The Lipoprotein Composition of Dog Lymph
By IRVINE H. PAGE. M.D.,
L E N A A . LEWIS, P H . D . , AND GEORGE PLAHL,
M.D.
Deposition of lipids during development of atherosclerosis may come from extracellular fluid rather
than directly from plasma. The lipoprotein composition of lymph thus assumes importance in the
genesis of atherosclerosis. The predominant component found in thoracic duct lymph was the
—Si.21 4 and 7, with lesser amounts of the 23. It was similar qualitatively but not quantitatively
to dog's serum. The bulk of lymph lipoproteins derive from blood plasma. After fat feeding the
absorbed lipid is delivered by the lymph to the blood in large aggregates containing large quantities of — S > 70 component. The > 70 component seemed to reflect changes in lipid transport
in dogs more actively than the relatively more stable —S, 4, 7 and 23 components.
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Dogs weighing from 12 to 25 Kg. were given 300
ml. of milk, 300 ml. vegetable oil and 60 Gm. of
butter two to three hours before operation. General
anesthesia was induced with pentobarbital and the
trachea intubated. The external jugular vein was
exposed and followed down to below the level of
the subclavian vein. The thoracic duct was exposed
at its entrance into the innominate vein, the branches
of the jugular vein were tied off and then the vein
itself occluded just below the junction with the
subclavian vein. A plastic tube 3 mm. in diameter
was inserted through the upper part of the jugular
vein and the wound closed.
Lymph was collected at half-hour or hourly intervals for 12 to 24 horn's and centrifuged one hour
at 30,000 rpm without adjustment of the density.
After removal of the light fatty top layer, because
this interfered with the rest of the ultracentrifuge
pattern, the lymph was brought to a density of 1.21
by addition of potassium bromide as suggested by
Green, Lewis and Page1 and centrifuged in the
preparative centrifuge for 13 hours at 30,000 rpm.
The top 1 ml. was separated and subjected to flotation in the analytic ultracentrifuge. When radioiodine was studied, Geiger counts were also made
of the top 1 ml. and the whole untreated lymph.
Lymph and serum cholesterol were determined
by the method of Abell, Levy, Brodie and Kendall.2
The effect on lymph flow of intravenous or oral
administration of physiologic saline, oral glucose
(50 Gm. in 15 per cent solution) or casein (20 Gm.
in 10 per cent solution) was also studied.
Since there is currently no agreement on nomenclature we employ the abbreviation — S to represent
negative sedimentation or flotation at density 1.21.
Usually the I131 iodinated oil was given by stomach
tube in 100 ml. of vegetable oil. Measurements of
radioactivity were made by Dr. Otto Glasser on 0.5
ml. samples which were dried in stainless steel cups
and counted in an automatic sealer with a shielded
end-window Geiger tube.
From the Research Division of the Cleveland
Clinic Foundation, and the Frank E. Bunts Educational Institute, Cleveland, Ohio.
This investigation was supported in part by a
grant from the National Heart Institute, U. S.
Public Health Service. The radioactive iodine used
was supplied by Oak Ridge National Laboratory on
authorization from the Isotopes Division, U. S.
Atomic Energy Commission.
Received for publication Oct. 16, 1952.
First, the lipoprotein composition of lymph
was compared with that we had previously
found for dog's serum (Lewis and Page3). Dog's
serum contains a — S 23 component with an
average concentration of 44 mg. (range 28 to
75) per 100 ml. and a —S 4 or a —S 4, 7 component in concentrations of 285 mg. (range 235
I
IPOPROTEINS have not been described
as occurring in lymph. Since the compo_Jsition of lymph is similar to that of
extracellular fluid and since it is probably from
the latter that lipids are deposited in tissue to
produce atherosclerosis, its composition is of
interest to investigators of cardiovascular disease.
The ultracentrifuge provided the means of
characterizing the lipoproteins both in lymph
and in serum. They were studied under a variety
of conditions such as after administering I131
iodinated oil, casein, intravenous saline solution and pyrogen.
