116
Biochimica @ Elsevier
et Biophysics Acta, Scientific Publishing
409 (1975) 116-127 Company, Amsterdam
-
Printed
in The Netherlands
BBA 56667
BIOSYNTHESIS OF PHOSPHOGLYCERIDES IN SKELETAL MUSCLE IN CONTROL RATS AND IN RATS DEFICIENT IN ESSENTIAL FATTY ACIDS
F.A.
SHAMGAR*
and F.D.
The Russell Grimwade 3052 (Australia) (Received
April 24th,
COLLINS**
School
of Biochemistry,
University
of Melbourne,
Parkville,
Vicioria
1975)
Summary
1. The specific radioactivities of individual molecular species of phosphoglycerides in the skeletal muscles of control rats and of rats deficient in essential fatty acids have been determined 3 h after intraperitoneal injection of ortho[ 3 2 P] phosphate. 2. It has been demonstrated that the high average specific radioactivity of phosphoglycerides in muscles of rats deficient in essential fatty acids is due to both increased amounts and increased turnover of 1-palmitoyl-2-oleoyl phosphatidylcholine and phosphatidylethanolamine. 3. The l-stearoyL2arachidonoyl phosphatidylcholine was found to turn over faster than the 1-palmitoyl-2-arachidonoyl species. In rats deficient in essential fatty acids, the l-stearoyl-2-(5,6,11-eicosatrienoyl) phosphatidylcholine turned over more rapidly than the 1-palmitoyl-2-( 5,8,1 l-eicosatrienoyl) species. Both findings are in contrast with similar findings for liver.
Introduction The metabolism of phosphoglycerides is markedly affected by essential fatty acid deficiency and the incorporation of ortho[” 2 P] phosphate into liver phosphoglycerides in this condition was found to be increased [l-3]. The increased incorporation was associated with fundamental changes in composition: the levels of linoleic and arachidonic acids dropped whilst those of palmitoleic, oleic and 5,8,11-eicosatrienoic acids increased [ 1,4] . Even during prolonged deficiency of essential fatty acids appreciable amounts of these acids are retained in phosphoglycerides [ 51. * Present address: Department of Mediziv!, Road. Melbourne. Victoria 3004. Australia. * * Deceased.
Monash
University.
Prince
Henry’s
Hospital.
St. Kilda
117
Skeletal muscle in mammals represents a major store of phosphoglycerides and essential fatty acids [6]. Hence, it was thought to be relevant to follow the alterations in phosphoglyceride metabolism in skeletal muscle during essential fatty acid deficiency. The aim of the present study was to compare the specific radioactivities, with respect to ortho[j 2 P] phosphate, of muscle phosphatidylcholines and phosphatidylethanolamines from control rats with those from rats deficient in essential fatty acids. It was of particular interest to compare phosphoglyceride species containing arachidonic acid with those containing 5,8,11eicosatrienoic acid. A method previously described [7] for investigating the metabolism of molecular species of phosphoglycerides has been applied. Methods The experimental procedures used in the present study have been described previously [7]. Male Buffalo rats as weanlings were placed on either of TABLE
I
COMPOSITION Amounts
OF THE SYNTHETIC
are given as percentages
DIETS
of weight. Diet
component
Saturated fat (deficient essential fatty acids)
free)*
Safflower (control)
seed oil
63.0 24.6 3.8 2.1 1.9
63.0 24.6 3.8 2.1 1.9 4.6
Sucrose
Casein (fat and vitamin Salt mixture* Vitamin mixture* Cellulose Stearax* * Safflower seed oil
in
4.6
* Nutritional Biochemical Corp., Cleveland, Ohio, U.S.A. ** Saturated beef fat (Unilever Pty. Ltd, Melbourne, Australia). TABLE
II
FATTY
ACID COMPOSITION
Amounts are given the molecule, that shows the position acid, although it is Fatty
acid
OF THE LIPID COMPONENTS
as mol percentages. The number before the colon gives the number of carbon atoms in after the colon indicates the number of double bonds. The number in parentheses of the first double bond from the methyl end; 18 : 1 (n-7, n-9) is referred to as oleic a mixture of oleic acid and cis-vaccenic acid. Diet Saturated
14: 15
fat
Safflower
8.4 1.2
0 :0
16 : 0 17 : 0 (anteiso) 17 : 0
34.0 1.3 2.6
9.4
:0 : 1 (n-7, : 2 (n-6)
50.2 2.3
5.4 11.3 73.9
18 18 18
OF THE DIETS
n-9)
seed oil
118
the synthetic diets 3 weeks after birth and allowed continuous access to food and water. The animals were 6 months old when used. The composition of the diets is listed in Table I. The fatty acid composition of the lipid components of the diets is given in Table II. Results and Discussion Phosphatidylcholine and methyl esters of N2 Ph-phosphatidylethanol~ amine were prepared from hind leg muscles of control rats and of rats deficient in essential fatty acids. The animals had been injected intraperitoneally with ortho[’ 2 P] phosphate 3 h before sacrifice. The yields of phosphatidylcholine and phosphatidylethanolamine are given in Table III. Phosphatidylcholine and phosphatidylethanolamine were purified by thin-layer chromatography and the distributions of fatty acids at TABLE III THE YIELDS 0~ PH~SPHOGLYCERIDES OBTAINED FROM SKELETAL MUSCLE 0~ CONTROL RATS AND OF RATS DEFICIENT IN ESSENTIAL FATTY ACIDS Diet
__ .____ ._ _ _ .~.._ ...COntrOl Essential fatty acid deficient
Live weight of rat (g) 448 Muscle wet weight (g) 36.1 Total pgosphoglycerides (&mol/gl 8.7 N2Ph-phosphoglyee~des f@mol NZPhlgf 2.1 Choline phosphoglyeerides (ffmoligf 4.9 Non-choline phosphoglycerides (Ctmollg) 3.2 Phosphstidylcholine (mnol/g) 4.0 Phosphatidyletllanolamine (vmol/gl 1.1 Plasmslogens (in total phosphoglycerides. fimoligl 0.6 ___ __ .-.. _~~ .--. - .-. --- - ------
260 20.4 8.3 1.8 4.3 3.1 3.2 1.0 0.6
TABLE IV FATTY ACID COMPOSITIONS OF SUBFRACTIONS OF PHOSPHATIDYLCHOLINE ARGENTATION THIN-LAYER CHROMATOGRAPHY The phosphatidylcholine as mol percentages.
