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Effect of estrogens on the concentration and composition of arterial sterols and steryl esters in male White Carneau pigeons
59
Atherosclerosis, 27 (1977) 59-64 0 Elsevier/North-Holland Scientific
Publishers,
Ltd
EFFECT OF ESTROGENS ON THE CONCENTRATION AND COMPOSITION OF ARTERIAL STEROLS AND STERYL ESTERS IN MALE WHITE CARNEAU PIGEONS
M.T.
RAVI
SUBBIAH
Cardiovascular Research Rochester, Minn. 55901 (Received (Revised, (Accepted
and BETTY Unit, Mayo (U.S.A.)
7 July, 1976) received 8 October, 8 October, 1976)
A. DICKE Clinic and Mayo
Foundation,
1976)
Summary The effect of short-term (6 months) administration of conjugated equine estrogen (Premarin) on the content and composition of the aortic sterols in male White Carneau pigeons while they were on a cholesterol-free grain diet was investigated. Estrogen treatment resulted in a 38% increase (P < 0.05) in free sterol concentration, with a 28.8% concomitant decrease (P < 0.05) in the percent of cholesteryl esters. The total sterol concentration remained unchanged: This finding suggests that estrogens might influence the synthetic or hydrolytic (or both) processes that control the concentration of cholesteryl esters in the aorta. Fatty acid composition of steryl esters did not change significantly. The cholesterol content of plasma showed a mild reduction (14%) whereas the triglycerides increased significantly (30%). Key words:
Coronary atherosclerosis -- Estrogen - Myocardial infarclion Steryl esters - Trig’lycerides - White Carneau pigeons
- Sterol
esters ~~
Introduction Estrogens have long been used in the treatment of myocardial infarction and coronary atherosclerosis in humans [1,2], although a recent report [3] has questioned its beneficial effects. Pick et al. [4] demonstrated that, in cholesThis work was supported in part by Research the National Heart and Lung Institute.
Grant HL-14196
for a Specialized
Center
of Research
from
60
terol-fed cockerels, e&radio1 had a protective effect on the development of coronary atherosclerosis whereas aortic atherosclerosis was not affected. Studies by Prichard et al. [5] showed that estrogens decreased the severity of coronary atherosclerosis but not of aortic atherosclerosis in cholesterol-fed White Carneau pigeons. Other studies [6,7] showed that estrogens decreased spontaneous aortic atherosclerosis in young pigeons; in older pigeons, the effect was less pronounced. These authors did notice a decrease in the lipid-stainable material of the atherosclerotic lesions. However, nothing is known regarding the changes in sterol and steryl ester composition of spontaneous atherosclerotic lesions after estrogen treatment in the White Carneau pigeons. This study examines the changes in arterial sterol and steryl ester content in the aorta of male White Carneau pigeons on a cholesterol-free normal grain diet after 6 months of estrogen treatment. Data on the changes in plasma cholesterol and triglycerides after estrogen administration are also reported. White Carneau pigeons of 6 years of age were used for the study. At this age, all the birds of this breed have well-established spontaneous lesions [ 8,9]. Materials
and Methods
The pigeons were obtained from Palmetto Pigeon Plant, Sumter, South Carolina, or from our local colony. The birds were maintained on a cholesterol-free grain diet (Purina Pigeon Chow, Ralston Purina, St. Louis, MO.) during the experiments. Six-year-old male birds were divided into two groups of 20 pigeons each. One group received conjugated equine estrogen (Premarin), 0.252 mg/kg daily dissolved in their drinking water, for a period of 6 months. The control birds received plain drinking water. At the end of 6 months, the birds were killed following barbital anesthesia and after