Vol. 104, No. 1, 1982 January 15, 1982
VITAMIN
BIOCHEMICAL
RESEARCH COMMUNICATIONS Pages 187-192
K1 HYDROQUINONE FORMATION CATALYZED BY DT-DIAPHORASE Michael
J.
Fascot
and Louise
Division of Laboratories State Department of Health,
New York Received
AND BIOPHYSICAL
November
19,
M. Principett
and Research, Albany, New York
12201
1981
SUMMARY: Vitamin Kl hydroquinone has been identified as a metabolite of vitamin Kl biotransformation catalyzed by highly purified DT-diaphorase (NAD(P)H dehydrogenase, EC 1.6.99.2) isolated from livers of 3-methylcholanthrene induced rats. The hydroquinone was sufficiently stable to permit enzymatic reactions to be conducted under an atmosphere of air and quantitation of hydroquinone by high performance liquid chromatography. Based on kinetic data reported here, warfarin and probably dicoumarol at therapeutic levels do not appreciably affect M'-diaphorase catalyzed vitamin K hydroquinone formation. INTRODUCTION DT-diaphorase which
catalyzes
The enzyme it
have been
induced is
(NAD(P)H the
occurs
dehydrogenase,
reduction
primarily
detected
of a variety in the
in rats
electrophoretically
is
of redox-dyes
cytoplasm
in mitochondria
3 to 4 fold
EC 1.6.99.2)
of the
a flavoenzyme such as DCIPl(1).
liver
although
and microsomes(2).
by 3-methylcholanthrene
and immunologically
traces
DT-diaphorase and the
induced
indistinguishable
of is
enzyme
from the
native
farm(3). 4--Hydroxycoumarin are effective
inhibitors
of the
diaphorase
thesis
that
vitamin
and indanedione
K hydroquinone(4).
an extremely
poor
substrate
tTo whom correspondence
reduce role
menadione
of this Kl,
for
diaphorase
should
however,
to reduce
was initially
dicoumarol,
with
K3) led
in a number
the
ability
to the hypovitamin
K to
shown to be of in vitro
be addressed.
#To be submitted as part of a thesis by L.M.P. the Ph.D. requirement, Albany Medical College Albany, N.Y. 1The abbreviations performance liquid
coupled
(vitamin
enzyme is
Vitamin the
especially
This
of DT-diaphorase(1).
to rapidly
a physiological
anticoagulants,
DCIP, used are: chromatography.
in partial of Union
2,6-dichloroindophenol;
fulfillment University,
of
and HPLC, high
0006-291X/82/010187-06$01.00/0 187
Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 104, No. 1, 1982
Hydroquinone
assays(Z). vitamin
into
liposomes
purified
hepatic
Assays
utilized
chromogens
quantifies
the
presence purified
using
a direct
on vitamin ing
the
role
or enzymes
We recently vitamin
the
ferricyanide
have demonstrated
and/or have
which
K1 and NADH can proformation
in
quantitation
method.
Kl hydroquinone
formation
diaphorase
The effects
in vitamin
employed
indirect
formed
vitamin
were
DT-diaphorase
reducing
a technique
We report catalyzed
quantify
accept
reported
Kl hydroquinone
DT-diaphorase
of the
to potassium
of vitamin
formation
of dithiothreitol(8).
highly
presence
to detect
such as secondary
directly
reaction
by incorporating
protein.
Kl hydroquinone
hydroquinone.
achieved
dependent-y-carboxyglutamate
catalyzed-vitamin
from the
RESEARCH COMMUNICATIONS
of investigators(6,7)
in the
precursor
previously
the
a number
Kl hydroquinone
microsomal
was finally
and linking
DT-diaphorase
duce vitamin
AND BIOPHYSICAL
formation
Recently
reduction(5). that
BIOCHEMICAL
using
by hepatic
here
our
methods
equivalents HPLC which microsomes
investigations
in of
Kl hydroquinone
formation
of dicoumarol
and warfarin
determined K metabolism
with
the
and its
object function
of probin
coagulation. METHODS DT-diaphorase Preparation: Male Wistar rats (220+_1Og) from a colony maintained in this Division were administered 3-methylcholanthrene (5 mg/ml corn oil) ip at 25 mg/kg once daily for three days. Forty-eight hours after the last injection the rats were rendered unconscious with N2 and their livers perfused saline. The livers were removed, homo--in situ with physiological genized with two volumes by weight of 0.25 M sucrose and the homogenate centrifuged at 10,000 g for 30 min. Microsomes were separated from the supernatant by centrifugation at 105,000 g for 90 min and the protein content of the cytosol was adjusted to 6 to 7 mg/ml by dilution with 0.02 M Tris-HCl, 0.25 M sucrose buffer, pH 7.4. DT-diaphorase was purified from the cytosol by azodicoumarol and Sephracryl S-200 chromatography essentially as described by HiSjeberg et al.(g), except that the Sephracryl column was 2 x 70 cm and the flow rate was 10 ml/h. Protein contents were determined by the method of Bradford using Bio-Rad reagents. Assays: Assays of DT-diaphorase employing DCIP as substrate were performed essentially as described by Ernster(l), except that 0.01% (v/v) Emulgen 911 (Kao Atlas, Tokyo, Japan) was used as the detergent and the buffer was 0.2 M Tris-HCl, 0.15 M KCl, pH 7.4. Reactions were initiated by the addition of 0.29 ug of DT-diaphorase to 3 ml of buffered NADH-DCIP solution equilibrated at 250~. Rates of reactions were calculated using an extinction coefficient for reduced DCIP of 21 mM-'cm-' at 600 nm. Stock solutions of vitamin K were prepared at 20 mg/ml in 10% (v/v) Emulgen 911 as described previously( b ) and were diluted with water for use in metabolism studies. Reaction mixtures in 0.5 ml of 0.2 M Tris-HCl, 0.15 M KC1 buffer, pH 7.4, contained 2.6 ug DT-diaphorase, and NADH. Following incubation at 25Oc for 1 min, reactions were initiated by the addition of 20 ul of vitamin 188
BIOCHEMICAL
Vol. 104, No. 1, 1982
AND BIOPHYSICAL
I
I 2
0
MINUTES
Fig.