OBTAINED BY
was isolated from the leg muscles of rats fed the control diet. Amounts are given
~_._ ..._____..__ _ __._ __ _ ___ ._______...___ _~~ --.. -__-.___ ~~~~~ . . . .~~.I RecalRecslMonoenoicTetraTotal Fatty acid dienoic fraction _~~_-_._--.“--16 : 0 18: 0 18 : 1 (n-7, n-9) 18 : 2 (n-6) 20 : 4 (n-6)
--
~.______
Percentage of each frraction* * *
culated total*
enoic fraction
_~_~~-.____._..________- -__--.---- -~~~ ..-.-.-- -~ 35.8 7.0 18.9 38.3
38.6 7.1 4.3 50.0
54.8
37.3 6.8 12.4 21.1 22.4
37.1 7.0 12.3 21.0 22.6
45.2
* Recalculated from the fatty acid compositions of each of the subfractions. ** Recalculated from the computed amounts of molecular species (Table VIII). * * * Based on phosphorus content.
eulated total**
~~~ -31.7 7.2 10.8 21.3 23.0
119 TABLE
V
FATTY ACID COMPOSITIONS ARGENTATION THIN-LAYER
OF SUBFRACTIONS CHROMATOGRAPHY
The phosphatidylchollne was isolated from diet. Amounts are given as mol percentages.
OF PHOSPHATIDYLCHOLINE
the leg muscles
of rats fed the essential
OBTAINED
BY
fatty acid deficient ._
---_ Fatty acid
Monoenoic fraction
Trienoic fraction
Total
Recalculated total*
Recalculated total**
16 : 0 16 : 1 (n-7) 18 : 0 18 : 1 (n-7, n-9) 20 : 3 (n-9)
31.1 10.3 4.0 54.6
32.1
31.3 7.2 4.2 41.3 16.0
31.4 7.0 4.4 41.0 16.2
31.5 7.0 4.4 40.8 16.3
Percentage
67.6
32.4
of each fraction***
5.3 12.6 50.0
* Recalculated from the fatty acid compositions of each of the subfractions. ** Recalculated from the computed amounts of molecular species (Table VIII). *** Based on phosphorus content.
the l- and 2-positions determined. The purified phosphoglycerides were each fractionated using argentation thin-layer chromatography. Tables IV and V list the fatty acid compositions of the subfractions obtained from phosphatidylcholine and Tables VI and VII give similar information for phosphatidylethanolamine. Each of the subfractions obtained in sufficient quantity was subjected to countercurrent distribution (Figs 1 and 2) and the amount of each molecular species present was computed and listed in Table VIII (phosphatidylcholine) and Table IX (phosphatidylethanolamine) together with their relative specific radioactivities. The specific radioactivities are reported as percentages of the specific radioactivity of the Serum inorganic phosphate. This provides a
TABLE
VI
FATTY ACID COMPOSITIONS OF TIDYLETHANOLAMINE OBTAINED The phosphatidylethanolamine given as mol percentages.
SUBFRACTIONS OF METHYL ESTERS OF NZPh-PHOSPHABY ARGENTATION THIN-LAYER CHROMATOGRAPHY
was isolated
from the leg muscles
of rats fed the control
diet. Amounts
are
-__.__ Fatty acid
16 18: 18 18 20 22
:0 0
: 1 (n-7, : 2 (n-6) : 4 (n-6) : 6 (n-3)
Percentage
n-9)
Dienoic fraction
Tetraenoic fraction
Hexaenoic fraction
Total
Recalculated total*
Recalculated total* *
6.6 34.0 9.4 50.0
4.9 41.1 4.0
7.0 32.0 11.0
5.9 37.6 6.6 10.6 28.4 11.0
5.7 37.6 6.7 10.4 28.4 11.2
5.8 37.7 6.5 10.4 28.4 11.2
50.0 50.0 of each
fraction***
20.8
56.7
22.5
* Recalculated from the fatty acid compositions of each of the subfractions. * * Recalculated from the computed amounts of molecular species (Table IX). * * * Based on NZPh group content.
TABLE
VII
FATTY
ACID
COMPOSITIONS
TIDYLETHANOLAMINE The
OF
phosphatidylethanolaine
deficient
diet.
SUBFRACTIONS
OBTAINED
Amounts
was are given
BY
isolated
as mol
OF
ARGENTATION from
METHYL
ESTERS
THIN-LAYER
the
leg
muscles
of
OF
NZPh-PHOSPHA-
CHROMATOGRAPHY rats
fed
the
essential
fatty
acid
precentages. _~
Fatty
acid
Monoenoic
Trienoic
Tetraenoic
fraction
fraction
fraction
Recalculated total**
8.8
8.6
8.7
6.9
7.0
0
10.2
16
1 (n-7)
18.4
18
0
17.6
29.5
31.5
25.3
25.2
25.1
53.8
12.2
14.0
27.8
28.2
28.0
26.0
25.9
25.9
1 (n-7.
20
3 (n-9)
n-9)
20
4 (n-6)
50.0 50.0
Percentage
**
38.1
* Recalculated Recalculated
***
5.2
7.2
5.1
5.1
of each
fraction*
**
4.5
Recalculated total*
16
18
8.3
Total
Based
51.8
from
the
fatty
from
the
computed
on N2Ph
group
acid
10.1
compositions amounts
of each
of the
of molecular
subfractions.
species
(Table
IX).
content.
basis for comparing the specific radioactivities from control rats with those derived from rats deficient in essential fatty acids even though the body and muscle weights of the animals may have differed. The major molecular species present in muscle phosphatidylcholine from control rats were the linoleoyl and arachidonoyl species and from rats deficient in essential fatty acids, the oleoyl and 5,8,11-eicosatrienoyl species. The preTABLE A
VIII
COMPARISON
SPECIES
OF
IN ESSENTIAL
OF MUSCLE FATTY
THE
INCORPORATION
OF
PHOSPHATIDYLCHOLINE
ORTHOl”*PlPHOSPHATE FROM
CONTROL
INTO
RATS
AND
MOLECULAR
RATS
DEFICIENT
ACIDS
.~ Diet Fatty
acid
2
16
: 0
16
16
: 0
18
16
: 0
18
16
: 0
20
16
: 0
20
18 18
: 0 : 0
18 18
18
:
18
18
: 0
20
18
: 0
20
18
: 1 (n-7,n-9)
16
18
:1 :1 :1
18
18
fatty
acid
deficient
at position
1
18
Essential
Control
1 (n-7,n-9)
(n-7,n-9) (n-7,n-9)
20
(n-7,n-9)
20
:1 :1 :2 :3 :4 :1 :2 :1 :3 :4 :1 :2 :3 :4
Mel%
RSR*
(M,)
(R,)
8.8
1.59
14.0
31.3
1.03
32.2
35.3
0.33
11.6
0.52
4.1
Mc
(n-7) (n-7,n-9) (n-6)
(n-7,n-9) (n-6)
2.6 5.2
1
6.6
0.8
6.1
1.94
specific
radioactivity.
