Prostaglandins and thromboxanes biosynthesis in guinea-pig placenta infestigated by mass-spectrometry analysis.

257

International Journal of Mass Spectrometry and Ion Physics, 48 (1983) 257-260 Elsevier Scientific Publishkg Company, Amsterdam - Printed in The Netherlands

PROSTAGLANDINS

AND THROMBOXANES

BY MASS-SPECTROMETRY

D. ALBER Unite

J.C.

and

BIOSYNTHESIS

IN GUINEA-PIG

PLACENTA

INFESTIGATED

ANALYSIS.

HENRY

INSERM U 198,

Route

de Dole,

25000

BESANCON

(FRANCE)

INTRODUCTION The ability

of guinea-pig's

been rarely studied

has

In our laboratory a GC-MS for

we have

achieved

analysis

methods

for purification

(Water

silicic

acid

of reduced

to the

MATERIAL

(GSH)

belonging

AND

column

qualitative

Glutathion

derivatives

a study

and

to series

related

purification

of such

3 different Ass)

prostanoi'c derivatives

up to now.

simultaneous

Parallel

to synthetise

As far as an intensive

technic.

runing

placenta

derivatives

of placental

and reverse Indomethacine

we have

extracts

phase

investigation,

to that

subject

is absolutely

using

required

proceeded

: SE?-PAK

with cartridges

HPLC.

we have

studied

the influence

on the biosynthesis

of 3

2.

METHODS

Incubation Six series placenta

of duplicate

collected

at delivery.

30'

at 37"

GSH

(5 mM) is added

in tris

to series

incubations

HCl Buffer

Each

by acidification

fragment

pH 8. Series

to series

II, GSH

III, Homo-y-linolenic

are stopped

are realized

acid

with

fragments

is homogenized

I, IV and VI are

(5 mM)

and

with

incubated

not supplemented.

Indomethacine

(30 PM) is added

of the medium

and

of a same

(2 uM) are added

to series

0.1 M citric

V. Incubations

acid

(pH 4).

Purification Series

I, II, III.

For

recovery,

to the

incubation's

media

ether.

Each

is then

derivatives

extract are

eluted

by reverse

phase

HPLC

Isocratic

elution

rate

of 0.6.ml/mn

flow

which

with

tritium

are then

purified

labelled

extracted

with

a SEP-PAK

Methylformiate

(ref.1).

(Varian

5000 with

column

is run with

a mixture

of acidified

at 32°C.

Only

fractions

Prostaglandins

twice

with

3 volumes

cartridge. This

CIB Merck

The

fraction Lichrosorb

containing

TxB2,

0

is purified RP 18 IOU).

PGF2a

1983 Elsevier Scientific Publishing Company

of

prostanoi'c

water-acetonitrile

PGE2 are collected. OOZO-7381/83/00@3-OC100/$03.00

are added

(70:30) and

258 Series

IV, V _ Incubations's

and

purified

Two

fractions

through

by SEP-PAK Jnalysis After

elution,

We used

and

are

we measure (M-161) for

by a factor

the

the

two

In series

with cular

ng of PGF#

treated

used

as

: methylation,

the

samples

are

with

a data

ions

injected

is connected processing

PG’s biosynthesis

PGF2a

m/e

tissue

of 25 m. The to a

system.

detections.

variations

fresh

a SE 52 column

chromatograph

external

calculated

under

influence

in comparison

standard. 423.

and

ratio

to those

Concerning

Ions's

weight

relatively

influence

of any

we

TxB2

surfaces

are

recuperation

obtained

to this

added

with

ability

looked

detected

identified

incubation.

identified

a real

have

have

from

of we

use

corrected rate.

incubations

one which

exogenous

in

for TxB2, the

V with

PGDI,

the

intense

470

presence

achieved

from

to perform

None

permits

factor

on

the

PGF2a,

(= 468 SEP-PAK this

extracts

technique

of

acid

a clear

PGD2).

could

PGE2,

The

not

PGD2,

for

used

spectrum VI)

a quick

we were incubations

derivatives

6-0x0-Fla

and

is clearly

PGEl

as a precursor for m/e shows

The have

of

.

but

response

be shown.

