SHORT COMMUNICATIONS
635
BRA 53213
Biosynthetic pathways in the formation
of individual molecular
species of rat liver phospholipids
Recent investigations on the metabolism of the individual molecular species
of rat liver phospholipids suggested that the arachidonic acid present in phosphatidylcholine and pllosphatidylethanolamine
is introduced mainly through acylation
of the corresponding I-acyl 1ysoderivativesX-5. A de GOZXJ
synthesis as established
by KENNEDY@ would be more responsible for the formation of molecules containing
linoleic and oleic acid. In order to investigate the relative contributions of both
pathways to the synthesis of the various molecular species of phospholipids, rat liver
microsomes and slices were incubated in the presence of both [2-3H]glycerophosphate
or [@H]glycerol
and [x-l*C]fatty acids’.
Rat liver microsomes were isolated by differential cent~fugation as described
elsewhere7. About 5 mg of protein, as determined by the method of LOWRY et aL8,
were incubated with 50 nmoles of [3H]glycerophosphate and 20 nmoles of W-labelled fatty acids in the presence of 25 /moles ATP, 0.3 Lcmole CoA and 0.125 M KCl0.02
M Tris (pH 7.4). The total volume was 2 ml. Rat liver slices (ZOO mg) were
incubated under O,-CO, (95:5, v/v) atmosphere in 3 ml of Krebs-Ringer
solution
(Caz+ omitted) in the presence of zoo nmoles [z-3H]glycerol and potassium salts of
[r-W]fatty
acids, which were compIexed with albumins. After the indicated periods
of incubation the slices were extracted according to the procedure of BLIGH AND
DYERIO. Known
aliquots of the extracted
lipids were applied on thin-layer
plates
for the isolation of phosphatidylcholine,
pllosphatidylethanolamine,
diglycerides,
and phosphatidic acid as described beforeS+.
Phosphatidylcholine and phosphatidylethanolamine
were separated into their
different molecular classes by conversion with phospholipase C from Bacillus
cerem
and subsequent fractionation of the diglycerides formed, on silver nitrate-impregnated silica plates2. The various species of phosphatidic acid could be resolved by
argentation chromatography of their dimethyl derivatives6+.
After the elution of
the isolated products, radioactivity
incorporated was measured by means of a
Packard-Tricarb
instrument using an external standard for quenching corrections.
Table IA shows the ratios of [3H]glycerophosphate
and [%]fatty
acids incorporated by rat liver microsomes which had been incubated in both simultaneously.
The very low ratios observed for phosphatidylcholine
and phosphatidylethanolamine compared with the high ratios of phosphatidic acid and diglycerides demonstrate that in isolated microsomes, uptake of fatty acids in phosphatidylcholine and
phosphatidylethanolamine
proceeds mainly via acylation of endogenous lysophospholipids. Similar results were obtained when rat liver homogenates were used as
enzyme source. Homogenisation and isolation of cell particles may induce an impairment of the de nova synthesis or an increase of endogenous monoacylphosphoglycerides.
To investigate whether rat liver slices are still capahIe of the de E.OZVsynthesis,
slices were incubated for the indicated periods with [3H]glycerol and [i%]palmitate.
As shown in Table IB a relatively high uptake of glycerol into phosphatidic acid
could already be seen after 2.5 min, whereas phosphatidylcholine,
phosphatidylBiochim.
Biophys.
Acta,
176 (1969)
635437
SBORT COMMUNICATIONS
636
TABLE
I
THE SIMULTANEOUS INCORPORATION OF [I-‘%]FATTY
[2-3HjGLYCERoL
INTO
RAT
LIVER
hems AND [zJH
AFTER
PHOSPHOLIPIDS
INCUBATION
~GLYCEROPHOSPHATE
WITH
OR
(A)
MICROSOMES
AND
(B)
SLICES
-4. Nicvosonzes.20 nmoles of the [‘4C]fatty acid indicated and 50 nmoles of [3Hjglycerophosphate
were incubated with 5 mg of microsomal protein for 30 min. The ratios of incorporated glycerophosphate (3H) and fatty acids (‘4C) are presented.
-.
- -~
Fatty a&d
P~osphat~dylPhosphat~dyl&glycerides
P~osp~at~a~c
choline
etha~olam~ne
acid
-____.---.._____-~
16:o
0.07
0.17
1.60
I.76
_*
18:o
0.22
0.09
13.8
18:r
o.I6
0.27
6.0
7.8
18:2
0.20
0.11
2.6
2.6
~. -~
___.
B. Slices. 200 nmoles of [I%]palmitate and 200 nmoles of
of rat liver slices for the period of time indicated. The
fatty acids (‘“C) are presented.
~~..
~._ _____Incubation
time
PhosphatidylPhosphatidyl(min)
ChOliW
ethanolamine
___
~~_^
_~~~Phosphatidic
acid
Diglycerides
---
--. ~~.
-
2s
ethanolamine
and
however,
the ratio
acid
increases
and
the isotopic
ratios
min incubation
liver
diglycerides
for
work
tion
of the incorporated
microsomes
TABLE
AFTER
prolonged
fatty
acids
incubation,
for phosphatidic
phosphatidylethanolamine.
of phosphatidic
particles,
acid,
When
are capable
upon
after
it is evident
the addition
with
distribu-
of glyc~rophosphate
slices,
rat
de ‘MOVO
In agreement
et aL5 on the positional
of IIILL et al.3 on rat liver
15
that
of considerable
and phosphatidylethanolamine.
and the studies
LIVER
OF
SIMULTANEOUS
INCUBATION
__~
_~
[SH~GLYCEROL
PHOSPHATIDIC
The detailed composition
sed as nmoies.