26.4
2.21
76.5
Rd
Md.
R
M,
. Rz
26.4 62.5 -32.2
1.48
30.9
30.9 -11.6
5.2
1.95
10.1
4.1
0.61
1.61 1.6
21.9 5.6
10.1 21.9 5.6 -5.3
6.6
4.2
27.7
27.7 -11.8
11.8 8.2
1.23
10.1
10.1 -2.5
2.5 81.5
* Relative
3.57
Md’
5.3
(n-9)
Totals
7.4 34.6
3.5
(n-7)
(n-6)
(Rd)
13.6
(n-9)
(n-6)
RSR*
(Md)
-4.1
(n-7.m9) (n-6)
Mel%
20.9
(n-9) (n-6)
. Rc
209.0
127.7
121 TABLE IX A COMPARISON OF THE INCORPORATION OF ORTHO[“‘PlPHOSPHATE INTO MOLECULAR SPECIES OF MUSCLE PHOSPHATIDYLETHANOLAMINE FROM CONTROL RATS AND RATS DEFICIENT IN ESSENTIAL FATTY ACIDS Diet Fatty acid at position 2
1
~________~___. 16 : 0 16 18 16 18 18 16 18 18 18 16 18 18 16 18 18 16 18 18
: 1 (n-7,n-9) : 0 : 0 : 1 (n-7.m9)
:0 : 1 (n-7.m9) : 0 : 0 : 0 : 1 (n-7.m9)
:0 :0 : 1 (n-7,n-9) : 0 : 0 : 1 (n-7,n-9) : 0
16 18 16 18 18 18 18 18 20 20 20 20 20 20 22 22 22
Essential fatty acid deficient --__--~
contro1 __~_ ____~______ MoI%
RSR*
(M,)
(R,)
Mc
. Rc
MoI%
RSR*
(Md)
(Rd)
~--
-___.__ 3.4 ) 6.0 4.5 5.1 10.8 I
: 1 (n-7)
: 1 (n-7) : 1 (n-7.m9) : 1 (n-7) : 1 (n-7,n-9) : 2 (n-6) : 2 (n-6) : 1 (n-7,n-9) : 2 (n-6) : 3 (n-9) : 3 (n-9) : 3 (n-9) : 4 (n-6) : 4 (n-6) : 4 (n-6) : 6 (n-3) : 6 (n-3) : 6 (n-3)
2.9 4.6 1
1.09
8.2
0.62
8.2
5.7 4.3 1 46.7 2.9 4.2 1 15.4
Totals
0.26
2.6
0.13
6.1
0.57
4.0
0.17
Mc
1.86
17.5
17.5
3.09
63.0
63.0
.%
8.7 ) 12.8 30.0 0.9 2.8 6.4
1.82
15.1
15.1 -8.2
0.41
8.8
8.8
0.29
8.8
8.8 -2.6 4.2 -6.1
0.42
4.2
-4.0 -2.6
2.6 31.8
Md.Rd-
-8.2 8.3
13.3
Md’Md
117.5
85.7
* Relative specific radioactivity.
dominant species in muscle phosphatidylethanolamine were the arachidonoyl species in control rats and the 5,8,11-eicosatrienoyl species in deficient rats. An extremely small amount of arachidonic acid (less than 0.1%) was retained in phosphatidylcholine derived from deficient rats but a larger amount was found in phosphatidylethanolamine. No other fatty acids of the linoleoyl series were detected in the deficient animals. The average specific radioactivity of phosphatidylcholine was approximately double that of phosphatidylethanolamine irrespective of whether the animals from which the phosphoglycerides were isolated were controls or deficient in essential fatty acids. This finding indicates that phosphatidylcholine species, as a group, are turning over at a faster rate than phosphatidylethanolamine. This result is in marked contrast with that reported for liver [3] in which the average specific radioactivity of phosphatidylethanolamine was approx. five times that of phosphatidylcholine in both control and deficient rats. The average specific radioactivity of the muscle phosphatidylcholine from deficient rats was approx. three times that of phosphatidylcholine from control rats. A similar ratio of specific radioactivities was obtained for muscle phosphatidylethanolamine. These results suggest that certain phosphatidylcholines and phosphatidylethanolamines in the muscles of deficient rats were turning over at a faster rate than their equivalent species in muscles of control rats. In order to determine which species were responsible for this increased
122 PHOSPHATIDYLCHOLINES
PHOSPH’ATIOYLETHANOLAMINES
i&y/y
/&
::[X
y”
oiaD7f
0
170
190 210 230 TUBE NUMBER
250
270
50
70 TUBE
90 NVMBER
I10
l-30
I70
Yka I90
2:o --2&T TUBE NUMBER
~2%_3
Fig. 1. Countercurrent distributions of phosphoglyceride fractions obtained from contr 1 rats. The solvent (50 : systems were carbon tetrachloride/methanol/water (62 : 33 : 5, v/v) and heptane/m ??h anal/water 47.5 : 2.5, v/v) for phosphatidylcholine and methyl esters of NzPh-phosphatidylethanolamine, respectively [131. The symbols represent measured parameters and the curves show the recalculated distributions of radioactivity; b. the distributions of fatty parameters. a, shows the distribution of ortho[ 32Plphosphate acids at the l-position; c, the distributions of fatty acids at the 2-position.
turnover of both phosphatidylcholine and phosphatidylethanolamine in rats deficient in essential fatty acids Tables VIII and IX were constructed. In Table VIII, estimates of the incorporation of ortho[3 2 P] phosphate into each molecular species of phosphatidylcholine from control and deficient rats are compared. It is clear that the increased relative specific radioactivity of muscle phosphatidylcholine from deficient rats, compared with control rats, was due to the increase in both the amount and the relative specific radioactivity of the l-palmitoyl-2-oleoyl species. Table IX presents comparisons for phosphatidylethanolamine similar to those given for phosphatidylcholine in Table VIII. Although the relative specific radioactivities of several phosphatidylethanolamines are lacking from Table IX, it is clear that the increased relative specific radioactivity of phosphatidylethanolamine from rats deficient in essential fatty acids compared with
123 PHOSPHATIDYLCHOLINES
PHOSPHATIDYLETHANOLAMINES
Fig. 2. Countercurrent distributions of phosphoglyceride tial fatty acids. Other details are as for Fig. 1.