a sample of blood had been taken. The aortas were dissected quickly and the area of the lesion at the celiac branch [lo] of the aorta was removed from all the animals for chemical analysis. Chemical analysis was carried out on three separate pools of aortas (each pool consisting of six or seven birds) in each group of animals. Total lipids from each pool of aortas were extracted with 20 volumes of chloroform : methanol (2 : 1) as described by Folch et al. [ll]. Various lipids were separated by thinlayer chromatography on silica gel G using a solvent system of heptane : isopropyl ether : acetic acid (65 : 40 : 4, v/v/v) as described by Kuksis [ 121. In this solvent system, all the phospholipids stay at the origin. Bands corresponding to standards of free cholesterol, triglycerides, cholesteryl esters, and phospholipids were marked and scraped into vials. The bands corresponding to triglycerides and phospholipids were transmethylated with 10% H,SO, in methanol, and fatty acids were subjected to analysis by gas-liquid chromatoggraphy using methyl heptadecanoate as an internal standard [13]. The steryl ester fraction was saponified with 1 N NaOH in 20% ethanol [13]. The sterol and fatty acid fractions were separated by extraction before and after acidification. The fatty acids were then methylated and quantitated by gas-liquid chromatography as described above. The sterols (both free and esterified) were converted to trifluoroacetate derivatives and separated by gas---liquid chromatography using 501-cholestane
61
TABLE
1
PLASMA
CHOLESTEROL
AND
TRIGLYCERIDE
Body
CONTENT
weight
IN PIGEONS
Cholesterol
Triglycerides
mgjdl
mgldl
f SEM
a
136.0
-L 8.5
b
a
198.8
r 8.4
b
(kg)
f SEM
Control
618
?- 12
205.6
+ 8.8
Estrogen-treated ~-
635
f 16
178.6
+ 5.6
a 1’ < 0.05
for difference
b P < 0.001
for
between
difference
i SEM
groups.
between
groups.
as an internal standard [14]. This column completely separated cholestanol from cholesterol. The sterols were quantitated on the basis of the internal standard peak areas. Gas-liquid chromatography of fatty acid was carried out as described previously [13,15] in a Packard model 407 gas chromatograph on 4-f& 4-mm ID glass columns of 10% EGSS-X on Gas Chrom P (100-120 mesh). Column conditions were as follows: column, 185°C; injector, 240°C; detector, 240°C; and carrier gas, helium (50 ml/min). Sterols were analyzed in Packard Becker model 409 gas chromatograph on 4-ft, 4-mm ID glass columns of 10% SP-2401 on Supelport (100-120 mesh). Column conditions were as follows: oven, 220°C; injector, 220°C; detector, 300°C; and carrier gas, helium (50 ml/min). Fatty acids and sterols were identified by their retention times as compared with those of authentic standards. The plasma cholesterol was measured by the method of Levine and Zak [ 161. Plasma triglycerides were measured by the method of Kessler and Lederer [17] as modified by Ellefson and Caraway [18]. Protein content was determined by the method of Lowry et al. [19]. Results Table 1 shows the concentration of cholesterol and triglycerides in plasma of control and estrogen-treated pigeons. The plasma cholesterol level showed a mild reduction (about 14%) after estrogen treatment, whereas the triglyceride level showed an increase (about 30%) in the estrogen-treated group of pigeons. Chemical analysis of the aorta was then made to determine the content of
TABLE
2
STEROL Pigeon
AND
STERYL
ESTER
CONTENT
OF
PIGEON
AORTAS _~
group
Aortic
sterol
concentration
(mean
+ SEM)
a
-.___ Total
sterols
(mg/mg
Free
protein)
sterols
(mg/mg
protein)
% stery1
% Cholestanol
esters
(% of total
Control
0.21
+ 0.06
0.06
i 0.02
b
78.44
f 8.85
Estrogen-treated __-_
0.25
? 0.02
0.10
* 0.01
b
57.60
f 3.15’
a Mean
value
from
the
analysis
of three
different
b P <
0.05
for difference
between
groups.
