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Depletion of aortic free and ester cholesterol by dietary means in rhesus monkeys with fatty streaks
Atherosclerosis,
21 (1975) 195-203
195
CC) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
DEPLETION
OF
DIETARY
MOHAN STRONG
AORTIC
MEANS
IN
G. KOKATNUR,
Department of%thology,
FREE
AND
RHESUS
GRAY
ESTER
MONKEYS
T. MALCOM,
CHOLESTEROL WITH
DOUGLAS
BY
FATTY
A. EGGEN
STREAKS
JACK
AND
P.
Louisiana State University Medical Center, New Orleans, La. 70112 (U.S.A.)
(Received July 3rd, 1974) (Accepted October 28th, 1974)
SUMMARY
This
report
presents
specific fatty acids esterified
findings
concerning
to cholesterol
genie diet for 12 weeks followed
free and
esterified
in aortas of rhesus monkeys
by regression
regimen
cholesterol
and
fed an athero-
for 32 and 64 weeks.
Mean aortic total cholesterol of monkeys fed an atherogenic diet was more than twice that of the animals on a control diet. Esterified cholesterol showed a fourfold elevation while increase in free cholesterol was less than twofold. Free cholesterol and esterified
cholesterol
in the aorta
of animals
decreased
by about
30%
and
70%
respectively after 32 weeks on the regression diet. Very little additional changes occurred in the animals on the regression regimen for 64 weeks. In fatty acids esterified to cholesterol, the largest proportional increase was in stearic (18 :O) and oleic (18 : 1) acids and the least proportional increase was in linoleic ( 18 :2) and arachidonic (20 :4) acids after 12 weeks on the atherogenic diet. As a result of feeding the regression diet for a period of 32 weeks an overall depletion of about 85 % was observed from the levels in animals fed only an atherogenic diet. Cholesteryl stearate
and oleate
from levels observed
Key words:
returned
to near baseline
after feeding
levels with a reduction
the atherogenic
of about
90%
diet.
Aortic cholesterol - Cholesteryl esters - Fatty streaks - Regression
INTRODUCTION
Armstrong and Meganl demonstrated the effect of cholesterol-free diets on lipid depletion in coronary arteries of rhesus monkeys that were fed an atherogenic diet. This work was supported Heart and Lung Institute.
by U.S. Public Health Service Grant HL-08974 from the National
196
M. G. KOKATNUR, G. T. MALCOM, D. A. EGGEN, J. P. STRONG
Furthermore, Armstrong et al.” showed that dietary treatment caused regression of coronary atherosclerosis in these animals. The mechanism of removal of the deposited lipid, however,
is not clear. qualitative differences in early diet-induced fatty Tucker et al.3 reported streaks in rhesus monkeys after return to a basal diet for 16 weeks, even though grossly visible lesions did not show a striking decrease. In a subsequent experiment involving
a larger
number
of rhesus
monkeys
and longer
periods
of regression,
a
decrease in the extent of grossly visible arterial lesions and changes in cellular and extracellular components of the lesions were observedb. This report presents findings concerning
free and esterified
cholesterol
these rhesus monkeys fed an atherogenic ration for 32 and 64 weeks.
and specific cholesteryl diet for 12 weeks followed
ester fatty acids in by a commercial
MATERIALS AND METHODS
The design of this study is described
in detail elsewhere”.
Briefly, 36 young adult
male rhesus monkeys were fed the atherogenic diet shown in Table I for 12 weeks. After removal of 6 animals with highest and 6 with lowest mean serum cholesterol concentrations during the 12 weeks, the remaining 24 animals (with mean concentration ranging from 294 to 555 mg/dl) were divided into sextiles of four animals each. The animals within each block were assigned randomly to one of four groups, such that all groups
had similar
12 weeks on the atherogenic
mean serum cholesterol
levels (444-447mg/dl)
during
diet. Three of these groups (to be referred to as groups
the I,
11 and III) were selected randomly to be used in the present study. These 18 animals were returned to the basal commercial diet after the I?-week atherogenic diet period. Two additional groups of three animals each (groups IV and V) which had been selected randomly prior to beginning the 12-week atherogenic diet period, were fed only the
TABLE 1 COMPOSITION
OF THE ATHEROGENIC
Ingredients
Monkey chow” Casein Hegsted salt mixture Vitamin fortification mixture Butter (unsalted) Beef tallow Cholesterol (U&P.) Water Total
DlET
g/l00
g
52.55 8.50 1.10 0.50 15.00 4.50 0.35 17.50 100.00
8 Baked primate ration, Dietrich & Gambrill, Frederick, Md.
DEPLETION
TABLE LIPID
OF AORTIC
CHOLESTEROL
IN RHESUS
MONKEYS
197
2 ANALYSIS
OF BASAL
AND ATHEROtiENIC
DIET
Diet hnstrl
Total lipid Cholesterol /l-Sitosterol Fatty acids l2:O l4:O l6:O l6:l l8:O 18:l l8:2 l8:3
(m&g) (mg/g) (mg/g) (7: of total)
nthrrogmic
I3 0.02 0.45 T;’ T 21.8
164 3.4 0.23
1.3 18.8 54.2 3.9
2.2 9.8 31.8 1.4 17.9 30.4 6.5 0.4
100.0
100.0
T
Trace (-. 0.5%)
x T z
basal commercial
diet to serve as control
animals.