The similarity of lipoprotein composition of
serum and lymph was established. It is suggested that the lymph lipoproteins originate
chiefly from the blood and that the light coarse
aggregates of the — S > 70 class are less stable
than the — S 4, 7 and 23 components.
EXPERIMENTAL METHODS
RESULTS
S7
Circulation Research, Volume I, January 1953
LIPOPROTEIN OF LYMPH
to 356). The average values of the first samples
of lymph from nine dogs were qualitatively
similar. The — S 23 component was present in
average concentrations of 15 mg. per 100 ml.
(range 7 to 31) and the - S 4, 7 of 128 mg.
(range 33 to 213). Just as in serum, the — S 4
frequently resolved as a double peak at 4 and
7. The concentration of each lipoprotein fraction was usually somewhat less than half that
of serum.
of how much cholesterol is contained in the
top fraction of serum and lymph after centrifugation at 30,000 rpm for one hour. Analysis
for total cholesterol was made of serum and
lymph, the top fatty layer which constituted
about 1 ml. after centrifugation and the remainder of the tube after thorough mixing.
Table 2 shows the results.
As the experiment progressed, less cholesterol
was found in the lymph, although in some
LYMPH
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FIG. 1. Example of ultracentrifuge lipoprotein pattern of normal dog's lymph (upper frames)
and serum (lower frames) at density of 1.21.
When an unusual lipoprotein peak appears
in the serum of an animal, it will probably
also be found in the lymph. For example, in
dog A, (table 1) the unusual — S 9 component
was present in both serum and lymph.
The total concentration of cholesterol of the
first sample of lymph in the fat-fed dogs ranged
from 76 to 100 mg. per 100 ml. Removal of
the fatty top layer during the preliminary
centrifuging reduced this to a range of 16 to
91 mg. This finding brought up the question
experiments the concentration varied little
throughout the period of collection. The rate
of lymph flow was sometimes remarkably constant. For example, in dog B (table 1) it averaged about 12 ml. an hour for 24 hours. In
other dogs (for example, dog C, table 3) after
a few hours it decreased by half or more. The
rate was increased two to three times by intravenous infusion of 100 to 200 ml. of 0.9 per
cent sodium chloride. Despite the volume in-
89
I. H. PAGE, L. A. LEWIS AND G. PLAHL
crease, the concentration of lipoproteins remained almost unchanged (table 1, dog B).
Orally administered glucose, casein Or 0.9
per cent sodium chloride had little effect on
flow rate, lipoprotein pattern or concentration
of the lymph. Any effect they may have exerted
mg. per 24 hours). This is about the ratio of
the two components in serum.
In one animal (dog C, table 3) fever (1.5 G.)
produced by intravenous injection of pyrogen
(Pyromen, 100 or 200 gamma, Baxter Laboratories) was associated with increased lymph
TABLE 1.—Lipoproteins Cholesterol and Radioactivity of Thoracic Duct Lymph
Treatment
lime ol
Collection
Lymph
Vol.
- I
ml.
Radioactivity
count/0.5
ml. lymph
Cholesterol
mg./lOO ml. lymph
Uncentrifuged
After
prelim,
centrifuging
Lipoproteins mg./lOO ml.
Material
-S>70
23
9
7
4
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Dog A.
Thoracic duct cannulated
lymph collection
lymph collection
100 microcuries I131
iodinated oil
lymph collection
lymph collection
lymph collection
lymph collection
lymph collection
Serum
—
66
43
lymph
lymph
71 +
71 +
7
47
59
66
119
33
19
lymph
—
14
59
104
45
lymph
lymph
—
—
19
14
95
57
Serum
47
75
—
—
261
28
30
lymph
lymph
—
12
—
—
83
121*
25
lymph
—
14
—
—
100
120*
27
22
17
19
lymph
lymph
lymph
lymph
lymph
—
—
—
—
—
12
9
7
<7
<7
lymph
lymph
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
95
92
7S
83
52
55
57
10:00-11:00
11:00-12:00
12:00-1:00
1:00
16
14
—
1:00-2:40
2:40-3:38
3:38-4:30
4:30-5:30
5:30-6:30
15
3
13
0.5
1.7
2.0
8
5
2.9
—
4.0
110
119
133
133
91
356+
59
149+
71
127
Dog B.