’ P <
0.05
for difference
between
groups.
pools
with
aortas
from
six birds
’
in each
1.22
i 0.13
0.53
f 0.23
group.
free sterols)
b Others
a Mean
include
20
of the data
: 1, 22
: 2.
ESTERS
pools
i 1.9
+ 3.3
:0
of aorta
16.1
17.3
16
composition
etc.
acid
STERYL
different
: 0. 22
three
2 0.4
+ 0.5
3.2
3.3
Control
Estrogen-treated
% Fatty
OF
:0
from
COMPOSITION
14
group
ACID
FATTY
Pigeon
3
TABLE
with
(mean
OF
five
5.2
5.7
16
1
a
to seven
+_ 0.2 pigeons
AORTAS
? 0.3
:
? SEM)
PIGEON
9.2
9.0
18 1.5
in each
i 0.8
i
: 0
pool.
27.9
27.8
18
: * 1.0
k 3.4
1
25.1
26.0
18
:
t 1.0
+ 1.1
2
2.9
2.1
20
f 0.9
+ 0.6
:4
10.0
8.8
b
? 2.3
+ 1.7
Others
63
free and esterified sterols (Table 2). No difference in total sterol content was detected between the control and the estrogen-treated pigeons. The content of the free sterols was nearly 38% higher in the estrogen group. This correlated well with the significant reduction (P < 0.05) in the esterified sterol fraction noted in this study. There was no difference in the fatty acid composition of the steryl esters in the two groups (Table 3); this indicated that there is no decrease in any specific cholesteryl ester. Cholestanol, which has previously been found in pigeon aortas [13], showed a slight decrease in concentration its significance in atherosclerosis is still in the estrogen group. However, unknown. Discussion This study has shown that, although estrogens do not cause a change in the total sterol content of the aorta, they do significantly decrease the percent of cholesteryl esters with a resulting increase in free sterol concentration. Cholesteryl esters have been shown to increase during atherogenesis in the White Carneau pigeon [ 131. This investigation supports the earlier histologic study of Hanash et al. [7], wherein a decreased amount of lipid-stainable material in the aorta was observed after Premarin treatment. This might have been due to a decrease in the cholesteryl ester content of the aorta. What effect the longterm (1-2 years) administration of Premarin will have on the arterial sterols remains to be determined. The decrease in cholesteryl ester concentration in the aorta could be due to a decrease in cholesterol esterification or an increase in cholesteryl ester hydrolysis or both. These studies do point to further studies concerning the effect of estrogens on the enzymes responsible for the hydrolysis and synthesis of cholesteryl esters in the aorta. Our previous investigation [20] showed that the pigeon aorta contains at least two cholesteryl ester hydrolase activities (one in the microsomal fraction and the other in the supernatant). Preliminary studies in this laboratory suggest that cholesteryl ester hydrolase activity in the microsomal fraction is higher in estrogen-treated pigeons. Whether the synthesis of cholesteryl esters and the infiltration of cholesteryl esters from plasma is altered following estrogen treatment is not known. Further studies in this direction are needed to pinpoint this mechanism of decrease of cholesteryl esters in the aorta with estrogen treatment. The decrease in plasma cholesterol after estrogen treatment is fairly small, as noted in previous studies [ 6,7]. The plasma triglycerides increased significantly after estrogen treatment in these male pigeons in accordance with the results noted in other species [ 211. Acknowledgement The authors are indebted to Dr. Bruce A. Kottke for his continued support and interest. Thanks are due to Mr. James W. Penner and Miss Laurie K. Vongroven for their assistance during animal sacrifice and dissection of aortas. The authors are indebted to Ayerst Laboratory, New York, for a generous supply of Premarin used in the study.
