CHOLESTERYL ESTER METABOLISM IN ATHEROSCLEROTIC ARTERIAL TISSUE Richard W. St. Clair Department of Pathology and The Arteriosclerosis Research Center Bowman Gray School of Medicine Winston-Salem. North Carolina 27103
Associated with the development of atherosclerosis are several alterations in the metabolism of the arterial wall.' Although all of these metabolic changes contribute to the ultimate expression of the disease, their specific role in the pathogenesis of the atherosclerotic lesion is unclear. One of the hallmarks of atherosclerosis is the accumulation of lipids within the arterial wall. This phenomenon is particularly true for cholesterol and cholesteryl esters, which increase to a greater extent in the atherosclerotic artery than does any other single component. In the severely diseased artery, it is not unusual to see as much as a 10-fold increase in the content of unesterified cholesterol and more than a 50-fold increase in the cholesteryl ester content.' Due to this major increase in the cholesteryl ester content of the arterial wall, considerable emphasis has been placed on understanding the mechanism of its accumulation. In this paper, I will review the evidence from our laboratory, and others, that relates to the significance of stimulation of cholesterol esterification in the arterial wall to the pathogenesis of atherosclerosis.
METHODS In the results to be described, four types of experimental approaches to the study of cholesterol esterification have been used. Details of these procedures are included in the references cited; the approaches include, arterial p e r f u s i ~ n , ~cell-free .~ hom~genate,'.~ organ c ~ l t u r e ,and ~ . ~arterial smooth muscle cell in c u l t ~ r e . ~ * ~ Each of these methods has particular advantages and disadvantages, some of which will be pointed out in the following results. Nevertheless, the conclusions drawn from these various approaches are remarkably consistent in suggesting an important role of cholesterol esterification in the pathogenesis of atherosclerosis.
c HOLESTEROL
ESTERIFICATION I N NORMAL AND ATHEROSCLEROTIC ARTERIAL TISSUE
Studies with arterial tissue either perfused or incubated with radiolabeled acetate indicated that arterial tissue could synthesize considerable quantities of fatty acids, with their syntheses directly proportional to the severity of atherosclerosis.z This increase in fatty acid synthesis is located in the area of the atherosclerotic plaque rather than in adjacent normal tissue. More than 95% of these newly synthesized fatty acids are found esterified to phospholipids, triglycerides, and cholesteryl esters. In normal arterial tissue, as shown in TABLE1, typically less than 5-1075 of the newly synthesized fatty acids are
228
St. Clair: Cholesteryl Ester Metabolism
229
esterified to cholesterol, with more than 90% found incorporated into phospholipids and triglycerides. In the atherosclerotic artery, however, there is a major shift in esterification of fatty acids toward a greater esterification to cholesterol, as was first described by Lofland et a1.2.10.11 In severely diseased aortas, for example, as much as 60% of the newly synthesized fatty acids may be found esterified to cholesterol. Such a stimulation in cholesterol esterification occurs in the experimentally induced lesions of several experimental animals (TABLEl), and in the "naturally occurring" atherosclerotic lesions of rnan.I2 Although an increase in the synthesis of phospholipids and triglycerides is often seen in atherosclerotic tissue, the relative extent of this increase is usually much less than for stimulation of cholesterol esterification. In addition, a stimulation in the synthesis of phospholipids and triglycerides is not a prerequisite for increased cholesterol esterification. Increased cholesterol esterification has been shown to occur in TABLE1 ATHEROSCLEROSIS O N INCORPORATION OF [l-"C]ACETATE INTO LIPIDS OF PERFUSED AORTICSEGMENTS
INFLUENCE OF
Rabbit* Control Cholesterol fed
N
Atherosclerosis Index ('70)
12 10
82
White Carneau Pigeons Control 60 Cholesterol fed 42 Squirrel Monkeysq Control Cholesterol fed
4 13
0
0
28 18 35
Cholesteryl Phospholipids 8225 45,300t (5.91
Triglycerides 826 1122
(1.4)
Esters
2750 9700t (3.5)
52,000 124,000t (2.4)
35,000 73,000t (2.1)
3500 37,000t (10.6)
84,288 141,770 (1.7)
84,598 63,649 (0.9)
2827 14,9371. (5.3)
*dpm/mg of DNA after a 4-hr perfusion with [ I-14C]acetate. t p < 0.05 versus control. $Valuesin parentheses represent the relative change from controls. §dpm/mg of wet weight after a 4-hr perfusion with [ l-'*C]acetate. (Results include abdominal and thoracic aorta.