1
I
I
,
4 6
4
RESEARCH COMMUNICATIONS
I
AFTER TERMINATIONOFREACTION
Effect of time on vitamin K1 hydroquinone oxidation by air. The NADH and vitamin K1 concentrations were 0.9 mM and 40 UM Vitamin K1 respectively. Incubations were for 3 min at 25'C. hydroquinone concentrations were determined as described in METHODS at various times after termination of reaction by addition of dicoumarol. Insert: Effect of hydroquinone concentration and time Values were determined by repetitive on rates of oxidation by air. injections of the same sample.
Kl solution containing sufficient mulgen 911 to yield a final detergent conReactions were terminated by the addition of 20 ~1 of centration of 0.01%. dicoumarol solution (2 mg disodium salt/ml). A 0.1 fraction of the mixture was assayed for vitamin Kl hydroquinone concentration by HPLC essentially as described previously(8) except that the column was a 5 mm ID Radisl Pak Cl8 (Waters Associates, Milford, MA) and solvent B was acetonitrile/isopropsnol (4/l) and solvent A was water/solvent B (l/l). RESULTS AND DISCUSSION The product chromatographed the
of vitamin with
HPLC conditions
synthetically used.
back to vitamin
Kl thus
vides
direct
the
first
quinone
formation
function
in the
vitamin
establishing
and establishes
formation
in postribosomal
was previously
based
on results
for
with
the
to air
by Wallin purified
microsomes. 189
counder
the product
oxidized
Kl hydroquinone.
This
catalyzes
Kl hydro-
vitamin
has the
metabolic
carboxylase
precursor
by DT-diaphorase
Kl hydroquinone(8)
DT-diaphorase
K 2,3 epoxide
proposed
obtained
as vitamin
DT-diaphorase
that
K-vitamin
vitamin exposure
it that
cofactor
diaphorase
treated
Upon prolonged
evidence
vitamin
catalyzed
prepared
K hydroquinone
glutamate
(7)
Kl transformation
cycle
which
protein. et a1.(6) DT-diaphorase
potential
pro-
to
by providing
catalyzes
y-carboxy-
Such a role and Wallin and Triton
for
DT-
end Suttie X-100
Vol. 104, No. 1, 1982
-04
0
BIOCHEMICAL
0.4
02
0.8
I
AND BIOPHYSICAL
12
ym-'
03
[NADHI
Fig.
2
Fig.
3
Since were
vitamin
tions
determined
tration
and that
of samples (Fig. oxidation
rates
Based on these determined
within
rected
loss
for
within
in Fig.
five
oxidation
virtually hydroquinone
four
min after
due to oxidation.
that
oxidation
with initial
independent concentrations termination
range
affected
quanti-
from
that
of these
of the
Repetitive
values
to
concentraactual
concen-
HPLC analysis concentrations
of these
concentrations
of hydroquinone
concentration.
in incubation of reaction
three
at various
hydroquinone
hydroquinone the
experiments
and analyzed
95 percent time.
air,
obtained
Extrapolation
within
over
K1 in
contents
demonstrated
was linear
demonstrated
data
1.
min were
two different
were
to which
NADH and DT-diaphorase
of reaction
containing
1, insert)
Kl,
provided
of termination
extent
to vitamin
The hydroquinone
of vitamin are
oxidizes
the
hydroquinone.
thereafter time
Kl hydroquinone to determine
min incubations
the
at
DT-diaphorase catalyzed-vitamin K 1 hydroquinone formation at varying vitamin K concentrations in the absence (0) and presence of dicoumarol (o $ . The NADH concentration was 0.9 mM and the dicoumarol concentration was 26 PM. Incubations were for 3 min at 25OC. Other conditions were as described in METHODS. Slopes were calculated by linear regression analysis.