64
References 1 Stan&r, J., Pick, R., Katz, L.N., Pick, A., Kaplan, B.M.. Berkson, D.M. and Century, D., Effectiveness of estrogens for therapy of myocardial infarction in middle-age men, J. Am. Med. Assoc., 183 (1963) 632. 2 Stamler. J., Pick, R. and Katz, L.N., Experiences in assessing estrogen antiatherogenesis in the chick. the rabbit, and man. Ann. N.Y. Acad. Sci., 64 (1956) 596. 3 Jones, R.J., Klimt, C.R. and Stamler, J., The coronary drug project - A secondary prevention trial. In: F.G. Schettler and A. Weizel (Eds.), Atherosclerosis Ill, Springer-Verlag. Berlin, 1974. pp. 729747. 4 Pick, R., Stamler, J.. Rodbard, S. and Katz, L.N.. The inhibition of coronary atherosclerosis by estrogens in cholesterol-fed chicks, Circulation, 6 (1952) 276. 5 Prichard, R.W.. Clarkson, T.B. and Lofland, H.B.. Estrogen in pigeon atherosclerosis - Estradiol vale&e effects at several dose levels on cholesterol-fed male White Carneau pigeons, Arch. Path., 82 (1966) 15. atherosclerosis 6 Souadjian. J.V., Kottke, B.A. and Titus, J.L., Estrogen effect on spontaneous Experimental studies in White Carneau pigeon, Arch. Path., 85 (1968) 463. 7 Hanash, K.A.. Kottke, B.A., Greene, L.F. and Titus, J.L., Effects of conjugated estrogens on spontaneous atherosclerosis in pigeons. Arch. Path., 93 (1972) 184. 8 Clarkson, T.B., Prichard, R.W.. Netsky, M.G. and Lofland, H.B., Atherosclerosis in pigeons - Its spontaneous occurrence and resemblance to human atherosclerosis, Arch. Path.. 68 (1959) 143. 9 Subbiah, M.T.R.. White Carneau pigeon -- An ideal model for the study of progression and regression of atherosclerosis. Biochem. Exp. Biol., In press. 10 Lauper, N.T., Unni, K.K., Kottke, B.A. and Titus, J.L., Anatomy and histology of aorta of White Carneau pigeon, Lab. Invest., 32 (1975) 536. 11 Folch, J.. Lees, M. and Sloane Stanley, G.H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 12 Kuksis. A.. Quantitative lipid analysis by combined thin layer and gas-liquid chromatographic systems, Chromatogr. Rev., 8 (1966) 172. 13 Subbiah, M.T.R.. Kottke, B.A. and Carlo, LA., Studies in spontaneously atherosclerosis-susceptable and -resistant pigeons - Nature of plasma and aortic sterols. steryl esters, and free fatty acids, lnt. J. Biochem., 5 (1974) 63. 14 Klause, K.A. and Subbiah, M.T.R., Improved resolution of cholestanol and cholesterol by gas-liquid chromatography - Application to pigeon testicular sterols, J. Chromatogr.. 103 (1975) 170. 15 Subbiah, M.T.R., Unni. K.K., Kottke, B.A., Carlo, I.A. and Dinh. D.M., Arterial and metabolic changes during the critical period of spontaneous sterol accumulation in pigeon aorta, Exp. Mol. Path., 24 (1976) 287. 16 Levine, J.B. and Zak. B., Automated determination of serum total cholesterol, Clin. Chim. Acta, 10 (1964) 381. 17 Kessler. G. and Lederer. H., Fluorometric measurement of triglycerides. In: L.T. Skeggs, Jr. (Ed), Technicon Symposia 1965: Automation in Analytical Chemistry. Mediad Inc.. New York. N.Y., 1966, PP. 341-344. 18 Ellefson, R.D. and Caraway, W.C., Lipids in lipoproteins. In: N.W. Teitz (Ed.), Fundamentals of Clinical Chemistry, W.B. Saunders Company, Philadelphia, Pa.. 1976, p. 504. 19 Lowry, O.H., Rosebrough. N.J., Fan, A.L. and Randall. R.J., Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193 (1951) 265. 20 Subbiah, M.T.R. and Dicke, B.A., On the cholesteryl ester hydrolase activity in the microsomal and supernatant fractions of pigeon aorta, Atherosclerosis, 24 (1976) 575. 21 Renaud, S., Thrombosis and atherosclerosis prevention by estrogens in hyperlipemic rats, Atherosclerosis, 12 (1970) 467.