microsomal preparations of arterial tissue in which no change in phospholipid synthesis was seen.13 We have also demonstrated increased cholesterol esterification in both homogenates4 and organ cultures of arterial tissue' in which there was either no change or a decrease in phospholipid and triglyceride syntheses. Further evidence for the independence of stimulation of cholesterol esterification compared with esterification to other lipids is the fact that the former is increased a t a time in the early progression of atherosclerosis when there is no change in the syntheses of phospholipids and triglycerides.14 In the pigeon, for example, as shown in FIGURE1, a significant increase in cholesterol esterification occurs after only 2 weeks of cholesterol feeding, a time at which no grossly visible atherosclerotic lesions are seen.4 In severely atherosclerotic aorta, cholesterol esterification was stimulated up to 50 times that seen in normal controls.
A n n a l s N e w York Academy of Sciences
230
*.
ll*
- 16 -I4
-12
-10
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L
:
-8
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-2
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1
2
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5
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7
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Duration of Cholesterol Feeding (mo.)
F I C ~ ~ 1.R EInfluence of duration of cholesterol feeding on degree of atherosclerosis and rate of cholesterol esterification in cell-free preparation of White Carneau pigeon aorta. Results are
expressed as the change in incorporation of oleic acid into cholesteryl ester, phospholipid, and triglyceride relative to the rate observed in normal control aortas. For experimentaldetails, see Reference 4. Changes in the rate of cholesterol esterification are the result of the enzyme fatty acyl-CoA-cholesterol acyl transferase (ACAT). Although there was also a stimulation in incorporation of oleic acid into phospholipids and triglycerides, it occurred later and to only a fraction of the extent of that seen for cholesterol esterification. Similar results have been reported for rabbit aorta after only 3 days of cholesterol feeding.I5 The site of synthesis of cholesteryl esters within the arterial wall appears to be the smooth muscle cells or foam cells of the atherosclerotic lesion.16-1s The cholesteryl ester composition of foam cells is considerably different from that of the plasma cholesteryl esters, in that the foam cells a r e enriched with cholesteryl oleate a t the expense of cholesteryl lino1eate.19-2’ Presumably, a considerable proportion of the cholesteryl oleate of the foam cell is derived from local synthesis, because cholesteryl oleate synthesis is stimulated to the greatest extent in the atherosclerotic artery.3 Further evidence that a substantial proportion of the cholesteryl esters of the foam cells are derived from synthesis is found in the observation that foam cells nearly disappear from experimentally produced atherosclerotic lesions after removal of the atherogenic diet and the subsequent return to normal plasma cholesterol concentrations. When this procedure is performed, the cholesteryl ester concentration of the atherosclerotic artery decreases, with the decline occurring principally in foam cell cholesteryl esters. Apparently, there is little removal of extracellular cholesterol.22 Upon regression of atherosclerosis, there is a reduction in the rate of cholesterol esterification that parallels the loss of cholesteryl esters from the lesions and the disappearance of foam c e k 5
St. Clair: Cholesteryl Ester Metabolism MECHANISMS OF CHOLESTERYL ESTERSYNTHESIS
IN
23 1
ARTERIAL TISSUE
