The
Origin
Phospholipid
of Aortic
in Rabbit
Atheromatosis
By
D. B.
Radioactive
ZILVERSMIT,
phosphorus
aortic
derangement
of
phospholipid
found
itself rather than
to
in
PH.D.,
(P32)
was
MORIS
L.
injected
into
phospholipid
SHORE, B.A. AND
metabolism
normal
was
ACKERMAN,
M.D.
and cholesterol-fed rabbits. A marked
the cholesterol-fed animals. The
found in
the atheromatous
have been
aortas appeared to have been synthesized
deposited there from the plasma.
of atheromatosis has been
THE etiologyfrom
of
different
explored
R. F.
by the
aorta
active phosphorus revealed a major disturbance in the phospholipid metabolism of
the rabbit aorta when atheromata are present.
points
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
view." 2,3 Blood cholesterol has been
investigated intensively and found elevated
in a portion of the patients and in all experimental animals developing this condition.
Plasma phospholipids are usually elevated
when the cholesterol level is high. Some
workers, however, have related the increased
cholesterol-phospholipid ratio to the development of atheromata. More recently attention
has been focused on "physical hyperlipemia,"
that is, the presence of abnormal lipoproteins
and exaggerated chylomicronemia in the
plasma of atherosclerotics.
The increased cholesterol and phospholipid
content of the atheromatous aorta was noted
long ago. More recently Buck and Rossiter
studied the fatty materials of plaques in fresh
human aortas and reported that phospholipids, and, in particular, sphingomyelins constituted an important part of the plaque lipids.4
Chernick and co-workers5 have reported that
the normal rat aorta is capable of synthesizing
fatty acids and phospholipids in vitro but
further relation of this observation to the
mechanism of atherogenesis was not made.
In the present investigation we have attempted to evaluate the role of plasma and
aorta phospholipids in rabbit atheromatosis.
Analysis of specific activity data obtained
from phospholipid molecules labeled with radioFrom the Divisions of Physiology, Preventive
METHODS
White New Zealand rabbits were fed 100 Gm. of
Purina Rabbit Chow per day. In the experimental
animals, the Purina diet was supplemented with 1
Gm. of cholesterol* and 2.8 Gm. of Humko vegetable
fat per day for a period of five months. The total fat
content of the experimental diet amounted to 5.3 per
cent, as compared to 2.5 per cent for the normal diet.
At the end of the five-month period 0.5 millicuries of
radioactive phosphate (P32) was administered intravenously to control and experimental animals.
Blood samples were taken from each animal two,
four and six hours after injection. At the six-hour
interval the animals were sacrificed and the liver and
thoracic aorta were removed for analysis. Plasma
and tissues were analyzed for phospholipid p32 and
p31 by extracting twice for two hours with ethanol
at 60 C. The tissue residue was subjected to an overnight Soxhlet extraction with ethyl ether, and subsequently the alcohol-ether extract was taken almost
to dryness in vacuo under a carbon dioxide atmosphere. The phospholipids were re-extracted with
petroleum ether, and P3 and p31 were determined in
this extract. Where acid-soluble inorganic and
organic phosphates were determined, separate
tissue aliquots were homogenized in 5 cc. of 20 per
cent trichloroacetic acid. After centrifugation, the
supernatant liquid was removed and the inorganic
and organic acid soluble phosphates were separated
by precipitation with magnesium.6 Aortic cholesterol
was determined by the method of Sperry and
Webb.7
RESULTS
The analytic data are given in table 1. Liver
phospholipid concentrations in the experimental animals were not elevated significantly
above control levels, but plasma and aortic
*Kindly donated in part by Merck and Company.
Medicine and Medicine, The University of Tennessee
College of Medicine, Memphis, Tenn.
This study was supported in part by a grant from
the Life Insurance Medical Research Fund.
Mr. Shore is a Predoctoral Fellow of the Life
Insurance Medical Research Fund.
