Microsomal lipid peroxidation in human pregnant uterus and placenta

Vol. 79, No. 3, 1977

BIOCHEMICAL

MICROSOMAL LIPID

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PEROXIDATION IN HUMAN PREGNANT UTERUS AND PLACENTA

G. FALKAY,

J.

HERCZEGAND M. SAS

Department

of Obsterics

WHOCollaborative Human Reproduction,

and Gynecology

Centre in Clinical Research Univeristy Medical

on

School of Szeged, H-6701 Szeged, POB 438 Received

October

25,

1977

SUMMARY A study was made of the microsomal lipid peroxidation of the pregnant human uterus and placenta. It was found that the lipid peroxidation of the‘ 'microsomal fraction of the uterus is specific for prostaglandin formation: the lipid peroxidation was enhanced by arachidonic acid, and inhibited by antiprostaglandins. Accordingly, it is suitable as a screening test for the pharmacological examination of antiprostaglandin effects. The lipid peroxidation in the placenta is not specific. In both tissues examined the lipid peroxidation is linked to ascorbic acid.

INTRODUCTION On the basis it

is accepted

of the

of the most recent

that

the first

step

prostaglandins

/PGs/

is the

the precursor

fatty

acid

peroxides

by cycle-oxygenase

oxidative

cyclization

oxidation

/Fig,

vesicle,

transformation

is none other

acid/

to endo-

In fact, than

of

this

lipid

per-

1.1

In the inal

in the biosynthesis

larachidonic /l-3/.

findings

cases of

rabbit

renal

Copyright 0 I977 by Academic Press, Inc. AN rights of reproduction in any form reserved.

some animal medulla,

tissues

lung/,

/sheep

where the

semPG

843 ISSN

0006-291X

BIOCHEMICAL

Vol. 79, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ARACHIDONIC

cycl0

ACID

-0xygenost

I

EF*dw~;;-

-PGE2 .I “OH ENOOPEROXIOE

( PGH2

1

O&\” MALONOlALMHYDE

Fig.

1. Biosynthesis of prostaglandins from precursor acid.

synthesis

is

peroxidation formed

intensive, is

from the

isomerase, peroxides

it

specific

are formed

14-71.

undergo

reaction

with

All

produced

tissues these

with

spectrophotometrically

linear

with lipid

the

lipid

PGs are

/endoperoxide

while

malondialdehyde

quantitatively

hydro-

low PG synperoxides

and can be

thiobarbituric

acid

14, 81.

Two conditions the

thus,

in the

contents

of

PG formation:

reductas/,

thetase

specificity

that

peroxides

endoperoxide

determined

has been proved for

lipid

and malendialdehyde

lipid

must be satisfied peroxidation

for

in a tissue

the to be

proved: /l/

with

increasing

as PG substrate,

concentration the

lipid

of

arachidonic

peroxidation

increasej

a44

must

acid,

BIOCHEMICAL

Vol. 79, No. 3, 1977

/2/

the antiprostaglandins peroxidation

increase

labour

lipid

manner 171.

the PGs in the

significantly

in pregnancy

amniotic

and during

191.

biosynthesis human uterus is made to

the

the

is known that

No data

lipid

must inhibit

in a dose-dependent

It fluid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the microsomal or placenta. investigate