Lipid analysis
of the atherogenic
and basal diets is shown in Table 2. The animals housed in individual cages were offered food once every day in an amount that was just sufficient to maintain normal growth. Blood samples of the overnight-fasted animals were obtained by venipuncture under sedation with phencyclidine hydrochloride. Monkeys
from group I were autopsied
which time the mean serum cholesterol still about lesions Groups
level had dropped
twice the basal level. This group provided
existing
just prior to the period
II and III were maintained
respectively
2 weeks after returning
before being autopsied.
to 226 mg/dl,
a measure
when regression
which was
of the extent
was expected
on the basal diet for periods Three animals
to basal diet, at of the
to commence.
of 32 and 64 weeks
from diet control-group
IV were
autopsied at the same time as group I. Animals from diet control-group V were autopsied at the same time as group III. The descending thoracic and abdominal aorta removed at autopsy was carefully stripped of connective tissue and fat. The entire aorta was then divided sagittally into right and left halves. The right side was used for gross and microscopic study”; the left half was frozen and kept at -20°C until chemical analysis was carried out. The thawed aorta was dissected bluntly to separate the intima from the media and adventitia. It was observed that the intimal layer always included a small amount of the media. This layer of intima plus inner media was freeze-dried prior to lipid extraction. The weighed samples were homogenized in a tissue grinder, as described previously5, with a small amount of chloroform-methanol (2: 1) and lipid extracted by the
M. G. KOKATNUR, G. T. MALCOM, D. A. EGGEN, J. P. STRONG
198 method
of Folch et ~11.6. The extracts were evaporated
by means of a vacuum.
Aliquots
of the lipid extract were used to determine the total cholesterol and free cholesterol. Total cholesterol7 (after saponification with alkali) and free cholesterols were determined
by gas-liquid
chromatographic
analysis
of the trimethylsilyl
derivatives
using
cholestane as an internal standard. Esterified cholesterol was obtained by difference. Another aliquot was used to determine the fatty acids esterified to cholesterol. The cholesteryl
esters were separated
from
the other
lipids
on silica Gel G thin-layer
chromatography with a solvent system of petroleum ether-diethyl ether-acetic acid (90:10:l). The cholesteryl esters were then transmethylated with 6 % sulfuric acid in anhydrous
methanol
in Teflon-lined
esters were determined columns.
TABLE
by gas-liquid
screw cap tubes at 80°C for 15 hrs8. The methyl chromatography
on diethyleneglycol-succinate
3
CHOLESTEROL
CONTENT
Group No.
No. of animals
I
4
(MC/G
DRY
WEIGHT)
Weeks on diet athevogenie 12
basal
2
OF RHESUS
AORTAS
Cholesterol in individual animals ~ ester free
Mean by group” free
ester
total
9.6 13.9 13.9 14.9 25.1 20.4
9.55 (0.89)
6.75 (1.42)
16.30 (2.26)
II.0
3.3 5.4 5.0 4.9 12.5 9.4
6.3 8.5 8.9 10.0 12.6
II
6
12
32
7.1 6.6 7.8 7.2 5.7 6.7
1.3 2.0 2.5 2.1 1.8 3.0
8.4 8.6 10.4 9.3 7.5 9.7
6.85 (0.29)
2.12 (0.24)
8.98 (0.42)
III
6
12
64
5.9 6.4 6.3 5.8 8.3 5.4
1.3 1.7 1.3 1.1 3.6 2.6
7.2 8.1 7.6 6.9 11.9 8.0
6.35 (0.42)
I .93 (0.40)
8.28 (0.75)
IV
3
0
14
4.6 6.6 5.6
1.4 2.0 0.8
6.0 8.6 6.4
5.60 (0.58)
1.40 (0.35)
7.00 (0.81)
V
3
0
76
5.7 6.7 6.0
I .o 1.6 1.4
6.7 8.3 7.4
6.13 (0.30)
1.33 (0.18)
7.47 (0.46)
B Figures in parentheses represent S.E.M.
DEPLETION OF AORTIC CHOLESTEROL IN RHESUS MONKEYS
199
RESULTS
Cholesterol content of the aorta The cholesterol mental
groups
content
is shown
of the aortic intima-media
in Table
3. The aortic
preparations
lipid in groups
from the experi-
IV and V represent
control levels in animals fed only the basal diet. Aortic lipids in group I represent baseline lesions induced after 12 weeks on the atherogenic diet. Results from groups II and III show the effect of 32 or 64 weeks of the basal diet on the experimentallyinduced aortic lesions. At the end of the atherogenic
diet period,
mean
aortic
total
cholesterol
of
monkeys in group I was more than twice that of the diet controls (group IV or V). The increase in free cholesterol was less than two-fold, while esterified cholesterol showed more than a fourfold elevation. There was a mean net increase of 3.4 mg/g free cholesterol and 5.4 mg/g of esterified
cholesterol.
After 32 weeks on the basal diet (group II), the mean aortic total cholesterol decreased by about 45% from the level in group 1. Free cholesterol decreased by approximately
30”/,, whereas
this period of regression. cholesterol was observed (group III), these changes
esterified
cholesterol
decreased
by about
CholesteryI ester fatty acids in the aorta The mean values of the major fatty acids esterified to cholesterol TABLE
70% during
Although a slight reduction in free and esterified aortic during an additional 32-week period on the basal diet were not statistically significant.
in the aorta are
4
MASS OF SELECTED
Grortp
CHOLESTERYL
ESTERS
Weeks on diet
athero-
IN AORTAS
OF RHESUS
Micrograms
hasal
MONKEYS”
of cholesteryl
esters/g
dry weight”
16:O
16:l
18:O
18:l
18.2
IO:4
1193 (244)
472 (99)
663 (163)
2516 (652)
1443 (236)
t::,
genie -~
I
6
I2
2
II
6
I2
32
361 (36)
111 (17)
101 (14)
482 (66)
729 (129)
64 (7)
III
6
12
64
372 (76)
130 (29)
103 (8)
429 (93)
644 (180)
48 (10)
IV
3
0
14
230 (60)
83 (26)
III (19)
298 (91)
316 (111)
::,
222 (24)
76 (9)
264 (55)
420 (72)
V
3
0
a Since only selected cholesteryl
76
32 (8)
esters are presented in the table, the total is not quite equal to the chemically determined total aortic ester shown in Table 3. b Figures represent mean; number in parentheses indicates S.E.M.