581
Circulation,
Volume
IX, April, 1954
582
ORIGIN OF AORTIC PHOSPHOLIPID IN RABBIT ATHEROMATOSIS
TABLE 1.-Lipids and Acid Soluble Phosphates in Aorta, Liver, and Plasma
Number of
S.E.
Mean ±
,cv*
Animals
Per cent Increase
Over
Cont.
ChoL
Control
Cholesterol-Fed
6
6
9
9
2.6 q- 0.1
0.55 q- 0.09
2.9 q-0.1
0.90 ± 0.10
Aorta
Liver
Plasma
6
6
6
9
9
9
0.542 ± 0.030 1.12 ± 0.079
3.13 ± 0.10 3.59 ± 0.17
0.073 q- 0.004 0.368 ± 0.064
Total Lipid
Cholesterol
Phospholipid
6
6
6
9
9
9
Liver
Aorta
7
3
9
4
116
55.6
Organic Acid Sol. P. Specific Liver
Aorta
Activity X 103:
7
4
9
4
62.6
Phospholipid Specific Activity X 103l
6
6
6
9
9
9
8.05 ± 0.47
5.98 ± 1.00
4.80 ± 0.81
6
9
0.673
Rabbit Wt. (Kg.)
Whole Thoracic Aorta
Control
12
64
(Gm.)
Per cent Phospholipid§
Lipid per Whole Thoracic
Aorta
(mg.)
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Inorganic P. Specific Activity X 103
Liver
Aorta
Plasma
Per cent of Inj. p32 in Phospholipid of Entire
24.7 q- 4.2 89.4
0.746 ± 0.133 27.0
2.84 q- 0.33 10.5
64.4
- 9.1
- 4.8
- 1.7
106
15
404
262
3520
270
± 7.95 155
±28.1
±-18.7
51.0
± 4.72
33.6
-8.27
± 6.55
± 15.6
60.1
58.2
± 5.40
± 3.27
-3.99
-9.62
±
12.1 ± 1.7
10.6 ± 1.8
4.17 ± 0.62
0.131 4.39
±
1.00
50
77
-13
552
Thoracic Aorta X 103
*
S.E.
=
Standard error.
t Specific activity expressed as the percentage of the injected p32 per milligram of phosphorus.
§ Phospholipid P X 25, per 100 Gm. fresh tissue.
phospholipid concentrations were increased
400 per cent and 100 per cent, respectively.
The total amount of phospholipid in the whole
thoracic aorta was actually about four times
greater in the cholesterol-fed animals than in
the control animals. Although the total aortic
cholesterol was increased by an even greater
amount, the data emphasize that phospholipids constitute a significant portion of the
plaque. If the entire increase in aortic lipid
was due to the presence of fatty plaques,
phospholipids account for 14 per cent of the
plaque lipid.*
The p32 data in table 1 are the means of
specific activities, which are calculated as the
percentage of the administered p32 in the
phospholipid fraction per milligram of phosphoThis may not be entirely correct since some increase in adventitial fat may occur in the cholesterol*
fed animals.
lipid phosphorus. This quantity is a relative
the percentage renewal of phospholipid molecules provided the cholesterol feeding does not alter the rate of incorporation of
p32 into the phospholipid precursors. Apparently cholesterol feeding did not affect
these precursors, for the specific activities of
the inorganic and organic acid-soluble phosphates of the liver and aorta are essentially
the same in the experimental and control
measure of
animals.
The data in table 1 indicate that the specific
activity of liver phospholipids was not greatly
affected by the cholesterol feeding. In contrast, the amount of radioactive phospholipid
per cubic centimeter of plasma (specific activity
times concentration) was five times as large
in the cholesterol-fed animals as in the controls. Since all plasma phospholipids are presumably derived from the liver,8 it must be
D. B.
ZILVERSMIT,
M. L. SHORE AND R. F. ACKERMAN
concluded that in the cholesterol-fed rabbits
the
exchange
pholipids is
not
between liver
findings
cholesterol to rats
radioactive
In the aortas
of Perlman and
Specific Activity X
Animal
depressed
of the
in
lipid
pronounced stimulation of phosphosynthesis was observed (bottom row
table
1). Comparison
does
not
adequately
Average
increase in the turnover
since
the
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
thoracic
of
amount
the
phospholipids
phospholipids
in the
in the
an
p32
4.93
9.80
2.96
8.41
1.26
3.58
T
N.