peroxidation placenta,

are available

to

the

on the fraction

In this

see how specific

of

paper

intensity

in the pregnant

in vitro

PG

the pregnant

an attempt

of microsomal human uterus this

is

for

and PG

formation. MATERIAL AND METHOD

Uterus and placenta tissue samples were obtained at the time of Caesarean sections. Exciped myometrium strips /ca. 600-800 mg/ were cut from region of uterine corpus immediately adjacent to the low transverse incision and stored at -30 OC until processing /max. 24 hrl. The tissue samples were homogenized at 0 oC in 10 volumes of TRIS-HCl buffer /pH 7.81. The homogenizate was centrifuged for 10 min at lO,OOOxg, and the supernatant was recentrifuqed for 60 min at 105,OOOxg. The sediment /microsomal fraction/ was resuspended in TRIS-HCl buffer, and 2 ml of the suspension /2.0-2.5 mq microsomal protein/ was used for the incubation examinations. The protein contetnts of the individual samples were determined by the method of Lowry et al. /lo/. The 2 ml incubation mixture contained: O-200 ,umole arachidonic acid, 850,umole ascorbic acid, 30/umole Fe2+. After the incubation period 110 mint the reaction was stopped with 1 ml 20 % TCA. The mixture was centrifuged for 5 min at lO,OOOXg, and the supernatant was decanted off and boiled for 10 min on a 100 OC water-bath with 1 ml 0.67 % thiobarbituric acid. The red colour developing was measured spectrophotometrically in a 1 cm cell at 532 nm. A sample inactived for 5 min at 100 oC prior to the incubation was used as control. The malondialdehyde /MDA/ concentrations of the unknown samples were read off a calibration curve recorded with malondialdehyde-tetraethyl-acetal /K and K Lab. Inc., Plainview, USA./ /molar extinction coefficient 1.32x105/. The enzymatic activity is given in units of nmole MDA formed/mg protein/l0 min.

845

BIOCHEMICAL

Vol. 79, No. 3, 1977

Table

I.

Effect of peroxidase

cofactors activities.

Lipid Cofactors

on the microsomal

lipid

peroxidation

MDA formation

None n=5 Ascorbic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

nmole/mg protein/l0

Uterus

Placenta

3,0+0,17

4,9+0,26

min.

acid

850 amole n=5 Ascorbic acid 850 umole +

9,2+0,3

16,5+0,5

9,7+0,41

16,9+0,52

Fe3+ 30 ,umole n=5 Ascorbic acid 850 ,umole + Fe2 30 flumole

26,4+0,6

21,8+0,47

n=5

RESULTS

In the at first

37 OC under

employed,

the

aerobic

conditions

is

10 min in both

/Fig.

2.1.

acid,

Fe 2+

Table

activities acid

system

I.

the

uterus

illustrates

increased

the uterus the

linear

the effects lipid

and the placenta.

enzymatic

activity

846

during

the

and the placenta

and Fe3+ on the microsomal of

enzyme reaction

of

ascorbic

peroxidase Ascorbic

by approximately

BIOCHEMICAL

Vol. 79, No. 3, 1977

25

5

20

10 TIME

Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF

INCUBATION

(MIN

2. Lipid peroxidase activity of microsomal fraction of human uterus, as a function of the incubation time. Microsomal protein: 2.5 mg; arachidonic acid: 100 umole; cofactors: ascorbic acid: 850 umole, Fe2+:30 umole

3-4-fold.

The enzyme-induction

whereas Fe2+ markedly

of the

peroxidase

lipid

Figure lipid

peroxidase

centration

of the

arachidonic

acid.

not

the activity

of ascorbic

acid.

the change in the activity

of

of increasing

larachidonic

of the microsomal in proportion

elevated

presence

as a function

of substrate

peroxidation increases

in the

3.depicts

of Fe 3+ could

effect

be detected,

the

1

acid/.

fraction

to the increase

con-

The lipid

of the uterus in the

No change occurs

concentration

in the case of

the placenta. The inhibitory hibitors

/PGSIs/

effect

on the lipid

of the PG synthetase

peroxidation

in-

could be dgrpnstrated

Vol. 79, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

UTERUS

@

PLACENTA

0

20

50

loo

200

ARACHIDOMC

AaO jdhIOl

Fig.

3. Effect of arachidonic acid on lipid peroxidase activities of microsomal fractions of human uterus and placenta. Microsomal protein: 2.2 mg /uterus/; 2.0 mg /placenta/. Incubation:10 min at 37 OC.

only

in the myometrium.

increasing

concentration

the uterine roxenR each/

Arachidonic

corpus,

/Syntex/

and identical

and IndomethacinR

PG synthetase

between

the

enzymatic

arachidonic is

that

linear

41. The inhibitory

about

half

that

of

11 mmole

the

incubation the

double

reciprocal effect

vessel.

correlation and the

to be expected

kinetic:

/Fig.

/Chinoin/

concentration

of Michaelis

of

of Nap-

too,

acid

fraction

quantities

individual

inhibition

activity

was added in

to the microsomal

were added to every

During

acid

on the basis plot

is

of Naproxen

is

Indomethacin.