M. G. KOKATNUR, G. T. MALCOM, D. A. EGGEN, J. P. STRONG
200 presented
in Table 4. There was an absolute
increase
in all major fatty acid fractions
during the 12 weeks on the atherogenic diet (group 1) over the control animals IV and V). Cholesteryl oleate was the ester present in greatest concentration weeks on the diet. The largest (18:0) and oleic (18:l)
proportional
increase
(about
esters and the least proportional
linoleic (18 :2) and arachidonic (20 :4) esters. There was an absolute decrease in all cholesteryl
tenfold)
increase
(groups after 12
was in stearic
(threefold)
was in
ester fatty acids after a 32-week
period on the basal diet (group II). The effect of the basal diet on the cholesteryl
esters
of aortic intima varied for the different fatty acids. As indicated in Table 3, there was an overall depletion of about 70% in total cholesteryl ester concentration after 32 weeks on the regression diet. Cholesteryl stearate and oleate rapidly returned to baseline or near baseline concentration period (Table 4). A reduction
with a net reduction
of 50 % in cholesteryl
of more than 80 % during this
linoleate
and of 25 % in cholesteryl
arachidonate was also observed. There was little change in cholesteryl esters during the additional 32 weeks (compare group II and group III), except for a further small, though
not statistically significant, reduction in linoleic and arachidonic acid esters. The data of Tables 3 and 4 can also be examined in another way. If the concentration of lipid in the aortas of the control groups is considered as a base to which
induced lipid is added, then this induced lipid can be estimated as being the difference between the measured concentration for the 6 animals of the combined control groups (IV and V). These differences have been calculated and the means for groups 1, 11 and III are given in Table 5. When changes for groups (groups IV and V), were calculated mentally-induced
total
I, 11, and 111 from the basal values
from these data, approximately
cholesterol
was depleted
during
82 % of the experi-
the first regression
period.
From the experimentally-induced lipid, approximately 86 % of esterified cholesterol and 74% of free cholesterol was depleted during the 32-weeks regression period. There was little further change in cholesterol content of the aorta during the additional 32 weeks on the regression regimen. It is clear from the data presented in Table 5 that a larger absolute quantity and a much larger fraction
of the induced
cholesteryl
esters than the induced
free chol-
TABLE 5 MEAN
VALUES
FOR INDUCED
MENT
GROUPS
(Ill&
Group
DRY
LIPID
CONCENTRATION
IN AORTIC
INTIMA-MEDIA
OF RHESUS
FROM
3
TREAT-
WEIGHT)‘L
No. of Induced aortic cholesterol a/zimds totml .frw ester
Induced choksteryl rstw of fatty mid 18:O
IS:1
18:2
20:4
0.39 0.58 0.03 0.02 0.05 0.02 ___. iL Units are mg/g dry weight as against pg/g dry weight in Table 4. I) Number of carbon atoms: Number of double bonds.
2.30 0.20 0.15
I .08 0.36 0.28 ~~~~
0.04 0.02 0.005
1
II III
6 6 6
9.07 1.75 I .05
3.68 0.98 0.48
5.38 0.75 0.57
16:O”
0.97 0.14 0.15
16:l
DEPLETION
OF AORTIC
CHOLESTEROL
esterol has been removed
IN RHESUS
MONKEYS
from the intima-media
during
201 the 32- or 64-week regression
periods.
Essentially all of the cholesteryl stearate (I 8 :0) and more than 85 I:;, of the ( 16: 1) and oleic ( I8 : I ) acid induced cholesteryl esters of palmitic (16:0), palmitoleic have been lost by 32 weeks on the regression diet. However, for the polyunsaturated fatty acids, only 50 % (20:4) and 6.5 “/:,(I 8 :2) of the induced cholesteryl esters were lost during
this period.
The change
from group
for total, free, and ester cholesterol
I to group
II was statistically
(P < 0.01) and for all cholesteryl
significant
esters (P -c: 0.05)
with the exception of the ester of arachidonic acid (P > 0. I). The differences in induced free cholesterol or cholesteryl esters between groups II and III were not statistically significant (P : ’ 0.05) for any of these measures.
Rhesus monkeys a great reduction low-fat diet for communication3 when the animals be a time-lag of
with experimentally-induced
lipid deposition
in the aorta show
of aortic cholesterol and its esters when returned to a low-cholesterol, 32 weeks or longer. Chemical analyses not reported in an earlier indicated that there was little. if any, change in aortic cholesterol were returned to a basal diet for only I6 weeks. Thus, there seems to several months before appreciable net loss of the lipid occurs.
In the earlier
study3, changes
in the distribution
of the lipid between
intimal
cells and extracellular tissues were shown to occur as early as 16 weeks. These changes are more striking in animals of the present study that were returned to the basal diet for 32 or 64 weeks. The qualitative changes that occur in distribuition of lipid before net loss of lipid (chemically mechanism changes.
measured)
of lipid removal.
