Formation of an intermediate N-oxide in the oxidative demethylation of N,N-dimethylaniline catalyzed by liver microsomes

Vol. 15, No. 2, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FORMATION OF AN INTERMEDIATE OF N,N-DIMETHYLANILINE

N-OXIDE IN THE OXIDATIVE DEMETHYLATION CATALYZED BY LIVER MICROSOMES* + M. Ziegler and Flora H. Pettit

Daniel Clayton

Foundation

Biochemical The University

Institute and the Department of Texas, Austin, Texas

of Chemistry

Received February11,1964 Microsomes catalyzes molar

The N-oxide

tive

from mammalian

the demethylation

amounts

sidered

isolated

is demethylated

demethylation

sequence

tissue

and formaldehyde at a rate

intermediate

(Pettit

sufficiently

high

"r C6Hs-N(CH3)2

iii)

for

as indicated

i> C6H5-N(CH3) 2 NADPH O2 F

it

1963).

to be con-

dependent

by the

oxida-

following

0

r

C6H5-N(CH3) 2

C6H5-NCH3 + “2’ NADPH

C6H5-N(CH3)2

equi-

and Ziegler,

in the NADPH and oxygen

of N,N-dimethylaniline,

an enzyme that to yield

of reactions:

ii)

contain

of N,N-dimethylaniline-N-oxide

of N-methylaniline

as a possible

liver

C6H5-NW3

+ CH20

O2 However, the oxidative reaction

(i)

the over-all that

by rat

or pig

to establish

demethylation does

occur

reaction

the N-oxide

intermediate

*

in order

is

reaction, and at a rate

(iii). formed

The data

Supported in part GM-09044-03.

it

the N-oxide is

equal

to show that

to or greater

summarized

demethylation

is an intermediate

necessary

and at a sufficiently

in the oxidative liver

that

than

in this rapid

rate

for

from National

it

of N,N-dimethylaniline

Institutes

+ Work carried out during tenure as an Established American Heart Association, Inc.

188

of Health,

Investigator,

of

demonstrate

microsomes.

by a grant

partial

the rate

report

in

to be an catalyzed

BIOCHEMICAL

Vol. 15, No. 2, 1964 The microsome from

liver

homogenates

Hogeboom, out

in

fractions

--et al.

the manner

trations

were

prepared

(1948).

AND BIOPHYSICAL isolated

in 0.25

described

earlier

of NADPH, substrate,

by differential

g sucrose

The oxidative

RESEARCH COMMUNICATIONS

according

demethylation (Pettit

centrifugation

assays

and Ziegler,

and microsomal

to the method

protein

were

carried

1963). are

of

The concen-

given

in the

Table

and Figure, The following

method

aniline-N-oxide. perchloric

Aliquots acid,

solution

(final

aqueous

all

to give

5 minutes

a final

and then

acid

derivative mmolar

that

extinction

density

All

formed.

fresh

evident decrease interfere

their with

ability the

lates

in the medium.

liver

microsomes;

forms

for

is based

reduced

the yellow

by

p-nitroso

of the assay was found

the

that

small

with

there

and a corresponding

increase

the

to be in optical

fresh

liver

microsomes

in the reaction is a marked

decrease

increase

in

in the amount

synthesis

microsomes

as shown

in Figure

I.

cholate

does not

appreciably

with

the ratio

linear

N,N-dimethylaniline.

demethylation

Qualitatively

is

medium,

of N-oxide

to oxidize

however,

The assay

the conditions

for

and NaN02

was heated

quantitatively

of the N-oxide

the microsomes

subsequent

acid

water.

readily

Under

particles

The rate

disrupting

the pH of the

The solution

is

I demonstrate

and cholate-treated

that

diethylether

of NADPH.

aged or cholate-treated

formaldehyde

with

extraction,

g.

corrected

accumulation

of the N-oxide

both

are

shown in Table

accumulation

times

of E-nitrosodimethylaniline

in the absence

no appreciable

but with

1940).