of the
times
K,] Sum-
[“lTA$lN
DT-diaphorase catalyzed-vitamin K 1 hydroquinone formation at varying NADH concentrations in the absence (0) and presence of was 40 uM and the dicoumarol (0). The vitsYnin K concentration dicoumarol concentration was 2 k uM. Incubations were for 3 min 2'5OC. Other conditions were as described in METHODS. Slopes were calculated by linear regression analysis.
undertaken
tation
RESEARCH COMMUNICATIONS
mixtures
and were
were
not
cor-
Vol. 104, No. 1, 1982 Rates were
linear
tion
over
BIOCHEMICAL
of vitamin for the
Kl hydroquinone
three
min at 25'C
range
with
respect
NADH from
17.5
to an apparent
quinone the
Km from
centration tion
to 14.4
at any of the
2).
UM and the did
PM.
upon the
NADH concentra-
was a competitive
Kl
the
Km for
The Vmax was 5.6 uM/min.
inhibitor (Fig.
Vmax from not
NADH or vitamin
by DT-diaphorase
Dicoumarol
of 30.3
to vitamin
of 520 uM, warfarin
catalyzed
2) and at 26 UM increased
value
respect
RESEARCH COMMUNICATIONS
dependent
was an uncompetitive
with
64.1
and were
to NADH (Fig.
dicoumarol
formation
formation
of 1 to 10 uM (Fig.
inhibitor
In contrast,
AND BIOPHYSICAL
of vitamin
Kl hydro-
dicoumarol
decreased
3):
7.2 to 1.0 nM/min.
inhibit
vitamin
At a con-
Kl hydroquinone
Kl concentrations
forma-
used in these
investigations. Using
DCIP as substrate
and Ernster(l1)
previously
inhibitor
with
to DCIP.
We obtained
purified
respect
investigations
ever, none
when the formation
optimum
marol
for
Kl or DCIP. substrates
the
kinetic
is
than
dicoumarol
64.1
Vmax for
i)
less
similar
that
as a
occurs
formation
is
respect
PM; iii)
vitamin
DT-diaphorase
Kl hydroquinone catalyzed-reduction 191
has been
identified
of vitamin
at
7.2 uM/min
inhibitor to the
are very
a "physiological"
at
to NADH dicou-
competitive
In reference
how-
latter
much less rather
than
substrate.
In conclusion
for
of hydroqui-
23.3
warfarin
with
are observed,
DCIP is
with
used
the
of DCIP reduction
and iv)
and particularly
of DT-diaphorase
same for
maximum rate
hydroquinone
of DCIP reduction.
highly
The modes of in-
the
uM and for
effective
the
respect
to those
Kl.
the
with
with
differences
483 pM/min;
40 fold
as inhibitors
of the
Kl is
data
from vitamin
are compared:
vitamin
K, reduction
an "artificial"
to their
at 10 PM NADH whereas
Km for
that
was a competitive
are therefore
Major
Hollander
inhibitor
conditions
by dicoumarol
DCIP reduction
apparent
effective
lite
formation
was approximately
of vitamin
DT-diaphorase,
dicoumarol
DCIP similar
of hydroquinone
occurs the
for
NADH concentration
and that
is
data
experimental
two
purified that
under
vitamin
900 PM; ii)
'it
demonstrated
of DT-diaphorase
substrates
partially
to NADH and was an uncompetitive
DT-diaphorase
hibition
for
as a metabo-
Kl and a quanti-
Vol. 104, No. 1, 1982 tative
BIOCHEMICAL
HPLC assay
particularly
of its
warfarin
DCIP reduction. could but
function
formation
inhibit
Based
AND BIOPHYSICAL
on the
of the
in vitro
4-hydroxycoumarin
data
in the
diaphorase
developed.
Kl reduction
vitamin
physiologically
inhibition
has been
RESEARCH COMMUNICATIONS
much less
presented
production
is not
Dicoumarol
here,
of vitamin
a primary
and
effectively
than
DT-diaphorase K hydroquinone,
mode of action
of the
anticoagulants.
ACKNOWLEDGEMENT This National
work
was supported
Institutes
in part
by Grant
HL-19772
(MJF)
from the
of Health.
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Ernster, Pullman,
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Ernster, L., Danielson, Acta. 58, 171-188.
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Lind,
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Martius,
C. and Strufe,
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Martius,
C.,
6.
Wallin,
R.,
7.
Wallin,
R. and Suttie,
8.
Fasco,
B. (1981) Arch.
C. and Hsjeberg,
M.J.
Ganser,
M. (1962) Biochem.,
L. and Ljunggren,
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D. and Prydz, J.W.
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326, 24-25.
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194, 983-988. Biophys.
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Hsjeberg, Biochem.
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Bradford,
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Hollander,
560-567.
B., Blomberg, Biophys. 207, M.M. P.M.
K.,
Stenberg,
S. and Lind,
C. (1981) Arch.
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(1976) Anal. and Ernster,
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