fractions obtained from rats deficient in essen-
that from control rats was due to larger amounts of the l-palmitoyl-2oleoyl, the 1-stearoyl-2-palmitoleoyl and the 1,2-dioleoyl species. The muscle phosphatidylcholines with the fastest relative turnover were the l-palmitoyl-koleoyl and the I-oleoylS-linoleoyl species in control animals and the 1-palmitoyl-2-palmitoleoyl and the l-oleoyl-2-palmitoleoyl species in animals deficient in essential fatty acids. The species with the slowest turnover were the l-palmitoyl-2arachidonoyl phosphatidylcholine in control rats and l-oleoyl-2-( 5,8,1 l-eicosatrienoyl) phosphatidylcholine in deficient rats. The muscle phosphat~dyle~anol~~es with the fastest turnover were either the l-palmitoyl-2linoleoyl or the l-oleoyL24inoleoyl species in control rats and either the l-palmitoyl-2-oleoyl, the l-stearoyl-2-palmitoleoyl or the 1,2-dioleoyl species in deficient rats but the results are not conclusive due to the incomplete separation of certain mixtures during countercurrent distribution. The phosphatidylethanolamines with the slowest turnover were probably the l-ste~oyl-2~~achidonoyl species in control rats and the l-stearoyl-lr(5,8,11-eicosatrienoyl) species in rats deficient in essential fatty acids, but again conclusive results could not be obtained. Each arachidonoyl phosphatidylcholine species in control animals was turning over at a slower rate than the
124
TABLE
X
A COMPARISON
OF
THE
RELATIVE
PHOSPHATIDYLCHOLINES ll-EICOSATRIENOYL TIAL
FATTY
SPECIFIC
ISOLATED
RADIOACTIVITIES
FROM
CONTROL
PHOSPHATIDYLCHOLINES
ISOLATED
Mel%
:0 :0 :0 :0 :1 :1
18 18 18
THOSE
FROM
RATS
ARACHIDONOYL OF
MUSCLE
DEFICIENT
IN
5.
8.
ESSEN-
Essential
fatty
acid deficient
at position 2
18
MUSCLF.
Control acid
1
16
OF WITH
ACIDS
Diet Fatty
16
RATS
Relative
specific
Mel%
Relative
radioactivity
20
3 (n-9)
20
4 (n-6)
20
3 (n-9)
20
4 (n-6)
(n-7,
n-9)
20
3 (n-9)
(n-7,
n-9)
20
4 (n-6)
20.9 34.6
specific
radioactivity
1.48
0.33
6.5
0.8
4.0
0.61
3.5
1.6
8.2
1.23
corresponding 5,8,11-eicosatrienoyl species in deficient animals (Table X). With equivalent phosphatidylethanolamines, it was possible to conclude only that the l-stearoyl-2-(5,8,11-eicosatrienoyl) species in deficient ,rats was turning over more rapidly than the corresponding arachidonoyl species in control rats and that the average arachidonoyl phosphatidylethanolamine in deficient rats was turning over three times faster than that in control rats (Table XI). Trewhella and Collins [3] found that the liver phosphatidylcholine with the slowest turnover was the l-stearoyl-2-arachidonoyl species in control rats and the 1-stearoyl-2-( 5,8,11-eicosatrienoyl) species in rats deficient in essential fatty acids. The ratio of the relative specific radioactivities of liver species with the fastest and slowest turnover (1-palmitoyl-2-linoleoyl and l-stearoyl-2-arachidonoyl phosphatidylcholines, respectively) from control animals was 155 : 1, compared with 5.9 : 1 for equivalent species (1-oleoyl-2-linoleoyl and 1-palmitoyl-2-arachidonoyl phosphatidylcholines, respectively) in muscle. The relative specific radioactivity of liver 1-palmitoyl-2-linoleoyl phosphatidylcholine was 5.2 compared with 1.03 for this phosphatidylcholine in the muscle of control rats. Since muscle phosphoglycerides, in contrast to those of liver, are not
TABLE A
XI
COMPARISON
OF
THE
RELATIVE
SPECIFIC
PHOSPHATIDYLETHANOLAMINES ARACHIDONOYL ESSENTIAL
ISOLATED
FROM
PHOSPHATIDYLETHANOLAMINES FATTY
OF
CONTROL
RATS
ISOLATED
acid
~~
Mel%
2
:0
18
: 1 (n-7, :0
ARACHIDONOYL THOSE
RATS
OF
MUSCLE
DEFICIENT
IN
Essential
fatty
acid deficient
at position
1
16
WITH
FROM
Control
Fatty
MUSCLE
ACIDS
Diet
18
RADIOACTIVITIES
20 n-9)
20 20
Relative
specific
Mel%
:4 :4 :4
(n-6) (n-6) (n-6)
1
10.0
0.26
46.7
0.13
Relative
specific
radioactivity
radioactivity
0.15
10.1
0.42
125
exported phosphoglyceride metabolism in muscle reflects the synthesis and degradation of these compounds solely within this tissue. The higher specific radioactivities of muscle phosphatidylcholines compared with those of phosphatidyl~thanol~ines can be explained in terms of the precursor pool sizes and their specific radioactivities (e.g. of cytidine diphosphocholine compared with that of cytidine diphosphoethanolamine) but the pool sixes and the specific radioactivities of these precursors have not been determined. The ratio of the specific radioactivity of the average phosphatidylcholine to that of the average phosphat~dylethanol~~e obtained for control rats (2.7) was close to that for rats deficient in essential fatty acids (1.9). This suggests that the increase observed in specific radioactivities of phosphoglycerides in essential fatty acid deficiency was due to the increased specific radioactivity of a common precursor (i.e. inorganic phosphate) rather than of either of the phosphoglyceride classes. However, within each group, radical changes in composition are imposed by the deficiency in essential fatty acids. Fatty acids of the (n-6) series are replaced by fatty acids of the (n-9) series and an increase in the amount of fatty acids of the (n-7) series also occurs [3]. Collins [8] has suggested that the higher specific radioactivity of liver phosphatidylcholine in rats deficient in essential fatty acids could have been due to an increased rate of
SPEC,F,C
RAo,OACT,“,TY
OF
SERUM
INORGANIC
El E
PHOSPHATE
TAKEN
AS
IO0
PHOSPHORYLCHOLINE
CYTIDINE
DIP~~SPHOCHOLINE
Biochimica @ Elsevier
et Biophysics Acta, Scientific Publishing
409 (1975) 116-127 Company, Amsterdam
-
Printed
in The Netherlands
BBA 56667
BIOSYNTHESIS OF PHOSPHOGLYCERIDES IN SKELETAL MUSCLE IN CONTROL RATS AND IN RATS DEFICIENT IN ESSENTIAL FATTY ACIDS
F.A.