Studies
takes
place may give some clues as to the
are currently
in progress
to investigate
such
In the normal uninvolved aortas of young humans” and in the normal uninvolved arteries of monkeys’0 there is a higher concentration of free cholesterol than esterified cholesterol. With age and development of atherosclerosis, however, this pattern is changed and an increase in the concentration of esterified cholesterol occurs. In human aortas with fatty streaks and advanced lesionsg~ii as well as in animal aortas of esterified cholwith experimentally-induced atherosclerosis i3. the concentration esterol is characteristically higher than that of free cholesterol. Furthermore, there seems to be a preferential accumulation of certain fatty acid esters of cholesterol in the aortic intimag~ii~i~. In the present study there was a grelter increase in esterified cholesterol than free cholesterol in the aortic intima-media after induction of fatty streaks; however, the final concentration of free cholesterol remained greater than the concentration of esterified cholesterol. If the feeding period had been longer and more lesions had been induced, cholesteryl esters would probably have predominated over free cholesterol. Dietary
.fut and cholesteryl esters in lesions
Strong
and McGill13
observed
that the type of dietary
fat influenced
the fatly
M. G. KOKATNUR,
202 acid composition
of aortic lipid during
G. T. MALCOM,
the induction
D. A. EGGEN,
of experimental
J. P. STRONG
atherosclerosis
in baboons. The experimental diet in the present study was high in saturated fatty acids and oleic acid and low in linoleic acid. After the monkeys were fed the atherogenie diet, the largest relative increase over baseline values was observed for esters of the saturated and monounsaturated fatty acids and the lowest relative increase was observed for the esters of the polyunsaturated fatty acids. Therefore, the nature of the dietary fat seemed to be reflected in the fatty acid composition of the aortic lipid. When lipid was depleted
during the 32- and 64-week periods
on basal diet, the reduc-
tion in lipids was most striking in the specific cholesteryl esters which increased the most during induction of experimental lesions. After the 64-week regression the residual lipid had greater concentrations of all major cholesteryl ester fatty acids than the lipid of control
animals.
Depletion of clzolesteryl esters These observations were even more striking basal levels was examined.
Seven-eighths
when the concentration
in excess of
or more of the induced cholesteryl
esters of
saturated and monounsaturated fatty acids were removed during regression whereas only one-half to two-thirds of the induced esters of polyunsaturated fatty acids were removed. Whatever the mechanism for removal of cholesterol, it appears to be most effective for the esters of saturated and monounsaturated fatty acids and less effective for free cholesterol and polyunsaturated fatty acid cholesteryl esters. The selective accumulation and depletion of certain cholesteryl esters in aortic fatty streaks demonstrates the dynamic nature of cholesteryl ester metabolism in this experimental model. It is likely that the type and amount of dietary fat and metabolic activity of the aortic wall influence the selective accumulation of cholesterol and its esters.
Additional
studies
of the time course
occurring at an early stage of lesion regression understanding of the regression process.
of chemical
and
structural
are needed to obtain
changes
a more complete
REFERENCES I ARMSTRONG, M. L. AND MEGAN, M. B., Lipid depletion in atheromatous coronary arteries in Rhesus monkeys after regression diets, Circulut. Res., 30 (1972) 675. 2 ARMSTRONG, M. L., WARNER, E. D. AND CONNOR, W. E., Regression of coronary atheromatosis in Rhesus monkeys, Circulut. Rex, 27 (1970) 59. 3 TUCKER, C. F., CATSULIS, C., STRONG,J. P. AND EGGEN, D. A., Regression of early cholesterol Amer. J. P&z., 65 (1971) 493. induced aortic lesions in Rhesus monkeys, 4 EGGEN,D. A., STRONG,J. P., NEWMAN, III, W. P., CATSLJLIS,C., MALCOM, G. T. AND KOKATNLJR, M. G., Regression of diet-induced fatty streaks in rhesus monkeys, Lab. Invesf., 31 (1974) 294. 5 GEER, J. C. AND GUIDRY, M. A., Cholesteryl intima and fatty streaks,
ester composition
Exp. Molec. Pathol.,
and morphology
of human
normal
3 (1964) 485.
6 FOLCH, J., LEES, M. AND SLOANE-STANLEY, G. H., Simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 7 ABELL, L. L., LEVY, B. B., BRODIE, B. B. AND KENDAL, F. E., A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity, J. Biol. Chetn., 95 (1952) 357. 8 CHRISTIE, W. W., Lipid Analysis, Pergamon, Elmsford, N.Y., 1973.
IIEPLETION OF AORTIC CHOLESTEROL IN RHESUS MONKEYS
203
9 SMITH,E. B., The influence of age and atherosclerosis on the chemistry of aortic intima, J. Athero.scIer. Res., 5 (1965) 224. IO PORTMAN,0. W. AND ALEXANDER,M., Lipid composition of aortic intima plus inner media and other tissue fractions from fetal and adult monkeys, Arch. Bi&zen~. Biophy~‘., 117 (1966) 357. I I SMITH,E. B., EVANS, P. H. AND DOWNHAM,M. D., Lipid in the aortic intima. The correlation of morphological and chemical characteristics, J. Afherosckr. RPS., 7 (1967) 171. 12 PORTMAN,0. W., ALEXANDER,M. AND MARUFFO,C. A.. Nutritional control of arterial lipid composition in squirrel monkeys. Major ester classes and types of phospholipids, J. Nutrition, 91 (1967) 35. 13 STRONG, J. P. AND MCGILL, H. C., Diet and experimental atherosclerosis in baboons, Amer. J. Path..