Thomcic duct cannulated
lymph collection
lymph collection
50 microcuries I131
iodinated oil
lymph collection
lymph collection
lymph collection
lymph collection
lymph collection
lymph collection
lymph collection
lymph collection
lymph collection
200 ml. 0.9% NaCl
i.v.
4:00-5:00
5:00-5:30
5:30-6:00
7:45
12
12
7:45-8:45
8:45-9:45
9:45-11:00
11:00-12:00
12:00-2:00
2:00-3:00
3:00-5:00
5:00-7:00
7:00-8:30
8:30-10:00
11
10
10
10
12
12
12
8
7
O.S
1.3
2.7
8.0
8.6
7.5
2.8
4.8
3.8
115*
14
<7
27
9
7
52
* Serum
would probably be obscured by the "priming"
of the animals with the high fat meal.
The total amount of lipoprotein poured into
the circulation over a period of 24 hours was
calculated from measurements of lymph collected in four dogs over periods varying from
93^ to 24 hours. The — S 23 component was
much smaller (54, 18, 85 and 157 mg. per 24
hours) than the - S 4, 7 (434, 140, 608, 858
flow and decrease in cholesterol and lipoprotein concentration. After about two hours the
temperature returned to normal, along with
the cholesterol and lipoproteins to the control
levels.
Six dogs were given a high fat meal and
approximately six hours later I131 iodinated
oil given. The greatest concentration of radioactive -lipids was observed in the lymph six
90
LIPOPROTEIN OF LYMPH
to eight hours later, chiefly in the top layer
after centrifugation. Only a very small amount
of radioactivity was found in the serum, the
TABLE 2.—Total Cholesterol Content in mg./WO ml. of
"Top" and ' Bottom" Fractions of Serum
Original Dog's Serum
Cholesterol in
mg./lOO ml.
Cholesterol in mg./lOO ml.
Top Fraction
Bottom Fraction
238
90
207
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213
87
118
167
162
132
Dog's lymph
58
SO
44
TABLE
87
108
162
129
158
129
156
135
34
20
11
26
25
25
168
3.—Effects of I
Treatment
m
The lymph of two dogs which had not been
fed the high fat meal was collected. The ultracentrifuge pattern of the first sample showed
much lower concentrations of — S > 70 fraction. After ingestion of 300 ml. of skimmed
milk, a moderate increase occurred within an
hour, but by three to four hours it had greatly
increased. Concurrently the — S 23 and — S
4, 7 components remained unchanged. The
total cholesterol concentration increased about
20 per cent two hours after the administration
of skimmed milk.
Since the — S > 70 fraction had risen sharply
after feeding what was provided as "skimmed
milk," it was desirable to study the problem
further by feeding casein. Twenty grams was
given a dog and the lymph collected for four
hours. As table 4 shows, cholesterol and the
Iodvnated Oil and Pyrogen on Composition of 'Thoracic Duct Lymph
Time of
Collection
Lymph
ml.
Radioactivity
count/0.5
ml. lymph
Cholesterol mg./lOO ml.'
Lymph
unccntrifuged
Serum
Lipoproteins mg./lOO ml.