SHAMGAR*
and F.D.
The Russell Grimwade 3052 (Australia) (Received
April 24th,
COLLINS**
School
of Biochemistry,
University
of Melbourne,
Parkville,
Vicioria
1975)
Summary
1. The specific radioactivities of individual molecular species of phosphoglycerides in the skeletal muscles of control rats and of rats deficient in essential fatty acids have been determined 3 h after intraperitoneal injection of ortho[ 3 2 P] phosphate. 2. It has been demonstrated that the high average specific radioactivity of phosphoglycerides in muscles of rats deficient in essential fatty acids is due to both increased amounts and increased turnover of 1-palmitoyl-2-oleoyl phosphatidylcholine and phosphatidylethanolamine. 3. The l-stearoyL2arachidonoyl phosphatidylcholine was found to turn over faster than the 1-palmitoyl-2-arachidonoyl species. In rats deficient in essential fatty acids, the l-stearoyl-2-(5,6,11-eicosatrienoyl) phosphatidylcholine turned over more rapidly than the 1-palmitoyl-2-( 5,8,1 l-eicosatrienoyl) species. Both findings are in contrast with similar findings for liver.
Introduction The metabolism of phosphoglycerides is markedly affected by essential fatty acid deficiency and the incorporation of ortho[” 2 P] phosphate into liver phosphoglycerides in this condition was found to be increased [l-3]. The increased incorporation was associated with fundamental changes in composition: the levels of linoleic and arachidonic acids dropped whilst those of palmitoleic, oleic and 5,8,11-eicosatrienoic acids increased [ 1,4] . Even during prolonged deficiency of essential fatty acids appreciable amounts of these acids are retained in phosphoglycerides [ 51. * Present address: Department of Mediziv!, Road. Melbourne. Victoria 3004. Australia. * * Deceased.
Monash
University.
Prince
Henry’s
Hospital.
St. Kilda
117
Skeletal muscle in mammals represents a major store of phosphoglycerides and essential fatty acids [6]. Hence, it was thought to be relevant to follow the alterations in phosphoglyceride metabolism in skeletal muscle during essential fatty acid deficiency. The aim of the present study was to compare the specific radioactivities, with respect to ortho[j 2 P] phosphate, of muscle phosphatidylcholines and phosphatidylethanolamines from control rats with those from rats deficient in essential fatty acids. It was of particular interest to compare phosphoglyceride species containing arachidonic acid with those containing 5,8,11eicosatrienoic acid. A method previously described [7] for investigating the metabolism of molecular species of phosphoglycerides has been applied. Methods The experimental procedures used in the present study have been described previously [7]. Male Buffalo rats as weanlings were placed on either of TABLE
I
COMPOSITION Amounts
OF THE SYNTHETIC
are given as percentages
DIETS
of weight. Diet
component
Saturated fat (deficient essential fatty acids)
free)*
Safflower (control)
seed oil
63.0 24.6 3.8 2.1 1.9
63.0 24.6 3.8 2.1 1.9 4.6
Sucrose
Casein (fat and vitamin Salt mixture* Vitamin mixture* Cellulose Stearax* * Safflower seed oil
in
4.6
* Nutritional Biochemical Corp., Cleveland, Ohio, U.S.A. ** Saturated beef fat (Unilever Pty. Ltd, Melbourne, Australia). TABLE
II
FATTY
ACID COMPOSITION
Amounts are given the molecule, that shows the position acid, although it is Fatty
acid
OF THE LIPID COMPONENTS
as mol percentages. The number before the colon gives the number of carbon atoms in after the colon indicates the number of double bonds. The number in parentheses of the first double bond from the methyl end; 18 : 1 (n-7, n-9) is referred to as oleic a mixture of oleic acid and cis-vaccenic acid. Diet Saturated
14: 15
fat
Safflower
8.4 1.2
0 :0
16 : 0 17 : 0 (anteiso) 17 : 0
34.0 1.3 2.6
9.4
:0 : 1 (n-7, : 2 (n-6)
50.2 2.3
5.4 11.3 73.9
18 18 18
OF THE DIETS
n-9)
seed oil
118
the synthetic diets 3 weeks after birth and allowed continuous access to food and water. The animals were 6 months old when used. The composition of the diets is listed in Table I. The fatty acid composition of the lipid components of the diets is given in Table II. Results and Discussion Phosphatidylcholine and methyl esters of N2 Ph-phosphatidylethanol~ amine were prepared from hind leg muscles of control rats and of rats deficient in essential fatty acids. The animals had been injected intraperitoneally with ortho[’ 2 P] phosphate 3 h before sacrifice. The yields of phosphatidylcholine and phosphatidylethanolamine are given in Table III. Phosphatidylcholine and phosphatidylethanolamine were purified by thin-layer chromatography and the distributions of fatty acids at TABLE III THE YIELDS 0~ PH~SPHOGLYCERIDES OBTAINED FROM SKELETAL MUSCLE 0~ CONTROL RATS AND OF RATS DEFICIENT IN ESSENTIAL FATTY ACIDS Diet
__ .____ ._ _ _ .~.._ ...COntrOl Essential fatty acid deficient
Live weight of rat (g) 448 Muscle wet weight (g) 36.1 Total pgosphoglycerides (&mol/gl 8.7 N2Ph-phosphoglyee~des f@mol NZPhlgf 2.1 Choline phosphoglyeerides (ffmoligf 4.9 Non-choline phosphoglycerides (Ctmollg) 3.2 Phosphstidylcholine (mnol/g) 4.0 Phosphatidyletllanolamine (vmol/gl 1.1 Plasmslogens (in total phosphoglycerides. fimoligl 0.6 ___ __ .-.. _~~ .--. - .-. --- - ------
260 20.4 8.3 1.8 4.3 3.1 3.2 1.0 0.6
TABLE IV FATTY ACID COMPOSITIONS OF SUBFRACTIONS OF PHOSPHATIDYLCHOLINE ARGENTATION THIN-LAYER CHROMATOGRAPHY The phosphatidylcholine as mol percentages.