50 (1967) 669.
21 (1975) 195-203
195
CC) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
DEPLETION
OF
DIETARY
MOHAN STRONG
AORTIC
MEANS
IN
G. KOKATNUR,
Department of%thology,
FREE
AND
RHESUS
GRAY
ESTER
MONKEYS
T. MALCOM,
CHOLESTEROL WITH
DOUGLAS
BY
FATTY
A. EGGEN
STREAKS
JACK
AND
P.
Louisiana State University Medical Center, New Orleans, La. 70112 (U.S.A.)
(Received July 3rd, 1974) (Accepted October 28th, 1974)
SUMMARY
This
report
presents
specific fatty acids esterified
findings
concerning
to cholesterol
genie diet for 12 weeks followed
free and
esterified
in aortas of rhesus monkeys
by regression
regimen
cholesterol
and
fed an athero-
for 32 and 64 weeks.
Mean aortic total cholesterol of monkeys fed an atherogenic diet was more than twice that of the animals on a control diet. Esterified cholesterol showed a fourfold elevation while increase in free cholesterol was less than twofold. Free cholesterol and esterified
cholesterol
in the aorta
of animals
decreased
by about
30%
and
70%
respectively after 32 weeks on the regression diet. Very little additional changes occurred in the animals on the regression regimen for 64 weeks. In fatty acids esterified to cholesterol, the largest proportional increase was in stearic (18 :O) and oleic (18 : 1) acids and the least proportional increase was in linoleic ( 18 :2) and arachidonic (20 :4) acids after 12 weeks on the atherogenic diet. As a result of feeding the regression diet for a period of 32 weeks an overall depletion of about 85 % was observed from the levels in animals fed only an atherogenic diet. Cholesteryl stearate
and oleate
from levels observed
Key words:
returned
to near baseline
after feeding
levels with a reduction
the atherogenic
of about
90%
diet.
Aortic cholesterol - Cholesteryl esters - Fatty streaks - Regression
INTRODUCTION
Armstrong and Meganl demonstrated the effect of cholesterol-free diets on lipid depletion in coronary arteries of rhesus monkeys that were fed an atherogenic diet. This work was supported Heart and Lung Institute.
by U.S. Public Health Service Grant HL-08974 from the National
196
M. G. KOKATNUR, G. T. MALCOM, D. A. EGGEN, J. P. STRONG
Furthermore, Armstrong et al.” showed that dietary treatment caused regression of coronary atherosclerosis in these animals. The mechanism of removal of the deposited lipid, however,
is not clear. qualitative differences in early diet-induced fatty Tucker et al.3 reported streaks in rhesus monkeys after return to a basal diet for 16 weeks, even though grossly visible lesions did not show a striking decrease. In a subsequent experiment involving
a larger
number
of rhesus
monkeys
and longer
periods
of regression,
a
decrease in the extent of grossly visible arterial lesions and changes in cellular and extracellular components of the lesions were observedb. This report presents findings concerning
free and esterified
cholesterol
these rhesus monkeys fed an atherogenic ration for 32 and 64 weeks.
and specific cholesteryl diet for 12 weeks followed
ester fatty acids in by a commercial
MATERIALS AND METHODS
The design of this study is described
in detail elsewhere”.
Briefly, 36 young adult
male rhesus monkeys were fed the atherogenic diet shown in Table I for 12 weeks. After removal of 6 animals with highest and 6 with lowest mean serum cholesterol concentrations during the 12 weeks, the remaining 24 animals (with mean concentration ranging from 294 to 555 mg/dl) were divided into sextiles of four animals each. The animals within each block were assigned randomly to one of four groups, such that all groups
had similar
12 weeks on the atherogenic
mean serum cholesterol
levels (444-447mg/dl)
during
diet. Three of these groups (to be referred to as groups
the I,
11 and III) were selected randomly to be used in the present study. These 18 animals were returned to the basal commercial diet after the I?-week atherogenic diet period. Two additional groups of three animals each (groups IV and V) which had been selected randomly prior to beginning the 12-week atherogenic diet period, were fed only the
TABLE 1 COMPOSITION
OF THE ATHEROGENIC
Ingredients
Monkey chow” Casein Hegsted salt mixture Vitamin fortification mixture Butter (unsalted) Beef tallow Cholesterol (U&P.) Water Total
DlET
g/l00
g
52.55 8.50 1.10 0.50 15.00 4.50 0.35 17.50 100.00
8 Baked primate ration, Dietrich & Gambrill, Frederick, Md.
DEPLETION
TABLE LIPID
OF AORTIC
CHOLESTEROL
IN RHESUS
MONKEYS
197
2 ANALYSIS
OF BASAL
AND ATHEROtiENIC
DIET
Diet hnstrl
Total lipid Cholesterol /l-Sitosterol Fatty acids l2:O l4:O l6:O l6:l l8:O 18:l l8:2 l8:3
(m&g) (mg/g) (mg/g) (7: of total)
nthrrogmic
I3 0.02 0.45 T;’ T 21.8
164 3.4 0.23
1.3 18.8 54.2 3.9
2.2 9.8 31.8 1.4 17.9 30.4 6.5 0.4
100.0
100.0
T
Trace (-. 0.5%)
x T z
basal commercial
diet to serve as control
animals.