Atherosclerosis, 27 (1977) 59-64 0 Elsevier/North-Holland Scientific
Publishers,
Ltd
EFFECT OF ESTROGENS ON THE CONCENTRATION AND COMPOSITION OF ARTERIAL STEROLS AND STERYL ESTERS IN MALE WHITE CARNEAU PIGEONS
M.T.
RAVI
SUBBIAH
Cardiovascular Research Rochester, Minn. 55901 (Received (Revised, (Accepted
and BETTY Unit, Mayo (U.S.A.)
7 July, 1976) received 8 October, 8 October, 1976)
A. DICKE Clinic and Mayo
Foundation,
1976)
Summary The effect of short-term (6 months) administration of conjugated equine estrogen (Premarin) on the content and composition of the aortic sterols in male White Carneau pigeons while they were on a cholesterol-free grain diet was investigated. Estrogen treatment resulted in a 38% increase (P < 0.05) in free sterol concentration, with a 28.8% concomitant decrease (P < 0.05) in the percent of cholesteryl esters. The total sterol concentration remained unchanged: This finding suggests that estrogens might influence the synthetic or hydrolytic (or both) processes that control the concentration of cholesteryl esters in the aorta. Fatty acid composition of steryl esters did not change significantly. The cholesterol content of plasma showed a mild reduction (14%) whereas the triglycerides increased significantly (30%). Key words:
Coronary atherosclerosis -- Estrogen - Myocardial infarclion Steryl esters - Trig’lycerides - White Carneau pigeons
- Sterol
esters ~~
Introduction Estrogens have long been used in the treatment of myocardial infarction and coronary atherosclerosis in humans [1,2], although a recent report [3] has questioned its beneficial effects. Pick et al. [4] demonstrated that, in cholesThis work was supported in part by Research the National Heart and Lung Institute.
Grant HL-14196
for a Specialized
Center
of Research
from
60
terol-fed cockerels, e&radio1 had a protective effect on the development of coronary atherosclerosis whereas aortic atherosclerosis was not affected. Studies by Prichard et al. [5] showed that estrogens decreased the severity of coronary atherosclerosis but not of aortic atherosclerosis in cholesterol-fed White Carneau pigeons. Other studies [6,7] showed that estrogens decreased spontaneous aortic atherosclerosis in young pigeons; in older pigeons, the effect was less pronounced. These authors did notice a decrease in the lipid-stainable material of the atherosclerotic lesions. However, nothing is known regarding the changes in sterol and steryl ester composition of spontaneous atherosclerotic lesions after estrogen treatment in the White Carneau pigeons. This study examines the changes in arterial sterol and steryl ester content in the aorta of male White Carneau pigeons on a cholesterol-free normal grain diet after 6 months of estrogen treatment. Data on the changes in plasma cholesterol and triglycerides after estrogen administration are also reported. White Carneau pigeons of 6 years of age were used for the study. At this age, all the birds of this breed have well-established spontaneous lesions [ 8,9]. Materials
and Methods
The pigeons were obtained from Palmetto Pigeon Plant, Sumter, South Carolina, or from our local colony. The birds were maintained on a cholesterol-free grain diet (Purina Pigeon Chow, Ralston Purina, St. Louis, MO.) during the experiments. Six-year-old male birds were divided into two groups of 20 pigeons each. One group received conjugated equine estrogen (Premarin), 0.252 mg/kg daily dissolved in their drinking water, for a period of 6 months. The control birds received plain drinking water. At the end of 6 months, the birds were killed following barbital anesthesia and after a sample of blood had been taken. The aortas were dissected quickly and the area of the