7.60
5.92
1.86
3.44
U
N.
7.43
7.94
1.70
5.40
Average
8.05
5.98
1.72
4.80
S.E.
0.47
1.00
0.28
0.81
fold. The comparison of the specific
activities of the phospholipids in the aorta,
plasma, and liver in table 2 shows that the
six
in the aorta of
specific activity of these
even
animals
cholesterol
the
of
a few
high
of
the
the
than
liver,
specific activity
greater
is
the most active
3.04
1.60
4.14
aorta
normally
3.38
4.70
6.30
indicates
phospholipids of the entire
of
perhaps
synthesis
phospholipid
which is
9.50
8.79
present
lipids
N.
N.
increase in
five to
F
nTea
0.948
N.
portion of atheromatous aortas was
as great as in the normal aortas.
percentage of the injected
8.69
P
four times
But the
N.
great
of these
the
E
R
specific activity
express
103
Aorta
and
liver
and
Plasma
Liver
experimental animals an
of the
Type
the appearance
phospholipids
of Aorta, Liver,
Phospholipids
co-
more
data
Activities
Plasma
plasma.
even
2*--Specific
accelerated. This observation does
confirm the
workers,9 who observed that the administration
of
of
TABLE
and plasma phos-
583
phospholipid
synthesizing tissue in the body. In all instances
in
the specific activity of the
or
the aorta is much higher than the average
terminal specific activity of plasma
This observation strongly suggests that
C
C.F.
17.8
D
C.F.
21.8
9.68
G
C.F.
10.2
7.98
H
C.F.
8.62
J
C.F.
7.14
K
N
C.F.
10.4
C.F.
14.5
C.F.
10.1
0
C.F.
Average
S.E.
*
24.0
1.47
7.49
0.983 4.03
1.92 6.98
1.36
4.58
1.28
4.42
0.917 3.04
1.03
2.79
8.30
0.975
8.80
10.4
8.18
10.5
8.20
0.95312.91
5.54
3.24
12.1
10.6
1.21
4.17
1.7
1.8
0.11
0.62
See footnote accompanying table 1.
C.F.
=
Cholesterol Fed.
N.
=
Normal.
phospholipids
phospho-
lipids.
the major portion of the
atheromatous plaque is
phospholipids
synthesized
in
rather than being derived from plasma
the
situ
in
phos-
pholipids or plasma lipoproteins.
One
might object that an alternate explanais possible. Our results by themmight be compatible with deposition of
tion of the data
selves
plasma
remove
phospholipids
selectively
if
one
the
aorta
were
not
component having a specific activity
of
considerably higher than that
phospholipids but
of the
be
It
pholipid.
plasma
specific activity
would
also
able
to
phospholipid
plasma
the
only
total
exceeding
combined
aortic
very difficult
to
the
phos-
rule
out
possibility completely, since there might
conceivably be present in plasma a particular
of very high specific activity. However, the studies of Turner and co-workers10
this
lipoprotein
indicate that the range of phospholipid specific
activities in the lipoproteins of human plasma
is rather narrow. In addition, our own data*
on the specific activity of plasma lecithin,
cephalin and sphingomyelin of atherosclerotic
rabbits have indicated that none of these fractions have sufficient p32 to account for the high
specific activity of aortic phospholipids on the
basis of deposition.
DISCUSSION
that the amount of phosphoobservation
The
whole
thoracic aorta of the cholipid in the
lesterol-fed rabbit is four times as great as in
control animals confirms the findings of Weinhouse and Hirsch in the rabbit1' and of Buck
and Rossiter in human autopsy material4 that
phospholipids constitute an appreciable fraction of the newly developed atheromatous
*
Unpublished preliminary observations.