Lipid analysis
of the atherogenic
and basal diets is shown in Table 2. The animals housed in individual cages were offered food once every day in an amount that was just sufficient to maintain normal growth. Blood samples of the overnight-fasted animals were obtained by venipuncture under sedation with phencyclidine hydrochloride. Monkeys
from group I were autopsied
which time the mean serum cholesterol still about lesions Groups
level had dropped
twice the basal level. This group provided
existing
just prior to the period
II and III were maintained
respectively
2 weeks after returning
before being autopsied.
to 226 mg/dl,
a measure
when regression
which was
of the extent
was expected
on the basal diet for periods Three animals
to basal diet, at of the
to commence.
of 32 and 64 weeks
from diet control-group
IV were
autopsied at the same time as group I. Animals from diet control-group V were autopsied at the same time as group III. The descending thoracic and abdominal aorta removed at autopsy was carefully stripped of connective tissue and fat. The entire aorta was then divided sagittally into right and left halves. The right side was used for gross and microscopic study”; the left half was frozen and kept at -20°C until chemical analysis was carried out. The thawed aorta was dissected bluntly to separate the intima from the media and adventitia. It was observed that the intimal layer always included a small amount of the media. This layer of intima plus inner media was freeze-dried prior to lipid extraction. The weighed samples were homogenized in a tissue grinder, as described previously5, with a small amount of chloroform-methanol (2: 1) and lipid extracted by the
M. G. KOKATNUR, G. T. MALCOM, D. A. EGGEN, J. P. STRONG
198 method
of Folch et ~11.6. The extracts were evaporated
by means of a vacuum.
Aliquots
of the lipid extract were used to determine the total cholesterol and free cholesterol. Total cholesterol7 (after saponification with alkali) and free cholesterols were determined
by gas-liquid
chromatographic
analysis
of the trimethylsilyl
derivatives
using
cholestane as an internal standard. Esterified cholesterol was obtained by difference. Another aliquot was used to determine the fatty acids esterified to cholesterol. The cholesteryl
esters were separated
from
the other
lipids
on silica Gel G thin-layer
chromatography with a solvent system of petroleum ether-diethyl ether-acetic acid (90:10:l). The cholesteryl esters were then transmethylated with 6 % sulfuric acid in anhydrous
methanol
in Teflon-lined
esters were determined columns.
TABLE
by gas-liquid
screw cap tubes at 80°C for 15 hrs8. The methyl chromatography
on diethyleneglycol-succinate
3
CHOLESTEROL
CONTENT
Group No.
No. of animals
I
4
(MC/G
DRY
WEIGHT)
Weeks on diet athevogenie 12
basal
2
OF RHESUS
AORTAS
Cholesterol in individual animals ~ ester free
Mean by group” free
ester
total
9.6 13.9 13.9 14.9 25.1 20.4
9.55 (0.89)
6.75 (1.42)
16.30 (2.26)
II.0
3.3 5.4 5.0 4.9 12.5 9.4
6.3 8.5 8.9 10.0 12.6
II
6
12
32
7.1 6.6 7.8 7.2 5.7 6.7
1.3 2.0 2.5 2.1 1.8 3.0
8.4 8.6 10.4 9.3 7.5 9.7
6.85 (0.29)
2.12 (0.24)
8.98 (0.42)
III
6
12
64
5.9 6.4 6.3 5.8 8.3 5.4
1.3 1.7 1.3 1.1 3.6 2.6
7.2 8.1 7.6 6.9 11.9 8.0
6.35 (0.42)
I .93 (0.40)
8.28 (0.75)
IV
3
0
14
4.6 6.6 5.6
1.4 2.0 0.8
6.0 8.6 6.4
5.60 (0.58)
1.40 (0.35)
7.00 (0.81)
V
3
0
76
5.7 6.7 6.0
I .o 1.6 1.4
6.7 8.3 7.4
6.13 (0.30)
1.33 (0.18)
7.47 (0.46)
B Figures in parentheses represent S.E.M.
DEPLETION OF AORTIC CHOLESTEROL IN RHESUS MONKEYS
199
RESULTS
Cholesterol content of the aorta The cholesterol mental
groups
content
is shown
of the aortic intima-media
in Table
3. The aortic
preparations
lipid in groups
from the experi-
IV and V represent
control levels in animals fed only the basal diet. Aortic lipids in group I represent baseline lesions induced after 12 weeks on the atherogenic diet. Results from groups II and III show the effect of 32 or 64 weeks of the basal diet on the experimentallyinduced aortic lesions. At the end of the atherogenic
diet period,
mean
aortic
total
cholesterol
of
monkeys in group I was more than twice that of the diet controls (group IV or V). The increase in free cholesterol was less than two-fold, while esterified cholesterol showed more than a fourfold elevation. There was a mean net increase of 3.4 mg/g free cholesterol and 5.4 mg/g of esterified
cholesterol.
After 32 weeks on the basal diet (group II), the mean aortic total cholesterol decreased by about 45% from the level in group 1. Free cholesterol decreased by approximately
30”/,, whereas
this period of regression. cholesterol was observed (group III), these changes
esterified
cholesterol
decreased
by about
CholesteryI ester fatty acids in the aorta The mean values of the major fatty acids esterified to cholesterol TABLE
70% during
Although a slight reduction in free and esterified aortic during an additional 32-week period on the basal diet were not statistically significant.
in the aorta are
4
MASS OF SELECTED
Grortp
CHOLESTERYL
ESTERS
Weeks on diet
athero-
IN AORTAS
OF RHESUS
Micrograms
hasal
MONKEYS”
of cholesteryl
esters/g
dry weight”
16:O
16:l
18:O
18:l
18.2
IO:4
1193 (244)
472 (99)
663 (163)
2516 (652)
1443 (236)
t::,
genie -~
I
6
I2
2
II
6
I2
32
361 (36)
111 (17)
101 (14)
482 (66)
729 (129)
64 (7)
III
6
12
64
372 (76)
130 (29)
103 (8)
429 (93)
644 (180)
48 (10)
IV
3
0
14
230 (60)
83 (26)
III (19)
298 (91)
316 (111)
::,
222 (24)
76 (9)
264 (55)
420 (72)
V
3
0
a Since only selected cholesteryl
76
32 (8)
esters are presented in the table, the total is not quite equal to the chemically determined total aortic ester shown in Table 3. b Figures represent mean; number in parentheses indicates S.E.M.