(series

after

eluates

be characterized,

gives

for

However

prostanoi-c

types

dihomo-y-linolenic

of TxBl

of M I D technique

of the 9 derivatives

certitude.

different

detection

by m&an

to synthetise

IV, PGEI and PGD1 cannot

of

possibility

50

with

mean of surfaces

to any

not ben we

ion. The

directly

is just

of GSH.

are

prior

in series

a very

for

for

the

experiment

easily

in search

The

in the PGF~B

and

relative

reveals

series

identify

(ref.2).

comments PGs

placenta are

to DRAY

PGs studied.

could

the

508

to

indexes

In a previous in situ

260°C.

surface's

from

addition

of

and

ions

accounts

without other

to

by multiple

affected

biosynthesis

PGFIa

series

steps

Finally

equipped

Mag R la-10s)

m/e

with

in three

(Riber

the

which

100 is

is run

realized

obtained

PGE2

to appreciate

the

described

3:7.

of variations

ion 423

searching

according

of this

trimethylsilylation.

holded

analyser

of GSH,

Results

Acid

supplemented

30 Chromatograph

is

estimation

The

are

of BSTFA-Hexane

Identifications

realized

series

then

a Girdel

quadripolar

Index

sample

each

Derivatization

temperature

301,

of Silicic

as previously

GC-MS

all

standard.

in a mixture

m/e

extracted

cartridge.

methoxymation

For

extraction,

by coupling

external

column

are

are collected.

VI . After

Series

oven's

an open

media

380,

a M+=

and 470,

541 mole-

P4 I D analysis demonstrated

qualitative

510

analysis.

the

In

259 spite

of a high

suitable

efficiency

for a quantitative

To some

extent

columns

of Silicic

purification substances

this

remark

After

is known

The

results

(Index

to obtain

of our experiment from

Index

(Index

Concerning

cleared

from

with

neat

related

Mass-

PGE2

the results

(ref.3).

this fact.

Among

is considerably

of PGF~D. is also

TxB2

any

open

pre-

80 per cent.

biosynthesis

isomerases,

Formation

using

a SEP-PAK

particulary

of

agents.

technique

with

are over

in agreement

microsomal

= 435).

= 178).

rates

to be not

contaminating

HPLC

extracts

stimulation are

it appears

purification only

and giving

recuperation of

colums

of numerous

for

pure

of interfering HPLC

the 3 PGs biosynthesized rate

because

is available

to be a factor

(medium

capillary

In our experiment,

acid.

susceptible

GSH

increased

assay

, has allowed

Chromatograms.

a lower

of glass

stimulated

are

but

contradictory

= 104).

The

addition

(Index

= 16).

of Indomethacine (see

Fig.

1

!

F]

+ GSH

strongly

inhibits

the

biosynthesis

1)

400

-

300

_

.

. . .

5mM

+ INDOMET. 2uM

of PGE2

. l. . * . .

200_

b* l

. 1

.77;1

TxB 2

PGF 2 Fig.1.

Diagram

under

the

influence

Index without The

showing 100

of reduced

: mean

of TxB2,

Glutathion

of the surfaces

(5mM) ratios

PGF~cx and PGE2 and

biosynthesis

Indomethacine

obtained

from

(2uM).

incubations

supplementation. obtained

precision

is 3.5 % for Tx32, To

the variations

PGk2

conclude,

placenta

of

8.8 % for

the fragmentographic PGF2"

our experiment

to synthesize

PGs

from

has

and

method

(surfaces

ratios)

11 % for PGE2 (n = 5 for each sample).

demonstrated

precursors

such

the ability

of guinea-pig

as dihomo-y-linolenic

acid

260 (serie

1)

obviously to

PGS,

and

arachidonic

In

identify. it

is

possible

bioconversion

coming

The

question

arises

the

formation

of

substance

acid the

to

in

if

: We thank

gift

Prostaglandins.

standard

of

but

for

reduced of

splitting

TxBl

that

we could

Glutathion,

enzymic

PGs reflects of

only

a non

intermediate

if

this

factor

it

acts

as

a revelator

of

PIKE

{UPJOHN

Company)

this

ACKNOWLEDGEMENTS of

2)

production a random

know

PGE3 or

synthetized

absence

that from

(serie

stimulates

not activator specific

endoperoxydes.

on

a non

the

main

specific

way

prostano’ic

tissue.

Dr.

J.

for

his

generous

REFERENCES 1

2 3

W.S. POWELL, Prostaglandins, 20 (1980) F. DRAY, B. CHARBONNEL and J. MACLOUF, 311-313. A. YOSHIf1OTO, H. IT0 and K. TOMITA, J.

947-957. Europ. Biochem.,

J.

Clin. 68

Invest.,

(1970)

5

487-499.

(1975)