of the readings

The data is

and Fuson,

with supernatant

3 g trichloroacetic

at pH 2.5 the N-oxide

coefficient

obtained

there

three

at 420 mn against

which

deproteinized

The deproteinized

of 0.009

to N,N-dimethylaniline (Shriner

8.2 cm2.

read

N,N-dimethyl-

were

After

to 2.5 with

concentration

on the observation nitrous

0.3 $.

to pH 9.4 and extracted

was adjusted

at 60°,

quantitatively

mixture

of the N,N-dimethylaniline.

solution

added

of the reaction concentration

was adjusted

to remove

was used to measure

similar

results

are

of

time It

with

is

does,

however,

which

then

accumu-

observed

with

pork

of the N-oxide

of the rates 189

It

with

the

of N-oxide

to formaldehyde

Vol. 15, No. 2, 1964 production

is

BIOCHEMICAL

usually

somes and may vary

higher with

than

AND BIOPHYSICAL RESEARCH COMMUNICATIONS that

different

obtained

with

preparations

fresh

of fresh

rat

pork

liver liver

micromicro-

somes. Table Products

I

Formed by the Oxidation of N,N-dimethylaniline by Liver Microsomes

Complete(a) Medium ,t

Microsomes fresh

II

ml.unoles/min/mg protein N-oxide Formaldehyde

rat

liver

microsomes

0.5

4.6

1.3

2.3

3.7 1.8

0.8

3.2

pork liver microsomes frozen 1 week

2.8

2.1

rat liver microsomes frozen 24 hours rat liver pretreated fresh

microsomes with cholate

pork

liver

(b)

microsomes

"

minus

NADPD

pork

liver

microsomes

0.00

0.00

n

minus

NADPH

pork

liver

microsomes

0.00

0.00

+ HZ02

(4

The complete assay medium contained nmoles/mlr potassium phosphate, N,N-dimethylaniline-3.0; NADPH-1.0 PD 7-O,+ -200; semicarbizide-1.0; and NAD -1.0. The concentration of microsomal protein, 2.6-3.3 mg/ml. Incubation time lo', Temp.-38O.

(b) Fresh microsomes (30 mg/ml) prefncubated cholate (0.2 mg/mg microsomal protein).

The N-oxide

is not

replaced

by substrate

generating

systems

In order the reaction material

formed

amounts

such as the D-amino

mixture

is

and deproteinized 0.5 g NaOH.

Aliquots

that

identical

was chromatographed

ing method.

in the absence of hydrogen

to demonstrate

The deproteinized

acid

potassium

or hydrogen

oxidase. assaying

as the

paper

of the reaction

N-oxide

190

solution

in

the using

mixture

ml of 20% ZnS04 followed supernatant

be

peroxide

N,N-dimethylaniline-N-oxide,

six ml) 0.45

at O" with

of NADPE, and NADPE cannot

peroxide

the material with

hours

on Whatman No. 1 filter

(usually

by adding

six

the ascendwere

by 0.85

was concentrated

removed

ml of under

Vol. 15, No. 2, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MINUTES Figure

I.

. 0 c-a

o

Rates of N-oxide and formaldehyde production by fresh and cholate-treated rat liver microsomes. Assay conditions as given in Table I. Concentration of microsomal protein 2.1 mg per ml mpmoles moles s+moles moles

l

G-4

vacuum

to approximately

anhydrous under

formaldehyde N-oxide with formaldehyde N-oxide with

0.1

and approximately

chromatography.

The paper

about

20 to 30 minutes

front

had moved about

chromatograms

with

was reduced

deep red

solution

the N-oxide 5 moles color

gives

reagent:

enough

and used a bright

the

strips

sodium

acid

immediately.

on paper however,

color

On paper on a brown

can be detected not

permanent 191

the

phase

the

removed

by spraying

one or two minutes

blue

were

ml

for

the vapor

to give

solvent

and dried

nitroa clear

was added

to destroy

solution

was diluted

sprayed background. by this

for

the paper

g sodium

borohydride

acetic

strips

After

10 ml of 0.04

0.8 ml of 15

After

with

be located

solid

2.0 ml of to 0.05

to paper

the chromatogram.

following

and then

is,

starting

with

was concentrated

equilibrated

could

with

of the N-oxide differential

were

The N-oxide

borohydride.