-S>70
23
9
7
4
Dog C
Lymph collection 100 MC
I131
iod. oil 2:30
100 7 Pyromen
i.v. 9:30
200 y Pyromen
i.v. 11:00
1:20-2:30
12
2:30-3:30
3:30-4:30
4:30-5:30
5:30-6:30
6:30-8:00
8:00-9:00
9:00-10:15
10:15-12:00
12:00-2:00
8:00-9:00
18
25
20
15
10
8
20
12
35
15
9:30-10:00
10:00-10:30
10:30-11:00
17
5
11:00-11:30
11:30-12:00
12:00-12:30
12:30-1:00
—
91
—
—
—
—
—
0.2
—
4.3
108
110
112
112
104
101
126
104
126
66
70
8
39.5*
40.5*
40.0*
11
11
17
17
40.0*
41.0*
40.5*
40.0*
90
75
177
183
194
163
169
169
33
12
24
17St
30
320f
47
36
7
260f
219f
<7
66
38
<1S
<18
9
7
47
41
83
55
7
<10
7
71
12
12
95
64
95
95
78
78
sit
S5t
17
47
38
114
71
28
97
109
• Body temperature, degrees centigrade.
t Separation of —S 4 and —S 7 not clear.
highest count occurring seven to eight hours
after iodinated oil administration. After 12
hours the count of the lymph had decreased
to half the maximum (dog B, table 1).
— S 23, 28 and — S 4 components changed
little but a moderate rise in — S > 70 occurred.
Since the volume of lymph increased, the total
of the cholesterol and lipoproteins carried in
I. H. PAGE, L. A. LEWIS AND G. PLAHL
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the lymph increased about 20 per cent for two
hours following casein ingestion.
Six hours after the skimmed milk, the high
lipid meal was given. The concentration of
— S > 70 increased so greatly that the lymph
had the appearance of heavy cream. For this
reason, preliminary removal of the "top layer"
was necessary to obtain a satisfactory sample
for ultracentrifugal analysis.
Samples collected hourly for eight hours after
the high lipid meal failed to show increase in
concentration of — S 23 and — S 4, 7 components. Total cholesterol increased within two
hours after the fat meal to approximately 40
per cent above the fasting level. As found in
the other animals fed fat, more than half the
TABLT
Lymph
4.—The Effect of Feeding Casein on Lymph
Flow and Lipoprotein Composition
Volume Cholesterol - S >70 23,28
ml. mg./lOOml.
Time
4
mg./WO ml.
Serum
1:15-2
20 gin.
p.m.
2:00-3
3:00-4
4:00-5
5:00-6
3:15
4:45
6:15
p.m. 30
112
casein in H2O at 2
p.m.
p.m.
p.m.
p.m.
67
75
55
35
23
92
107
96
170
170
5
IS
140
12
7
23
58
16
14
14
14
130
140
140
134
185
cholesterol was present in the "top layer" after
centrifugation.
It has never been established that the top
layer after centrifugation really contains lipoprotein. To this end we fed a dog one pint of
heavy cream and after eight hours took a
sample of blood. After spinning the serum one
hour at 30,000 rpm the top 1 ml. was removed.
Analysis showed 360 mg. total N in 100 ml.
and 150 mg. of cholesterol. The remainder of
the 9 ml. of serum was shaken thoroughly and
found to contain 1200 mg. total N and 194
mg. of cholesterol. Thus even in these very
fatty sera the light top layer material contains
some nitrogen and cholesterol.
DISCUSSION
The results show that lymph and serum of
dogs have similar lipoprotein patterns but differ
91
in concentration. Characteristic of both is the
small content of the material which floats rapidly on centrifugation, — S > 70, — S 23 as
compared with the slower floating — S 1 to
10 components. It is evident that after large
fat meals, the absorbed lipid is delivered into
the blood stream by the lymph predominantly
in large aggregates of lipid.
Since most of the radioactive iodine appeared
in the fatty top layer rather than in whole
serum, absorption of iodinated oil probably is
through lymphatics. The small specific activity of the serum is due to removal of the radioactive iodine by lymph, combined with small
absorption through the systemic circulation.
The presence of the highest I131 activity in
the top layer after centrifugation and little
in the more sharply defined lipoprotein components which is characteristic of dog's lymph
(—S 23 and — S 4) indicates that iodinated
011 initially is not transported as lipoprotein.