DISCUSSION

On the that activity

the pregnant specific

basic

of the

results

human uterus for

possesses

PG formation,

a48

it

since

may be said lipid with

peroxidase ara-

BIOCHEMICAL

Vol. 79, No. 3, 1977

0

CONTROL

x

1 mM

INDOlr(EfNAClN

o

1mM

NAPROXEN

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lo

20

30

( ARACHIDON

Fig.

m Mel

1-l

4. Double reciprocal plot of MDA formations as a function of the arachidonic acid concentration in the presence and in the absence of inhibitors. Incubation: 10 min at 37 OC.

chidonic

acid

and with

PGSIs it

the enzymatic

peroxidation

there

is no PG biosynthesis

also

is not

supported

chidonic

acid

the presence as regards

by the

This

in the negative

inactivation earlier

enhanced

suggests

the that

human placenta. This 14C-araresults of

examinations. is

jhydroxylationj by Ernster

However, important

of drugs,

and Nordenbrand

/ll/

liver. Hochstein

ically

specific.

of the enzyme in the placenta

as demonstrated

presence

can be

In the placenta

- PG incorporation

the

in the rat

activity

can be inhibited.

lipid

is

SAV

40

of

et al.

Fe2+ or Fe3+ is

induced

peroxidation

/12/

pointed

essential of

849

out for

that

the

the enzymat-

the microsomal

lipids.

Vol. 79, No. 3, 1977

However, only

BIOCHEMICAL

since

with

an inducing

of

the rat

and Nordenbrand

is

effect

Fe*+ in the presence

the basis

uterus

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

liver

/ll/

it

and placenta

typically

Since human uterus

the

experiments

for

lipid

linked lipid

peroxidase

conditions.

There

experimental

We consider for

the

screening

potentially

the

examination

drug

can be employed

with

for

spontaneous

abortion

appreciable

risk

the

progress

are well

studied

in vitro this

treatment

to be suitable

number of

in the

of the

present

supported

effect danger

without

that of

currently

of

the in

by the demonstrated

action.

ACKNOWLEDGEMENT This

work was Supported

in part

by WHO.

REFERENCES 1. Hamberg, M. and Samuelsson, B. 119731 Proc. Acad. Sci. U.S.A. 70, 899.

850

work,

any

The results

examinations

mechanism of molecular

in

research.

an antiprostaglandin

side-effects.

clinical-pharmacological

newly

compounds.

in gynaecology

of

of

for

of a large

the

provides

the method elaborated

antiprostaglandin

first

under

phase of drug

NaproxenR /Syntex/, is

of

effect

a possibility

/laboratory/

acid.

activity

PGSIs this

compounds to be examined rarely

the human

to the ascorbic

with

on

peroxidation

synthetized

the

that

the antiprostaglandin

is

acid,

of Ernster

may be stated

can be inhibited

a possibility

be detected

of ascorbic

microsomal

a reaction

could

Natl.

Vol. 79, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

T., and 2. Hamberg, M. Svensson, J., Wakabayashi, Samuelsson, B. 119741 Proc. Natl. Acad. Sci. U.S.A. 71, 345. D.H. and Hazelhof, E. 119731 Biochem. 3. Nugteren, Biophys. Acta 326, 448. 4. Sih, C.J. and Takegushi, C.A. 119731 Prostaglandins vol. 1. /Ed. Romwell/ p. 83. Plenum Press, New York, London. 5. Flower, R.J., Cheung, H.S. and Cushman, D.W. 119731 Prostaglandins 4, 325. 6. Robak, J., Dembinska-Kiec, A. and Gryglewski, R. 119751 Biochem. Pharmac. 24, 2057. 7. Robak,

J. and Sobanska, B. 119761 Biochem. Pharmac. 25, 2233. 8. Hamberg, M. and Samuelsson, B. 119671 J. Biol. Chem. 242, 5336. and 9. Dray, F. and Frydman, R. /1976/ Am. J. Obstet. Gynec. 126, 13. 10. Lowry, O-H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. 119511 J. Biol. Chem. 193, 265. 11. Ernster,

L. and Nordenbrand, K. 119671 Methods Enzymology Vol. 10. p. 574. 12. Hochstein, P., Nordenbrand, K. and Ernster, L. Biochem. Biophys. Res. Commun. 14. 323.

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