OBTAINED BY
was isolated from the leg muscles of rats fed the control diet. Amounts are given
~_._ ..._____..__ _ __._ __ _ ___ ._______...___ _~~ --.. -__-.___ ~~~~~ . . . .~~.I RecalRecslMonoenoicTetraTotal Fatty acid dienoic fraction _~~_-_._--.“--16 : 0 18: 0 18 : 1 (n-7, n-9) 18 : 2 (n-6) 20 : 4 (n-6)
--
~.______
Percentage of each frraction* * *
culated total*
enoic fraction
_~_~~-.____._..________- -__--.---- -~~~ ..-.-.-- -~ 35.8 7.0 18.9 38.3
38.6 7.1 4.3 50.0
54.8
37.3 6.8 12.4 21.1 22.4
37.1 7.0 12.3 21.0 22.6
45.2
* Recalculated from the fatty acid compositions of each of the subfractions. ** Recalculated from the computed amounts of molecular species (Table VIII). * * * Based on phosphorus content.
eulated total**
~~~ -31.7 7.2 10.8 21.3 23.0
119 TABLE
V
FATTY ACID COMPOSITIONS ARGENTATION THIN-LAYER
OF SUBFRACTIONS CHROMATOGRAPHY
The phosphatidylchollne was isolated from diet. Amounts are given as mol percentages.
OF PHOSPHATIDYLCHOLINE
the leg muscles
of rats fed the essential
OBTAINED
BY
fatty acid deficient ._
---_ Fatty acid
Monoenoic fraction
Trienoic fraction
Total
Recalculated total*
Recalculated total**
16 : 0 16 : 1 (n-7) 18 : 0 18 : 1 (n-7, n-9) 20 : 3 (n-9)
31.1 10.3 4.0 54.6
32.1
31.3 7.2 4.2 41.3 16.0
31.4 7.0 4.4 41.0 16.2
31.5 7.0 4.4 40.8 16.3
Percentage
67.6
32.4
of each fraction***
5.3 12.6 50.0
* Recalculated from the fatty acid compositions of each of the subfractions. ** Recalculated from the computed amounts of molecular species (Table VIII). *** Based on phosphorus content.
the l- and 2-positions determined. The purified phosphoglycerides were each fractionated using argentation thin-layer chromatography. Tables IV and V list the fatty acid compositions of the subfractions obtained from phosphatidylcholine and Tables VI and VII give similar information for phosphatidylethanolamine. Each of the subfractions obtained in sufficient quantity was subjected to countercurrent distribution (Figs 1 and 2) and the amount of each molecular species present was computed and listed in Table VIII (phosphatidylcholine) and Table IX (phosphatidylethanolamine) together with their relative specific radioactivities. The specific radioactivities are reported as percentages of the specific radioactivity of the Serum inorganic phosphate. This provides a
TABLE
VI
FATTY ACID COMPOSITIONS OF TIDYLETHANOLAMINE OBTAINED The phosphatidylethanolamine given as mol percentages.
SUBFRACTIONS OF METHYL ESTERS OF NZPh-PHOSPHABY ARGENTATION THIN-LAYER CHROMATOGRAPHY
was isolated
from the leg muscles
of rats fed the control
diet. Amounts
are
-__.__ Fatty acid
16 18: 18 18 20 22
:0 0
: 1 (n-7, : 2 (n-6) : 4 (n-6) : 6 (n-3)
Percentage
n-9)
Dienoic fraction
Tetraenoic fraction
Hexaenoic fraction
Total
Recalculated total*
Recalculated total* *
6.6 34.0 9.4 50.0
4.9 41.1 4.0
7.0 32.0 11.0
5.9 37.6 6.6 10.6 28.4 11.0
5.7 37.6 6.7 10.4 28.4 11.2
5.8 37.7 6.5 10.4 28.4 11.2
50.0 50.0 of each
fraction***
20.8
56.7
22.5
* Recalculated from the fatty acid compositions of each of the subfractions. * * Recalculated from the computed amounts of molecular species (Table IX). * * * Based on NZPh group content.
TABLE
VII
FATTY
ACID
COMPOSITIONS
TIDYLETHANOLAMINE The
OF
phosphatidylethanolaine
deficient
diet.
SUBFRACTIONS
OBTAINED
Amounts
was are given
BY
isolated
as mol
OF
ARGENTATION from
METHYL
ESTERS
THIN-LAYER
the
leg
muscles
of
OF
NZPh-PHOSPHA-
CHROMATOGRAPHY rats
fed
the
essential
fatty
acid
precentages. _~
Fatty
acid
Monoenoic
Trienoic
Tetraenoic
fraction
fraction
fraction
Recalculated total**
8.8
8.6
8.7
6.9
7.0
0
10.2
16
1 (n-7)
18.4
18
0
17.6
29.5
31.5
25.3
25.2
25.1
53.8
12.2
14.0
27.8
28.2
28.0
26.0
25.9
25.9
1 (n-7.
20
3 (n-9)
n-9)
20
4 (n-6)
50.0 50.0
Percentage
**
38.1
* Recalculated Recalculated
***
5.2
7.2
5.1
5.1
of each
fraction*
**
4.5
Recalculated total*
16
18
8.3
Total
Based
51.8
from
the
fatty
from
the
computed
on N2Ph
group
acid
10.1
compositions amounts
of each
of the
of molecular
subfractions.
species
(Table
IX).
content.
basis for comparing the specific radioactivities from control rats with those derived from rats deficient in essential fatty acids even though the body and muscle weights of the animals may have differed. The major molecular species present in muscle phosphatidylcholine from control rats were the linoleoyl and arachidonoyl species and from rats deficient in essential fatty acids, the oleoyl and 5,8,11-eicosatrienoyl species. The preTABLE A
VIII
COMPARISON
SPECIES
OF
IN ESSENTIAL
OF MUSCLE FATTY
THE
INCORPORATION
OF
PHOSPHATIDYLCHOLINE
ORTHOl”*PlPHOSPHATE FROM
CONTROL
INTO
RATS
AND
MOLECULAR
RATS
DEFICIENT
ACIDS
.~ Diet Fatty
acid
2
16
: 0
16
16
: 0
18
16
: 0
18
16
: 0
20
16
: 0
20
18 18
: 0 : 0
18 18
18
:
18
18
: 0
20
18
: 0
20
18
: 1 (n-7,n-9)
16
18
:1 :1 :1
18
18
fatty
acid
deficient
at position
1
18
Essential
Control
1 (n-7,n-9)
(n-7,n-9) (n-7,n-9)
20
(n-7,n-9)
20
:1 :1 :2 :3 :4 :1 :2 :1 :3 :4 :1 :2 :3 :4
Mel%
RSR*
(M,)
(R,)
8.8
1.59
14.0
31.3
1.03
32.2
35.3
0.33
11.6
0.52
4.1
Mc
(n-7) (n-7,n-9) (n-6)
(n-7,n-9) (n-6)
2.6 5.2
1
6.6
0.8
6.1
1.94
specific
radioactivity.