5 ml of water

strips

extracted

ml transferred

10 cm up the paper,

the

prusside

with

and then extract

0.025

before

at room temperature.

the excess

to 0.2 ml,

The chloroform

chloroform.

vacuum

with fresh microsomes fresh microsomes with cholate treated microsomes cholate treated microsomes

with

this

reagent

As little reagent.

and the chromatogram

as The

oxidizes

Vol. 15, No. 2, 1964 to a uniform the

other

ture

BIOCHEMICAL

light

blue

materials

after

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

exposure

present

to air

for

in the chloroform

about

minutes.

30

extract

None of

of the reaction

mix-

of the complete

reac-

microsomes

1~3

interfere. The material

tion

mixture

days)

present

(incubated

had the

for

in chloroform,

chloroform

and 0.35

The data N-oxide

is

ation predicted

soluble the

of oxidative

cholate

fact

that

1963)

of the N-oxide

is

to the

support

formed

N,N-dimethyl-

mixture

remained

or when

were

it

As little reaction

at

of methanol-

No spots

was

as

medium

Cholate,

in the

could

turnover

rate

of the N-oxide

fold

small

greater

amounts

by fresh,

at the

levels

demethylase

were

associated

the over-all

ing

synthesis

the high

this

can be detected or

the synthesis

does not

accumulates inhibit

laboratory).

Km value

the demethylation

Aging

the N-oxide

the enzyme

be

rate

microsomes.

used,

with

demethyl-

demethylase

of the N-oxide

so that

an

As could

the enzyme catalyzing

demethylase

preclude

1958).

than

"intact"

N-oxide

would

that

oxidative

Brodie,

experiments,

of the N-oxide,

enzymic

(cf.

(unpublished closely

the hypothesis

compounds

dissociate

the N-oxide

medium.

demethylase

1:l

added

demethylase

N-oxide

in

sulfate.

N-oxide

the

aged

in methanol,

the zinc

several

catalyzed

apparently from

in the reaction

the

only

mixture

treatment

front

of 0.82

report

product

demethylation,

in the reaction

solvent

N-oxide

N-methyl

and Ziegler,

as synthetic

of acetone-methanol.

after

in this

the intermediate

from

liver

chromatogram.

presented

of lipid

(Pettit

mixture

on a paper

rat

when the NADW was omitted

per ml of synthetic

be detected

the

in a 327 mixture

to the reaction

25 moles

with

and gave Rf's

on the chromatograms

extracts

properties

Both moved with

the origin

added

10 minutes

same chromatographic

aniline-N-oxide.

observed

in the chloroform

the

Unless

the

system

catalyz-

(193 x 10 -3 9

of the

of small

amounts

of

N-oxide.

We are ing

indebted

and characterizing

to Dr.

Rowland

Pettit

of our Department

the N,N-dimethylaniline-N-oxide

192

used

for in this

synthesizstudy.

BIOCHEMICAL

Vol. 15, No. 2, 1964 We wish

to express

our appreciation

for

assistance

in obtaining

his

AND BIOPHYSICAL

to Mr. pork

Roberts

RESEARCH COMMUNICATIONS of the Municipal

Abattoir

livers.

References Brodie,

B. B.,

Ann. --

Hogeboom, G. H-, 172, 619 (1948). Pettit, F. H., 193 (1963). Shriver Sons,

Rev- Biochem ., 57,

427

(1958).

Schneider,

W. C.,

and Palade,

and Ziegler,

D. M.,

Biochem.

and Fuson, "Identification New York, Second Inc.,

Edition,

G. P., Biophys.

J. --Biol--Res

of Organic Compounds," pp. 148-9 (1940).

193

Chem-

-Comm. a, John Wiley

and