lesion at the celiac branch [lo] of the aorta was removed from all the animals for chemical analysis. Chemical analysis was carried out on three separate pools of aortas (each pool consisting of six or seven birds) in each group of animals. Total lipids from each pool of aortas were extracted with 20 volumes of chloroform : methanol (2 : 1) as described by Folch et al. [ll]. Various lipids were separated by thinlayer chromatography on silica gel G using a solvent system of heptane : isopropyl ether : acetic acid (65 : 40 : 4, v/v/v) as described by Kuksis [ 121. In this solvent system, all the phospholipids stay at the origin. Bands corresponding to standards of free cholesterol, triglycerides, cholesteryl esters, and phospholipids were marked and scraped into vials. The bands corresponding to triglycerides and phospholipids were transmethylated with 10% H,SO, in methanol, and fatty acids were subjected to analysis by gas-liquid chromatoggraphy using methyl heptadecanoate as an internal standard [13]. The steryl ester fraction was saponified with 1 N NaOH in 20% ethanol [13]. The sterol and fatty acid fractions were separated by extraction before and after acidification. The fatty acids were then methylated and quantitated by gas-liquid chromatography as described above. The sterols (both free and esterified) were converted to trifluoroacetate derivatives and separated by gas---liquid chromatography using 501-cholestane
61
TABLE
1
PLASMA
CHOLESTEROL
AND
TRIGLYCERIDE
Body
CONTENT
weight
IN PIGEONS
Cholesterol
Triglycerides
mgjdl
mgldl
f SEM
a
136.0
-L 8.5
b
a
198.8
r 8.4
b
(kg)
f SEM
Control
618
?- 12
205.6
+ 8.8
Estrogen-treated ~-
635
f 16
178.6
+ 5.6
a 1’ < 0.05
for difference
b P < 0.001
for
between
difference
i SEM
groups.
between
groups.
as an internal standard [14]. This column completely separated cholestanol from cholesterol. The sterols were quantitated on the basis of the internal standard peak areas. Gas-liquid chromatography of fatty acid was carried out as described previously [13,15] in a Packard model 407 gas chromatograph on 4-f& 4-mm ID glass columns of 10% EGSS-X on Gas Chrom P (100-120 mesh). Column conditions were as follows: column, 185°C; injector, 240°C; detector, 240°C; and carrier gas, helium (50 ml/min). Sterols were analyzed in Packard Becker model 409 gas chromatograph on 4-ft, 4-mm ID glass columns of 10% SP-2401 on Supelport (100-120 mesh). Column conditions were as follows: oven, 220°C; injector, 220°C; detector, 300°C; and carrier gas, helium (50 ml/min). Fatty acids and sterols were identified by their retention times as compared with those of authentic standards. The plasma cholesterol was measured by the method of Levine and Zak [ 161. Plasma triglycerides were measured by the method of Kessler and Lederer [17] as modified by Ellefson and Caraway [18]. Protein content was determined by the method of Lowry et al. [19]. Results Table 1 shows the concentration of cholesterol and triglycerides in plasma of control and estrogen-treated pigeons. The plasma cholesterol level showed a mild reduction (about 14%) after estrogen treatment, whereas the triglyceride level showed an increase (about 30%) in the estrogen-treated group of pigeons. Chemical analysis of the aorta was then made to determine the content of
TABLE
2
STEROL Pigeon
AND
STERYL
ESTER
CONTENT
OF
PIGEON
AORTAS _~
group
Aortic
sterol
concentration
(mean
+ SEM)
a
-.___ Total
sterols
(mg/mg
Free
protein)
sterols
(mg/mg
protein)
% stery1
% Cholestanol
esters
(% of total
Control
0.21
+ 0.06
0.06
i 0.02
b
78.44
f 8.85
Estrogen-treated __-_
0.25
? 0.02
0.10
* 0.01
b
57.60
f 3.15’
a Mean
value
from
the
analysis
of three
different
b P <
0.05
for difference
between
groups.