M. G. KOKATNUR, G. T. MALCOM, D. A. EGGEN, J. P. STRONG
200 presented
in Table 4. There was an absolute
increase
in all major fatty acid fractions
during the 12 weeks on the atherogenic diet (group 1) over the control animals IV and V). Cholesteryl oleate was the ester present in greatest concentration weeks on the diet. The largest (18:0) and oleic (18:l)
proportional
increase
(about
esters and the least proportional
linoleic (18 :2) and arachidonic (20 :4) esters. There was an absolute decrease in all cholesteryl
tenfold)
increase
(groups after 12
was in stearic
(threefold)
was in
ester fatty acids after a 32-week
period on the basal diet (group II). The effect of the basal diet on the cholesteryl
esters
of aortic intima varied for the different fatty acids. As indicated in Table 3, there was an overall depletion of about 70% in total cholesteryl ester concentration after 32 weeks on the regression diet. Cholesteryl stearate and oleate rapidly returned to baseline or near baseline concentration period (Table 4). A reduction
with a net reduction
of 50 % in cholesteryl
of more than 80 % during this
linoleate
and of 25 % in cholesteryl
arachidonate was also observed. There was little change in cholesteryl esters during the additional 32 weeks (compare group II and group III), except for a further small, though
not statistically significant, reduction in linoleic and arachidonic acid esters. The data of Tables 3 and 4 can also be examined in another way. If the concentration of lipid in the aortas of the control groups is considered as a base to which
induced lipid is added, then this induced lipid can be estimated as being the difference between the measured concentration for the 6 animals of the combined control groups (IV and V). These differences have been calculated and the means for groups 1, 11 and III are given in Table 5. When changes for groups (groups IV and V), were calculated mentally-induced
total
I, 11, and 111 from the basal values
from these data, approximately
cholesterol
was depleted
during
82 % of the experi-
the first regression
period.
From the experimentally-induced lipid, approximately 86 % of esterified cholesterol and 74% of free cholesterol was depleted during the 32-weeks regression period. There was little further change in cholesterol content of the aorta during the additional 32 weeks on the regression regimen. It is clear from the data presented in Table 5 that a larger absolute quantity and a much larger fraction
of the induced
cholesteryl
esters than the induced
free chol-
TABLE 5 MEAN
VALUES
FOR INDUCED
MENT
GROUPS
(Ill&
Group
DRY
LIPID
CONCENTRATION
IN AORTIC
INTIMA-MEDIA
OF RHESUS
FROM
3
TREAT-
WEIGHT)‘L
No. of Induced aortic cholesterol a/zimds totml .frw ester
Induced choksteryl rstw of fatty mid 18:O
IS:1
18:2
20:4
0.39 0.58 0.03 0.02 0.05 0.02 ___. iL Units are mg/g dry weight as against pg/g dry weight in Table 4. I) Number of carbon atoms: Number of double bonds.
2.30 0.20 0.15
I .08 0.36 0.28 ~~~~
0.04 0.02 0.005
1
II III
6 6 6
9.07 1.75 I .05
3.68 0.98 0.48
5.38 0.75 0.57
16:O”
0.97 0.14 0.15
16:l
DEPLETION
OF AORTIC
CHOLESTEROL
esterol has been removed
IN RHESUS
MONKEYS
from the intima-media
during
201 the 32- or 64-week regression
periods.
Essentially all of the cholesteryl stearate (I 8 :0) and more than 85 I:;, of the ( 16: 1) and oleic ( I8 : I ) acid induced cholesteryl esters of palmitic (16:0), palmitoleic have been lost by 32 weeks on the regression diet. However, for the polyunsaturated fatty acids, only 50 % (20:4) and 6.5 “/:,(I 8 :2) of the induced cholesteryl esters were lost during
this period.
The change
from group
for total, free, and ester cholesterol
I to group
II was statistically
(P < 0.01) and for all cholesteryl
significant
esters (P -c: 0.05)
with the exception of the ester of arachidonic acid (P > 0. I). The differences in induced free cholesterol or cholesteryl esters between groups II and III were not statistically significant (P : ’ 0.05) for any of these measures.
Rhesus monkeys a great reduction low-fat diet for communication3 when the animals be a time-lag of
with experimentally-induced
lipid deposition
in the aorta show
of aortic cholesterol and its esters when returned to a low-cholesterol, 32 weeks or longer. Chemical analyses not reported in an earlier indicated that there was little. if any, change in aortic cholesterol were returned to a basal diet for only I6 weeks. Thus, there seems to several months before appreciable net loss of the lipid occurs.
In the earlier
study3, changes
in the distribution
of the lipid between
intimal
cells and extracellular tissues were shown to occur as early as 16 weeks. These changes are more striking in animals of the present study that were returned to the basal diet for 32 or 64 weeks. The qualitative changes that occur in distribuition of lipid before net loss of lipid (chemically mechanism changes.
measured)
of lipid removal.
