Vitamin K1 hydroquinone formation catalyzed by DT-diaphorase

Vitamin K1 hydroquinone formation catalyzed by DT-diaphorase

Vol. 104, No. 1, 1982 January 15, 1982 VITAMIN BIOCHEMICAL RESEARCH COMMUNICATIONS Pages 187-192 K1 HYDROQUINONE FORMATION CATALYZED BY DT-DIAPHOR...

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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,

4.

Martius,

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Martius,

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R.,

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B. (1981) Arch.

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M.J.

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194, 983-988. Biophys.

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B., Blomberg, Biophys. 207, M.M. P.M.

K.,

Stenberg,

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