26.4
2.21
76.5
Rd
Md.
R
M,
. Rz
26.4 62.5 -32.2
1.48
30.9
30.9 -11.6
5.2
1.95
10.1
4.1
0.61
1.61 1.6
21.9 5.6
10.1 21.9 5.6 -5.3
6.6
4.2
27.7
27.7 -11.8
11.8 8.2
1.23
10.1
10.1 -2.5
2.5 81.5
* Relative
3.57
Md’
5.3
(n-9)
Totals
7.4 34.6
3.5
(n-7)
(n-6)
(Rd)
13.6
(n-9)
(n-6)
RSR*
(Md)
-4.1
(n-7.m9) (n-6)
Mel%
20.9
(n-9) (n-6)
. Rc
209.0
127.7
121 TABLE IX A COMPARISON OF THE INCORPORATION OF ORTHO[“‘PlPHOSPHATE INTO MOLECULAR SPECIES OF MUSCLE PHOSPHATIDYLETHANOLAMINE FROM CONTROL RATS AND RATS DEFICIENT IN ESSENTIAL FATTY ACIDS Diet Fatty acid at position 2
1
~________~___. 16 : 0 16 18 16 18 18 16 18 18 18 16 18 18 16 18 18 16 18 18
: 1 (n-7,n-9) : 0 : 0 : 1 (n-7.m9)
:0 : 1 (n-7.m9) : 0 : 0 : 0 : 1 (n-7.m9)
:0 :0 : 1 (n-7,n-9) : 0 : 0 : 1 (n-7,n-9) : 0
16 18 16 18 18 18 18 18 20 20 20 20 20 20 22 22 22
Essential fatty acid deficient --__--~
contro1 __~_ ____~______ MoI%
RSR*
(M,)
(R,)
Mc
. Rc
MoI%
RSR*
(Md)
(Rd)
~--
-___.__ 3.4 ) 6.0 4.5 5.1 10.8 I
: 1 (n-7)
: 1 (n-7) : 1 (n-7.m9) : 1 (n-7) : 1 (n-7,n-9) : 2 (n-6) : 2 (n-6) : 1 (n-7,n-9) : 2 (n-6) : 3 (n-9) : 3 (n-9) : 3 (n-9) : 4 (n-6) : 4 (n-6) : 4 (n-6) : 6 (n-3) : 6 (n-3) : 6 (n-3)
2.9 4.6 1
1.09
8.2
0.62
8.2
5.7 4.3 1 46.7 2.9 4.2 1 15.4
Totals
0.26
2.6
0.13
6.1
0.57
4.0
0.17
Mc
1.86
17.5
17.5
3.09
63.0
63.0
.%
8.7 ) 12.8 30.0 0.9 2.8 6.4
1.82
15.1
15.1 -8.2
0.41
8.8
8.8
0.29
8.8
8.8 -2.6 4.2 -6.1
0.42
4.2
-4.0 -2.6
2.6 31.8
Md.Rd-
-8.2 8.3
13.3
Md’Md
117.5
85.7
* Relative specific radioactivity.
dominant species in muscle phosphatidylethanolamine were the arachidonoyl species in control rats and the 5,8,11-eicosatrienoyl species in deficient rats. An extremely small amount of arachidonic acid (less than 0.1%) was retained in phosphatidylcholine derived from deficient rats but a larger amount was found in phosphatidylethanolamine. No other fatty acids of the linoleoyl series were detected in the deficient animals. The average specific radioactivity of phosphatidylcholine was approximately double that of phosphatidylethanolamine irrespective of whether the animals from which the phosphoglycerides were isolated were controls or deficient in essential fatty acids. This finding indicates that phosphatidylcholine species, as a group, are turning over at a faster rate than phosphatidylethanolamine. This result is in marked contrast with that reported for liver [3] in which the average specific radioactivity of phosphatidylethanolamine was approx. five times that of phosphatidylcholine in both control and deficient rats. The average specific radioactivity of the muscle phosphatidylcholine from deficient rats was approx. three times that of phosphatidylcholine from control rats. A similar ratio of specific radioactivities was obtained for muscle phosphatidylethanolamine. These results suggest that certain phosphatidylcholines and phosphatidylethanolamines in the muscles of deficient rats were turning over at a faster rate than their equivalent species in muscles of control rats. In order to determine which species were responsible for this increased
122 PHOSPHATIDYLCHOLINES
PHOSPH’ATIOYLETHANOLAMINES
i&y/y
/&
::[X
y”
oiaD7f
0
170
190 210 230 TUBE NUMBER
250
270
50
70 TUBE
90 NVMBER
I10
l-30
I70
Yka I90
2:o --2&T TUBE NUMBER
~2%_3
Fig. 1. Countercurrent distributions of phosphoglyceride fractions obtained from contr 1 rats. The solvent (50 : systems were carbon tetrachloride/methanol/water (62 : 33 : 5, v/v) and heptane/m ??h anal/water 47.5 : 2.5, v/v) for phosphatidylcholine and methyl esters of NzPh-phosphatidylethanolamine, respectively [131. The symbols represent measured parameters and the curves show the recalculated distributions of radioactivity; b. the distributions of fatty parameters. a, shows the distribution of ortho[ 32Plphosphate acids at the l-position; c, the distributions of fatty acids at the 2-position.
turnover of both phosphatidylcholine and phosphatidylethanolamine in rats deficient in essential fatty acids Tables VIII and IX were constructed. In Table VIII, estimates of the incorporation of ortho[3 2 P] phosphate into each molecular species of phosphatidylcholine from control and deficient rats are compared. It is clear that the increased relative specific radioactivity of muscle phosphatidylcholine from deficient rats, compared with control rats, was due to the increase in both the amount and the relative specific radioactivity of the l-palmitoyl-2-oleoyl species. Table IX presents comparisons for phosphatidylethanolamine similar to those given for phosphatidylcholine in Table VIII. Although the relative specific radioactivities of several phosphatidylethanolamines are lacking from Table IX, it is clear that the increased relative specific radioactivity of phosphatidylethanolamine from rats deficient in essential fatty acids compared with
123 PHOSPHATIDYLCHOLINES
PHOSPHATIDYLETHANOLAMINES
Fig. 2. Countercurrent distributions of phosphoglyceride tial fatty acids. Other details are as for Fig. 1.