Phosphatidic
acid
Phosphatidylcholine
Phosphatidylethanolamine
Biochim.
and
After
acid decreases
and phosphatidylethanolamine
that
to isolated
labelled.
to fatty
_..__-_
to rat
it was found
that
II
INCORPORATION
RAT
yet
of SCHERPHOF*~ and POSSMAYER
liver
THE
with
of phosphatidylcholine
the previous
not
glycerol
phosphatidylcholine
are compared
10.9
6.2
4.8
3.3
4.5
_ _.__~
of phosphatidylcholine
slices, in sharp contrast
synthesis
were
of incorporated
12.1
_*
1.42
=.59
3.54
~.__~.
1.28
5
10
1.65
1.82
I5
._~
________ ---.~
* Not determined.
OF
[3H]glycerol were incubated with 200 mg
ratios of incorporated glycerol (3H) and
Biophys.
AND
ACID,
[14C]~~~~~~~~
WITH
RAT
of the incubation
Acta.
Tot&
____~.
T&ae%o?c
species
SpeCieS
3.12
0.94
3.32
IX.61
II.39
1.63
6.30
3.54
I.78
.-__
0.28
0.10
176 (1969) 635-637
AND
~~A~OR~OLE~~LARSPECIES
PHOSPHATIDYLETHANOLA~IINE
LIVER
SLICES
FOR 45 min
mixture is described in the text. Results are expres-
~_.
[3H]Glycerol
[W]Stearate
Ratio “H/I%
[31i]Glycerol
[%]Stearate
Ratio 3H/Y
[“H]Glycerol
[I% !Stearate
Ratio “H/‘*C
INTOTHE
PHOSPNATIDYLCHOI..INE
~~
.~
----.
,~~o~o&~~~~~CDisafwvated
species
species
.____~~ ____
Species
_. ~-.
1.21
0.89
0.26
0.2I
0.20
0.27
4.45
2.86
0.79
3.62
I.OI
0.95
0.80
2.80
;:Z
2%
1.15
I.35
I.53
0.89
~_...
Dienoic
2.20
3.92
2.69
0.84
3.20
0.62
1.29
0.18
0.2;
3.73
~~___.
0.31
_
0.58
~.
_..-
_
637
SHORTCOMMUNICATIONS
glycerol, after conversion into glycerophosphate,
is esterified in a nonrandom
fashion.
into
Table II presents the amounts of [SH]glycerol and [14C]stearate incorporated
the individual molecular species of phosphatidic
acid, phosphatidylcholine
and phosphatidylethanolamine.
It is clear from the data presented that the arachidonic acidcontaining
species of phosphatidylcholine
and phosphatidylethanolamine
have a
much lower 3H/14C ratio than the mono- and dienoic molecules. This implies that
at least in rat liver slices, a de nova synthesis via phosphatidic
acid is highly operative
for the formation
of mono- and dienoic molecular species of phosphatidylcholine
and phosphatidylethanolamine.
However, as may be concluded from the relatively
low incorporation
of glycerol, the polyunsaturated
molecules are synthesized mainly
by acylation
of endogenous
lysophospholipids.
Further work on the contributions
of both pathways to phospholipid synthesis in other tissues and other animal species
is in progress.
The collaboration
of Mrs. H. I. P. van Huis-Fokkinga
is greatly appreciated.
Laboratory
Utrecht
of Biochemistry,
(The
The State
University,
L.M.G.
Netherlands)
VAN GOLDE
G.L.SCHERPHOF
L.L.M.VAN
DEENEN
I A. CATALA AND R. R. BRENNER, Lipids, z (1967) 84.
2 L. M. G. VAN GOLDE, W. A. PIETERSON AND L. L. M. VAN DEENEN, Biochim.
Biophys.
152 (1968) 84.
3 E. E. HILL, D. R. HUSBANDS AND W. E. M. LANDS, J. Biol. Chem., 243 (1968) 4440.
4 J. B. MUDD, L. M. G. VAN GOLDE AND L. L. M. VAN DEENEN, B&him.
Biophys. Acta,
press.
5 F. POSSMAYER, G. L. SCHERPHOF, T. M. A. R. DUBBELMAN, L. M. G. VAN GOLDE AND L.
VAN DEENEN, Biochim. Biophys.
Acta, 176 (1969) 95.
6 E. P. KENNEDY, Federation Proc., 20 (1961) 934.
7 G. L. SCHERPHOF AND L. L. M. VAN DEENEN, Biochim. Biophys.
Acta, 113 (1966) 417.
8 0. H. LOWRY, N. J. ROSERROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193
Acta.
in the
L. M.
(1951)
26’5.
g M. VAVRECKA, R. POLEDNE AND R. PETRASEK, Biochim. Biophys. Acta, 125 (1966) 176.
IO E. G. BLIGH AND W. J. DYER, Can. J. Biochem. Physiol.,
37 (1959) 911.
II 0. RENKONEN, Biochim. Biophys. Acta, 152 (1968) 114.
12 G. L. SCHERPHOF, Thesis, Utrecht, 1967.
Received
December
z@h, 1968
Biochim.
Biophys.
Acta,
176 (1969) 635-637
BBA 53209
Aldosterone
biosynthesis by human fetal adrenal in vitro
Though adrenal tissue from a 3a-week-old human fetuswas capable of converting
labelled progesterone
to labelled aldosterone,
incubations
of adrenals of younger
fetuses were not able to make this transformationl.
More recently PASQUALINI et al.2
reported the presence of [3H]aldosterone
in the adrenals of a ao-week-old fetus perfused with [r,z-3H,]corticosterone.
We have investigated
the conversion of [4-l%]Biochim.
Biophys.
Acta,
176 (1969) 637-640