It is of interest that the top fatty layer of
lymph after preliminary centrifugation contained large amounts of cholesterol; in this it
resembles the serum top fatty layer. Chylomicron material contains only about 3 per cent
of free cholesterol and 97 per cent of neutral
fat according to Swank and Wilmot,4 but when
the entire fatty layer is included much more
total cholesterol is found, especially after feeding high fat meals. It also contains about one
third as much total nitrogen as the residue of
serum after centrifugation. The finding of large
amounts of cholesterol in lymph supports the
view of Biggs, Friedman and Byers5 and Chaikoff and associates6 that exogenous cholesterol
after its intestinal absorption is conveyed into
the systemic circulation by the lymph of the
thoracic duct.
The feeding of casein or skimmed milk was
associated with an increase in the — S > 701
fraction even though the lipid content of the
food was small. But the other lipoprotein components failed to change. Thus it appears that
the relatively more labile portion of the lipoprotein spectrum is a — S > 70 or "fast rising"
component. This is emphasized by feeding a
high fat meal which elicits a great increase in
the — S > 70 fraction but no concurrent increase in — S 23 or 4, 7 components.
92
LIPOPROTEIN OF LYMPH
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The high content of lipoproteins in lymph
seems to us of great interest in relation to the
problem of atherosclerosis.
Data gathered by one of us 7 ' 8 some years
ago showed the striking similarity of lipid
pattern in the blood vessel walls and the
plasma, leaving little doubt that the lipids in
the blood vessel walls were of plasma origin.
Now studying the lipoproteins, the similarity
of serum and lymph is demonstrated.
Since the cells of the blood vessels are probably in contact with plasma modified as extracellular fluid, the deposit probably comes directly from the extracellular fluid rather than
plasma. Analysis of extracellular fluid for lipoprotein has not so far been made. Lymph
resembles it in many respects, hence the finding
of the same lipoprotein pattern in lymph and
serum has added significance. It remains to be
shown whether the atherosclerotic vessel wall
also contains lipoproteins.
A recent communication of Forker, Chaikoff
and Reinhardt9 demonstrates the magnitude
of the circulation of protein from blood to
lymph. Approximately half of the dog's total
plasma protein traverses the thoracic duct in
a day. Even if only a part is bound to lipid,
the large turnover of lipoprotein by the lymph
is evident.
That lymph flow is increased in rats after
ingestion of sodium chloride solution was shown
by Reinhardt and Bloom.10 Further, Glenn,
Bauer and Cresson" observed an increase in
lymph lipids of dogs following fluids by mouth.
The only source of food was glucose or sucrose.
Intravenously administered sodium chloride
solution increases lymph flow, but our data
shows the concentration of lipoprotein remains
almost unchanged. Wasserman and Mayerson12
observed that saline infusions in dogs increases
the rate of disappearance of intravenously injected radioactive iodoalbumin with concurrent increase in albumin return by the lymph.
The infusion seemed merely to mobilize interstitial protein for return to the blood stream.
Further evidence substantiating this view has
been furnished by Shrewsbury and Reinhardt.13 Our results with lipoproteins on the
effect of increasing lymph flow with sodium
chloride solution appear to follow the same
pattern.
SUMMARY
1. Thoracic duct lymph of unfed dogs contains lipoproteins qualitatively similar but
quantitatively smaller than serum. The predominant component was the — S1.21 4 and 7
with lesser amounts of the 23 component.
2. When an unusual lipoprotein peak occurred in serum, it usually appeared concurrently in lymph.
3. The concentration of total cholesterol in
lymph was somewhat less than that in serum
and rose greatly after fat feeding. I131 iodinated
oil feeding resulted in concentration of radioactivity chiefly in the top fatty layer after
preliminary centrifugation.
4. Feeding skimmed milk or casein elicited
a moderate rise in the — S > 70 component of
lymph but little in the more stable — S 4, 7
and 23 components. After a high fat meal the
— S > 70 fraction rose to great heights, but
concurrent rise in the 4, 7 and 23 components
did not occur.