584
ORIGIN OF AORTIC PHOSPHOLIPID IN RABBIT ATHEROMATOSIS
plaques. The similarity in composition of the
early plaques to the lipid pattern of plasma
has prompted the idea that the lipids in the
plaque are derived from the plasma lipids.
This theory has received support from the
recent discovery that certain individuals with
a tendency toward atherosclerosis show the
presence of excessive amounts of abnormal
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
lipoproteins in plasma. Even though no direct
proof is available, the possibility that these
abnormal lipoproteins might be the precursors
of atheromatous deposits has been recognized.
Our studies in the rabbit, however, give a
different interpretation to the presence of large
amounts of phospholipids in the atheromatous
deposits. It appears that the atherosclerotic
rabbit has acquired the ability to synthesize
large amounts of phospholipids in the aorta.
The observation that the specific activity of
the phospholipids in the atheromatous plaques
is much higher than the average specific activity of the plasma phospholipids over a period
of six hours, excludes the possibility that an
appreciable fraction of the plaque's phospholipids was derived from the lipoproteins in the
bloodstream.
The relation of these observations to the
process of atherogenesis in rabbits has suggested two theories: (1) The atheromatous
plaque, that is, the cholesterol and phospholipid, are both derived from a local derangement of the lipid metabolism in the aorta.
The abnormal blood lipoproteins are not the
cause, but the result of a similar derangement
of lipid metabolism in the liver. (2) The cholesterol in the plaque is derived from the blood
plasma, but the phospholipids are synthesized
by the aorta. This possibility appears to be
supported by the findings of Biggs and Kritchevsky who observed that orally-administered
labeled cholesterol is found in the plaque of
rabbits whereas labeled water is not converted
to cholesterol in the artery.'2
A possible role of plasma phospholipids in
keeping cholesterol in solution has been postulated by many workers." s One might similarly
speculate that the accelerated phospholipid
synthesis by the aorta is an attempt to solubilize the cholesterol in the atheromatous plaque,
or a reflection of the intense activity of the
granulomatous inflammation
as it reacts to
cholesterol.
Whatever interpretation is put on the data
obtained in the cholesterol-fed rabbits, our
findings indicate that these animals suffer from
an alteration in the metabolism of the aorta.
The mechanism of atherogenesis in man might
be elucidated if a similar situation could be
discovered in the human artery. Our preliminary studies on human arteries have indicated that radioactive phospholipids do accumulate there after p32 administration.
SUMMARY
The incorporation of radioactive phosphorus
(P32) in the liver, plasma, and aorta phospholipid of cholesterol-fed and normal rabbits has
been measured. Increased amounts of phospholipids were found in the plasma and aorta of
the cholesterol-fed animals. Phospholipids in
the atheromatous aortas were synthesized five
times as fast as in the normal aortas. The major
amount of the plaque's phospholipid appeared
to be synthesized by the aorta, rather than
derived from plasma. The bearing of these
findings on atherogenesis is discussed.
ACKNOWLEDGMENT
The authors are indebted to Florence Blevins,
Lovell Lawrence, and Frances Carpenter for their
assistance with the analyses.
SUMARIO ESPAiOL
en
en
La incorporaci6n de f6sforo radioactivo (P32)
el higado, plasma y la fosfolipina de la aorta
conejos alimentados con colesterol ha sido
determinada. Cantidades aumentadas de fosfolipinas fueron encontradas en el plasma y la
aorta de los animales alimentados con colesterol.
Las fosfolipinas en las aortas ateromatosas fu6
sintetizada cinco veces mas rapidamente que
en las aortas normales. La mayor porci6n de la
fosfolipina en placas aparentemente fue sintetizada por la aorta en lugar de ser derivada del
plasma. El significado de estos hallazgos en
cuanto a la aterogenesis se discute.
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The Origin of Aortic Phospholipid in Rabbit Atheromatosis
D. B. ZILVERSMIT, MORIS L. SHORE and R. F. ACKERMAN
Circulation. 1954;9:581-585
doi: 10.1161/01.CIR.9.4.581
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