Studies
takes
place may give some clues as to the
are currently
in progress
to investigate
such
In the normal uninvolved aortas of young humans” and in the normal uninvolved arteries of monkeys’0 there is a higher concentration of free cholesterol than esterified cholesterol. With age and development of atherosclerosis, however, this pattern is changed and an increase in the concentration of esterified cholesterol occurs. In human aortas with fatty streaks and advanced lesionsg~ii as well as in animal aortas of esterified cholwith experimentally-induced atherosclerosis i3. the concentration esterol is characteristically higher than that of free cholesterol. Furthermore, there seems to be a preferential accumulation of certain fatty acid esters of cholesterol in the aortic intimag~ii~i~. In the present study there was a grelter increase in esterified cholesterol than free cholesterol in the aortic intima-media after induction of fatty streaks; however, the final concentration of free cholesterol remained greater than the concentration of esterified cholesterol. If the feeding period had been longer and more lesions had been induced, cholesteryl esters would probably have predominated over free cholesterol. Dietary
.fut and cholesteryl esters in lesions
Strong
and McGill13
observed
that the type of dietary
fat influenced
the fatly
M. G. KOKATNUR,
202 acid composition
of aortic lipid during
G. T. MALCOM,
the induction
D. A. EGGEN,
of experimental
J. P. STRONG
atherosclerosis
in baboons. The experimental diet in the present study was high in saturated fatty acids and oleic acid and low in linoleic acid. After the monkeys were fed the atherogenie diet, the largest relative increase over baseline values was observed for esters of the saturated and monounsaturated fatty acids and the lowest relative increase was observed for the esters of the polyunsaturated fatty acids. Therefore, the nature of the dietary fat seemed to be reflected in the fatty acid composition of the aortic lipid. When lipid was depleted
during the 32- and 64-week periods
on basal diet, the reduc-
tion in lipids was most striking in the specific cholesteryl esters which increased the most during induction of experimental lesions. After the 64-week regression the residual lipid had greater concentrations of all major cholesteryl ester fatty acids than the lipid of control
animals.
Depletion of clzolesteryl esters These observations were even more striking basal levels was examined.
Seven-eighths
when the concentration
in excess of
or more of the induced cholesteryl
esters of
saturated and monounsaturated fatty acids were removed during regression whereas only one-half to two-thirds of the induced esters of polyunsaturated fatty acids were removed. Whatever the mechanism for removal of cholesterol, it appears to be most effective for the esters of saturated and monounsaturated fatty acids and less effective for free cholesterol and polyunsaturated fatty acid cholesteryl esters. The selective accumulation and depletion of certain cholesteryl esters in aortic fatty streaks demonstrates the dynamic nature of cholesteryl ester metabolism in this experimental model. It is likely that the type and amount of dietary fat and metabolic activity of the aortic wall influence the selective accumulation of cholesterol and its esters.
Additional
studies
of the time course
occurring at an early stage of lesion regression understanding of the regression process.
of chemical
and
structural
are needed to obtain
changes
a more complete
REFERENCES I ARMSTRONG, M. L. AND MEGAN, M. B., Lipid depletion in atheromatous coronary arteries in Rhesus monkeys after regression diets, Circulut. Res., 30 (1972) 675. 2 ARMSTRONG, M. L., WARNER, E. D. AND CONNOR, W. E., Regression of coronary atheromatosis in Rhesus monkeys, Circulut. Rex, 27 (1970) 59. 3 TUCKER, C. F., CATSULIS, C., STRONG,J. P. AND EGGEN, D. A., Regression of early cholesterol Amer. J. P&z., 65 (1971) 493. induced aortic lesions in Rhesus monkeys, 4 EGGEN,D. A., STRONG,J. P., NEWMAN, III, W. P., CATSLJLIS,C., MALCOM, G. T. AND KOKATNLJR, M. G., Regression of diet-induced fatty streaks in rhesus monkeys, Lab. Invesf., 31 (1974) 294. 5 GEER, J. C. AND GUIDRY, M. A., Cholesteryl intima and fatty streaks,
ester composition
Exp. Molec. Pathol.,
and morphology
of human
normal
3 (1964) 485.
6 FOLCH, J., LEES, M. AND SLOANE-STANLEY, G. H., Simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 7 ABELL, L. L., LEVY, B. B., BRODIE, B. B. AND KENDAL, F. E., A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity, J. Biol. Chetn., 95 (1952) 357. 8 CHRISTIE, W. W., Lipid Analysis, Pergamon, Elmsford, N.Y., 1973.
IIEPLETION OF AORTIC CHOLESTEROL IN RHESUS MONKEYS
203
9 SMITH,E. B., The influence of age and atherosclerosis on the chemistry of aortic intima, J. Athero.scIer. Res., 5 (1965) 224. IO PORTMAN,0. W. AND ALEXANDER,M., Lipid composition of aortic intima plus inner media and other tissue fractions from fetal and adult monkeys, Arch. Bi&zen~. Biophy~‘., 117 (1966) 357. I I SMITH,E. B., EVANS, P. H. AND DOWNHAM,M. D., Lipid in the aortic intima. The correlation of morphological and chemical characteristics, J. Afherosckr. RPS., 7 (1967) 171. 12 PORTMAN,0. W., ALEXANDER,M. AND MARUFFO,C. A.. Nutritional control of arterial lipid composition in squirrel monkeys. Major ester classes and types of phospholipids, J. Nutrition, 91 (1967) 35. 13 STRONG, J. P. AND MCGILL, H. C., Diet and experimental atherosclerosis in baboons, Amer. J. Path..
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