5. Fever produced by injection of bacterial
pyrogen increased lymph flow and decreased
cholesterol and lipoprotein concentration; both
returned to normal after the fever subsided.
6. The rate of lymph flow was usually fairly
constant. Intravenous infusion of salt solution
increased volume with little change in lipoprotein concentration.
7. It is suggested that small amounts of
lymph lipoproteins may be synthesized in tissues drained by the thoracic duct, notably the
intestine, but the bulk of them derive from the
blood plasma. After heavy fat meals, the absorbed lipid is delivered to the blood in large
aggregates containing — S > 70, nitrogen and
cholesterol. Even after such nonlipid substances
as casein feeding, some increase in — S > 70
components may occur.
8. The lipoproteins with flotation greater
than 70 reflect changes in lipid transport and
metabolism in dogs more actively than the
relatively more stable — S 4, 7 and 23 components.
9. The high content of lipoproteins in lymph,
I. H. PAGE, L. A. LEWIS AND G. PLAHL
especially after alimentation, suggests that if
extracellular fluid is of similar composition,
deposition of lipids during development of
atherosclerosis may come directly from it rather
than plasma.
REFERENCES
1
GREEN, A. A., LEWIS, L. A., AND PAGE, I. H.:
A method for the ultracentrifugal analysis of
alpha and beta serum lipoproteins. Federation
Proc. 10: 191, 1951.
2
ABELL, L. L., LEVY, B. B., BRODIE, B. B., AND
KENDALL, F. E.: A simplified method for the
estimation of total cholesterol in serum and
demonstration of its specificity. J. Biol. Chem.
195: 357, 1952.
3
LEWIS, L. A., AND PAGE, I. H.: Ultracentrifuge
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KIYASU, J. Y., REINHARDT, W. 0., DAUBEN,
W. G., AND EASTHAM, J. F.: C-14 cholesterol.
1. Lymphatic transport of absorbed cholesterol4-C-14. J. Biol. Chem. 194: 407, 1952.
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PAGE, I. H.: Some aspects of the nature of the
chemical changes occurring in atheromatosis.
Ann. Int. Med. 14:1741,1941.
8
—: Arteriosclerosis and lipid metabolism. In
Moore, R. A., ed.: Ageing and Degenerative
Diseases. Biological Symposia 11: 43, 1945.
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FORKER, L. L., CHAIKOFF, I. L., AND REINHARDT,
W. 0.: Circulation of plasma proteins—their
transport to lymph. J. Biol. Chem. 197: 625,
1952.
10
REINHARDT, W. 0., AND BLOOM, B.: Voluntarily
ingested sodium chloride as a lymphagogue in
the rat. Proc. Soc. Exper. Biol. & Med. 72:
551, 1949.
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lipoprotein pattern of normal hypertensive and
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SWANK, R. L., AND WILMOT, V.: Chylomicra:
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Am. J. Physiol. 167: 403, 1951.
S. L.: Experimental thoracic duct fistula. Blood
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WASSERMAN, K., AND MAYERSON, H. A.: Mechanism of interstitial protein mobilization by saline
infusions. Federation Proc. 10: 142,1951.
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BIGGS, M. W., FRIEDMAN, M., AND BYERS, S. 0.:
Intestinal lymphatic transport of absorbed cholesterol. Proc. Soc. Exper. Biol. & Med. 78:
641, 1951.
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CHAIKOFF, I. L., BLOOM, B., SIPERSTJSIN, M. D.,
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GLENN, W. W. L., BAUER, F. X., AND CRESSON,
SHREWSBURY, M. S., JR., AND REINHARDT, W. 0.;
Comparative metabolic effects of ingestion of
water or 1 per cent sodium chloride solution in
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J. Physiol. 168: 366, 1952.
The Lipoprotein Composition of Dog Lymph
IRVINE H. PAGE, LENA A. LEWIS and GEORGE PLAHL
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Circ Res. 1953;1:87-93
doi: 10.1161/01.RES.1.1.87
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1953 American Heart Association, Inc. All rights reserved.
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