number of substances
with 2-methyl-1,4-naphthoquinone (VK3) as a
common structure. Structurally, these substances
of unsaturation bonded to the carbon at position
3. VK in nature is roughly divided into two types,
M. Kawano et al / Indo J Pharm 1 (2020) 7-13
8
VK1 and VK2. VK1 is contained in plant foods such
as broccoli, where it is known as phylloquinone.
VK1 thus comprises the majority of human VK
intake and is generally considered to account for
other hand, VK2, which is also called menaquinone
(MK), is mainly present in the liver of mammals
and comprises a group of 14 substances (MK1
units constituting the prenyl side chain [17]. Two
forms of MK consumed by humans are MK4,
mainly found in chicken and egg yolk, and MK7,
produced mainly by nattokinase and found in natto.
Human intestinal bacteria synthesize MK10 and
MK11 and, to a lesser extent, MK7, MK8, MK9,
and MK12 [18]. However, because the amount of
MK synthesized by enterobacteria is minuscule
compared to the VK requirement, dietary intake
of VK is considered essential [19]. The absorption
rate from the intestinal tract and elimination half-
during the small intestine absorption process and
then converted to MK4 in peripheral tissues [21,22].
dependent blood coagulation factor generation,
the VKOR metabolic activities for VK1-O
and MK4-O were evaluated in an in vitro
study using human liver microsomes (HLMs).
2. Methods
2.1 Materials
AgNO, dithiothreitol (DTT),
Tris(hydroxymethyl)aminomethane, and MK4
Chemical Corporation (Osaka, Japan), whereas
phylloquinone (VK1), phylloquinone epoxide
(VK1-O), and menaquinone-4 epoxide (MK4-O)
were purchased from Sigma Aldrich Japan (Osaka,
Japan). MK5 was generously supplied by Eizai Co.,
Ltd. (Tokyo, Japan). Pooled HLMs (Lot #. 88114)
were purchased from Nippon Becton Dickinson
Co., Ltd. (Tokyo, Japan). All other reagents were
obtained from commercial sources and were
HPLC-grade or special-grade reagents.
VK1, MK4, MK5, VK1-O, and MK4-O were
solutions of VK1, MK4, and MK5, were prepared
by dissolving each compound in ethanol at 10 mg/
mL and then diluted in methanol to the appropriate
concentration before use. Stock solutions of VK1-O
and MK4-O were prepared by dissolving each
compound in isopropanol at 10 mg/mL and then
diluted in methanol to the appropriate concentration
and diluted in water to the appropriate concentration
before use.
2.2 Enzymatic reduction of VK-O by HLMs
The reaction mixture was prepared by mixing
35 µL distilled water, 2.5 µL methanol, 25 µL
for the assay of VK1-O and MK4-O, respectively),
mM Tris(hydroxymethyl)aminomethane (pH
5 min using a block incubator as preincubation and
cold 0.05 M AgNO:isopropanol (5:9) solution was
added to stop the reaction. MK5 (500 µL) was used
n-hexane was added, the samples were vigorously
shaken for 15 min with a mechanical shaker, and the
mixture was centrifuged at 13,000 rpm for 5 min.
The upper organic layer was transferred to another
polyethylene tube, and evaporated to dryness under
reduced pressure and dark conditions. The residue
the solution was used for LC-MS/MS analysis.
The HLM concentration, reaction time and
initial concentration of substrates were determined
according to the results of a preliminary study.
M. Kawano et al / Indo J Pharm 1 (2020) 7-13
9
concentration range of 0.05–0.2 mg/mL when the
initial concentration of VK1-O and the reaction
For MK4, MK4 production increased with an
concentration range of 0.005–0.02 mg/mL when
the initial concentration of MK4-O and the reaction
VK1-O and MK4-O metabolic studies were set at
0.1 and 0.01 mg/mL, respectively (Fig. 1 A, C).
increasing reaction time in the range of 5–15 min
when the initial concentration of VK1-O and the
mg/mL, respectively. MK4 production increased
with an increasing reaction time in a range of 5–15
mg/mL when the initial concentration of MK4-O
0.1 µM and 0.01 mg/mL, respectively. Therefore,
the reaction time for both the VK1-O and MK4-O
metabolic studies was set at 10 min (Fig. 1 B, D).
The initial substrate concentration was set to ranges
including concentrations to obtain both of the
maximum reaction rate and 1/2 of the maximum
reaction rate.
2.3 Enzyme kinetic analysis
The reaction rate was obtained by measuring
the amount of VK1 or MK4 produced from VK1-O
50 µM) as substrates. The reaction rate parameter
was calculated by applying the Michaelis-Menten
equation (Eq. 1) to the relationship between the
substrate concentration and the reaction rate.
v = Vmax × [S]/(Km + [S]), Eq. 1
where v, Vmax, [S], and Km represent the
enzymatic reaction rate, maximum enzymatic
reaction rate, concentration of substrate, and
the nonlinear least squares method was used. The
Equation 2.
CLint = Vmax / Km Eq. 2
2.4 Analysis of VKs and VK-Os
Tandem quadrupole MS was used to analyze
was used with the following ionization parameters:
capillary voltage, 1000 V; desolvation temperature,
following transitions were monitored: 451/187
Sample cone voltages and collision energies were
V for VK1-O, 22 V and 22 V for MK4, 24 V and
For separation analysis, LC was performed with an
®
UPLC
®
BEH C18 column (2.1 mm × 50 mm, 1.7
conditions were as follows: column temperature,
times of VK1, VK1-O, MK4, MK4-O, and MK5
Good linearity was found within the ranges of
5–1000 ng/mL and 0.5–50 ng/mL for VK1 and
were under 15% and the accuracy were over 85%.