’ P <
0.05
for difference
between
groups.
pools
with
aortas
from
six birds
’
in each
1.22
i 0.13
0.53
f 0.23
group.
free sterols)
b Others
a Mean
include
20
of the data
: 1, 22
: 2.
ESTERS
pools
i 1.9
+ 3.3
:0
of aorta
16.1
17.3
16
composition
etc.
acid
STERYL
different
: 0. 22
three
2 0.4
+ 0.5
3.2
3.3
Control
Estrogen-treated
% Fatty
OF
:0
from
COMPOSITION
14
group
ACID
FATTY
Pigeon
3
TABLE
with
(mean
OF
five
5.2
5.7
16
1
a
to seven
+_ 0.2 pigeons
AORTAS
? 0.3
:
? SEM)
PIGEON
9.2
9.0
18 1.5
in each
i 0.8
i
: 0
pool.
27.9
27.8
18
: * 1.0
k 3.4
1
25.1
26.0
18
:
t 1.0
+ 1.1
2
2.9
2.1
20
f 0.9
+ 0.6
:4
10.0
8.8
b
? 2.3
+ 1.7
Others
63
free and esterified sterols (Table 2). No difference in total sterol content was detected between the control and the estrogen-treated pigeons. The content of the free sterols was nearly 38% higher in the estrogen group. This correlated well with the significant reduction (P < 0.05) in the esterified sterol fraction noted in this study. There was no difference in the fatty acid composition of the steryl esters in the two groups (Table 3); this indicated that there is no decrease in any specific cholesteryl ester. Cholestanol, which has previously been found in pigeon aortas [13], showed a slight decrease in concentration its significance in atherosclerosis is still in the estrogen group. However, unknown. Discussion This study has shown that, although estrogens do not cause a change in the total sterol content of the aorta, they do significantly decrease the percent of cholesteryl esters with a resulting increase in free sterol concentration. Cholesteryl esters have been shown to increase during atherogenesis in the White Carneau pigeon [ 131. This investigation supports the earlier histologic study of Hanash et al. [7], wherein a decreased amount of lipid-stainable material in the aorta was observed after Premarin treatment. This might have been due to a decrease in the cholesteryl ester content of the aorta. What effect the longterm (1-2 years) administration of Premarin will have on the arterial sterols remains to be determined. The decrease in cholesteryl ester concentration in the aorta could be due to a decrease in cholesterol esterification or an increase in cholesteryl ester hydrolysis or both. These studies do point to further studies concerning the effect of estrogens on the enzymes responsible for the hydrolysis and synthesis of cholesteryl esters in the aorta. Our previous investigation [20] showed that the pigeon aorta contains at least two cholesteryl ester hydrolase activities (one in the microsomal fraction and the other in the supernatant). Preliminary studies in this laboratory suggest that cholesteryl ester hydrolase activity in the microsomal fraction is higher in estrogen-treated pigeons. Whether the synthesis of cholesteryl esters and the infiltration of cholesteryl esters from plasma is altered following estrogen treatment is not known. Further studies in this direction are needed to pinpoint this mechanism of decrease of cholesteryl esters in the aorta with estrogen treatment. The decrease in plasma cholesterol after estrogen treatment is fairly small, as noted in previous studies [ 6,7]. The plasma triglycerides increased significantly after estrogen treatment in these male pigeons in accordance with the results noted in other species [ 211. Acknowledgement The authors are indebted to Dr. Bruce A. Kottke for his continued support and interest. Thanks are due to Mr. James W. Penner and Miss Laurie K. Vongroven for their assistance during animal sacrifice and dissection of aortas. The authors are indebted to Ayerst Laboratory, New York, for a generous supply of Premarin used in the study.
64
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