fractions obtained from rats deficient in essen-
that from control rats was due to larger amounts of the l-palmitoyl-2oleoyl, the 1-stearoyl-2-palmitoleoyl and the 1,2-dioleoyl species. The muscle phosphatidylcholines with the fastest relative turnover were the l-palmitoyl-koleoyl and the I-oleoylS-linoleoyl species in control animals and the 1-palmitoyl-2-palmitoleoyl and the l-oleoyl-2-palmitoleoyl species in animals deficient in essential fatty acids. The species with the slowest turnover were the l-palmitoyl-2arachidonoyl phosphatidylcholine in control rats and l-oleoyl-2-( 5,8,1 l-eicosatrienoyl) phosphatidylcholine in deficient rats. The muscle phosphat~dyle~anol~~es with the fastest turnover were either the l-palmitoyl-2linoleoyl or the l-oleoyL24inoleoyl species in control rats and either the l-palmitoyl-2-oleoyl, the l-stearoyl-2-palmitoleoyl or the 1,2-dioleoyl species in deficient rats but the results are not conclusive due to the incomplete separation of certain mixtures during countercurrent distribution. The phosphatidylethanolamines with the slowest turnover were probably the l-ste~oyl-2~~achidonoyl species in control rats and the l-stearoyl-lr(5,8,11-eicosatrienoyl) species in rats deficient in essential fatty acids, but again conclusive results could not be obtained. Each arachidonoyl phosphatidylcholine species in control animals was turning over at a slower rate than the
124
TABLE
X
A COMPARISON
OF
THE
RELATIVE
PHOSPHATIDYLCHOLINES ll-EICOSATRIENOYL TIAL
FATTY
SPECIFIC
ISOLATED
RADIOACTIVITIES
FROM
CONTROL
PHOSPHATIDYLCHOLINES
ISOLATED
Mel%
:0 :0 :0 :0 :1 :1
18 18 18
THOSE
FROM
RATS
ARACHIDONOYL OF
MUSCLE
DEFICIENT
IN
5.
8.
ESSEN-
Essential
fatty
acid deficient
at position 2
18
MUSCLF.
Control acid
1
16
OF WITH
ACIDS
Diet Fatty
16
RATS
Relative
specific
Mel%
Relative
radioactivity
20
3 (n-9)
20
4 (n-6)
20
3 (n-9)
20
4 (n-6)
(n-7,
n-9)
20
3 (n-9)
(n-7,
n-9)
20
4 (n-6)
20.9 34.6
specific
radioactivity
1.48
0.33
6.5
0.8
4.0
0.61
3.5
1.6
8.2
1.23
corresponding 5,8,11-eicosatrienoyl species in deficient animals (Table X). With equivalent phosphatidylethanolamines, it was possible to conclude only that the l-stearoyl-2-(5,8,11-eicosatrienoyl) species in deficient ,rats was turning over more rapidly than the corresponding arachidonoyl species in control rats and that the average arachidonoyl phosphatidylethanolamine in deficient rats was turning over three times faster than that in control rats (Table XI). Trewhella and Collins [3] found that the liver phosphatidylcholine with the slowest turnover was the l-stearoyl-2-arachidonoyl species in control rats and the 1-stearoyl-2-( 5,8,11-eicosatrienoyl) species in rats deficient in essential fatty acids. The ratio of the relative specific radioactivities of liver species with the fastest and slowest turnover (1-palmitoyl-2-linoleoyl and l-stearoyl-2-arachidonoyl phosphatidylcholines, respectively) from control animals was 155 : 1, compared with 5.9 : 1 for equivalent species (1-oleoyl-2-linoleoyl and 1-palmitoyl-2-arachidonoyl phosphatidylcholines, respectively) in muscle. The relative specific radioactivity of liver 1-palmitoyl-2-linoleoyl phosphatidylcholine was 5.2 compared with 1.03 for this phosphatidylcholine in the muscle of control rats. Since muscle phosphoglycerides, in contrast to those of liver, are not
TABLE A
XI
COMPARISON
OF
THE
RELATIVE
SPECIFIC
PHOSPHATIDYLETHANOLAMINES ARACHIDONOYL ESSENTIAL
ISOLATED
FROM
PHOSPHATIDYLETHANOLAMINES FATTY
OF
CONTROL
RATS
ISOLATED
acid
~~
Mel%
2
:0
18
: 1 (n-7, :0
ARACHIDONOYL THOSE
RATS
OF
MUSCLE
DEFICIENT
IN
Essential
fatty
acid deficient
at position
1
16
WITH
FROM
Control
Fatty
MUSCLE
ACIDS
Diet
18
RADIOACTIVITIES
20 n-9)
20 20
Relative
specific
Mel%
:4 :4 :4
(n-6) (n-6) (n-6)
1
10.0
0.26
46.7
0.13
Relative
specific
radioactivity
radioactivity
0.15
10.1
0.42
125
exported phosphoglyceride metabolism in muscle reflects the synthesis and degradation of these compounds solely within this tissue. The higher specific radioactivities of muscle phosphatidylcholines compared with those of phosphatidyl~thanol~ines can be explained in terms of the precursor pool sizes and their specific radioactivities (e.g. of cytidine diphosphocholine compared with that of cytidine diphosphoethanolamine) but the pool sixes and the specific radioactivities of these precursors have not been determined. The ratio of the specific radioactivity of the average phosphatidylcholine to that of the average phosphat~dylethanol~~e obtained for control rats (2.7) was close to that for rats deficient in essential fatty acids (1.9). This suggests that the increase observed in specific radioactivities of phosphoglycerides in essential fatty acid deficiency was due to the increased specific radioactivity of a common precursor (i.e. inorganic phosphate) rather than of either of the phosphoglyceride classes. However, within each group, radical changes in composition are imposed by the deficiency in essential fatty acids. Fatty acids of the (n-6) series are replaced by fatty acids of the (n-9) series and an increase in the amount of fatty acids of the (n-7) series also occurs [3]. Collins [8] has suggested that the higher specific radioactivity of liver phosphatidylcholine in rats deficient in essential fatty acids could have been due to an increased rate of
SPEC,F,C
RAo,OACT,“,TY
OF
SERUM
INORGANIC
El E
PHOSPHATE
TAKEN
AS
IO0
PHOSPHORYLCHOLINE
CYTIDINE
DIP~~SPHOCHOLINE