3. Results
The Michaelis-Menten plot of the reductive
reaction of VK1-O and MK4-O by the HLMs is
Figure 1. Vitamin K cycle and its inhibition by warfarin
M. Kawano et al / Indo J Pharm 1 (2020) 7-13
10
shown in Fig. 2 and the estimated Michaelis-
Menten parameter is listed in Table 1. The Km
value of MK4-O was around 7 times lower than
that of VK1-O and its Vmax value was around
4 times higher than that of VK1-O. The intrinsic
clearance—obtained by dividing the Vmax value
by the Km value—was about 30 times greater for
MK4-O than for VK1-O.
Table 1. Enzyme kinetic parameters of VK-O reduction
VK1-O MK4-O
Km (µM) 1.38 ± 0.37 0.201 ± 0.032
Vmax (pmol/min/mg
protein)
202.5 ± 9.11 819.2 ± 22.0
CL
int
(mL/min/mg protein) 0.147
The data of Km and Vmax are expressed as mean ±
standard deviation.
Although about 25% and about 12% of MK4-O
were reduced to MK4 after a 10-min reaction when
respectively, the amount of VK1 and MK4
produced was less than 10% of the substrate in
the VK1-O and MK4-O metabolic studies at other
concentrations (table 2).
Table 2. Ratio of metabolized VK-Os to reductants
Substrate
concentration (µM)
Reduction ratio (%)
VK1-O MK4-O
0.1 No data
0.5 No data
1 7.00 ± 1.35
5
10 1.93 ± 0.17 0.79 ± 0.11
50 0.38 ± 0.04 0.17 ± 0.02
100 0.19 ± 0.02 No data
The reduction ratios are shown as the ratio of VKs
produced to their complete reduction. The data are
expressed as mean ± standard deviation.
4. Discussion
VK is one of the fat-soluble vitamins essential
for the activation of VK-dependent proteins and
is involved not only in the production of blood
clotting factors, but also in the functional control
present in bone, cartilage, vascular smooth muscle,
brain/nerve tissue, and kidney [23,24]. As described
Figure 2. Basic experimental data for determining the experimental conditions. The symbols and error bars represent
the mean data and standard deviations, respectively.
M. Kawano et al / Indo J Pharm 1 (2020) 7-13
11
above, VK is roughly divided into two types—VK1
and in vivo elimination half-lives [20].
Km and maximum reaction rate shown as Vmax
of the MK4-O reduction by VKOR were around 7
and 4 times larger than that of VK1-O reduction.
Accordingly, the CLint value of the MK4-O
reduction by VKOR was around 30 times larger
than that of the VK1-O reduction (Fig. 2, Table 1).
Although about 90% of human VK intake is VK1
similar (0.044–1.357 and 0.074–0.759 ng/mL for
VK1 and MK4, respectively) [25], and most of the
VK in organs such as the brain, pancreas, and liver
VK-Os in human are known to be much lower level
than the that used in our study. So, the reaction of
VKOR in body is considered to be not saturated,
and the contribution of VK-Os as substrates should
MK4 intake should be greater than that of VK1.
Because the physiological substrate of VKOR in
mammal liver is reported to be MK4-O, the reuse
of VK1, which is not the original substrate, is likely
The interaction between warfarin and the VK1
found mainly in vegetables and seaweed has been
transient, disappearing within a few days in many
This phenomenon is thought to be due to the
consumed, most of the VK1, which would activate
VK-dependent blood clotting factors, disappears
from the body without reuse by VKOR reduction.
On the other hand, there are limited reports on the
interaction between warfarin and food containing a
individuals who ate VK2-containing foods was
reported to last for several weeks [27,28]. This
reduction and reuse of MK4 by VKOR. The
VKs is also consistent with our results.
rate of the VKOR reduction were somewhat larger
for MK4-O than for VK1, and the CLint of VKOR
was around 30 times higher for MK4-O than for
VK1-O. Therefore, under normal dietary intake
conditions, the production of VK-dependent
mainly by MK4-O, suggesting that individual
be suppressed by controlling the intake of MK4.
However, because we did not conduct a metabolic
study on the coexistence of VK1-O and MK4-O or
an interaction study between warfarin and VK1-O
a metabolic study of the coexistence of VK1-O and
MK4-O, an interaction study between warfarin and
VK1-O or MK4-O, and a clinical study of the blood
Figure 3. Reduction rate of VK-O by HLMs. The
symbols, error bars, and dotted lines represent
the mean data, standard deviations, and curve
circles show VK1 epoxide and MK4 epoxide,
respectively.
M. Kawano et al / Indo J Pharm 1 (2020) 7-13
12
concentration of VKs in patients taking warfarin.
5. Conclusion
maximum reaction rate of reduction by VKOR are
much larger for MK4-O than for VK1, and the CLint
of VKOR is around 30 times higher for MK4-O
than for VK1-O. These data suggest that individual
suppressed by controlling the intake of MK4.
6. Acknowledgement
This study was supported by a grant-in-aid for
Japan Society for the Promotion of Science.
Eisai Co., Ltd. (Tokyo, Japan). All other authors
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