Several lines of evidence strongly suggest that the sterol portion of the cholesteryl esters that accumulates in the atherosclerotic artery comes from the blood and is not synthesized to any significant extent in the arterial wall. Thus, the synthesis of cholesteryl esters in the arterial wall is the end result of esterification of cholesterol originally derived from the plasma with fatty acids derived either from the plasma or from local synthesis.' The following three mechanisms of cholesterol esterification have been described for arterial tissue: Lecithin-Cholesterol-Fatty Acyl Transferase ( L C AT ) LCAT catalyzes the transfer of 1 mol of fatty acid from the 2 position of lecithin to cholesterol to form cholesteryl ester plus lysolecithin. This enzyme has been reported to be present in arterial tissue from humans and rabbits, with an approximate 50% increase in its activity in atherosclerotic arterial Others, however, have failed to confirm this observation.4~'8~24-26 If LCAT is responsible for the esterification of cholesterol in atherosclerotic arterial tissue, cholesteryl esters should be formed from lecithin labeled in the 2 position with [ I-'4C]oleic acid. It is apparent from the results shown in TABLE 2 that only minimal activity of LCAT was observed. This finding is consistent with the observations of Hashimoto et al.24It seems apparent that if LCAT is present in arterial tissue, its activity is low, as in most tissues,*' and that LCAT probably does not account for a significant proportion of the stimulation in cholesterol esterification that occurs in the atherosclerotic artery. Cholesteryl Ester Synth etase (cholesterol es t erase) Cholesterol esterification in arterial tissue has also been reported to occur at acid pH by an enzyme that catalyzes the esterification of fatty acids to cholesterol. This enzyme does not require cofactors and is thought to be soluble rather than TABLE 2 INCORPORATION OF [l-'4c]oLEIC ACID AND 2-[l-"C~~leO~/]LECITHIN INTO CHOLESTERYL ESTERS BY CELL-FREE PREPARATIONS OF AORTA
Substrate [ 1-"CIOleate [ l-14C]Oleate (1 mM EDTA and 10 pM glutathione)
2-1l-L4C-o/eoyl]Lecithin 2-1 I - 14C-o/eoyl]Lecithin (1 m M EDTA and 10 pM glutathione)
White Carneau Pieeon Rabbit Control Chol-fed Control Chol-fed (dpm/mg of protein/hr) 9859*
36,285
1329
12,59 1
6043 22
32,283 120
1346 2
1 1,348
0
0
7
81
17
*Results are averages of duplicate determinations. For experimental details, see Reference 4.
Annals New York Academy of Sciences
232
membrane bound. An increase in the activity of the acid pH cholesteryl ester synthetase has been reported to occur in atherosclerotic rabbit and rhesus monkey aorta.28Recently, an acid cholesteryl ester hydrolase has been described in arterial tissue and is thought to be of lysosomal origin. This finding, along with the observation that the lysosomal cholesteryl ester hydrolase may be reversiblez9 and under certain conditions in vitro may act as a synthetase rather than as a hydrolase, leaves some question as to whether the acid pH cholesteryl ester synthetase of the arterial wall actually functions as a synthetase in vivo.
Fatty A cy/-CoA-CholesterolAcyl Transferase ( A CA T )
ACAT is a microsomal enzyme that catalyzes the esterification of a coenzyme A-activated fatty acid to cholesterol. I t is a particulate enzyme with a pH optimum of about 7.4. Oleic acid is the fatty acid most readily esterified by ACAT. With fatty acid used as the substrate, esterification requires ATP and coenzyme A and is stimulated by Mg2+.' This preference for esterification ofoleic acid may be of considerable significance to the pathogenesis of atherosclerosis, because cholesteryl oleate is the individual cholesteryl ester that accumulates to the greatest extent in the early atherosclerotic lesion. As is shown in FIGUREI , ACAT may be stimulated nearly 50-fold in atherosclerotic aortas from cholesterol-fed pigeons. Thus, it appears that the bulk of the stimulation in cholesterol esterification that occurs in the atherosclerotic artery is due to ACAT. The mechanism of this stimulation is unclear, however. Hashimoto et a/.*' and Brecher and ChobanianZRhave suggested that the increased esterification of cholesterol is due to increased amounts of enzyme (enzyme induction). Because the microsomal preparations from the atherosclerotic arteries used in these studies contained from 2.5 to 5 times as much cholesterol as did normal microsomes. one
f
$66000 2
. 5
46000
e
8
r o
cholesterol- fed control
r = 0 9 3 Pc.001 dpmCE'vs uq cholesterol
26000
B
B -e
14000
P 10000 P 6000 B
%
40
80
I20
160
200 240 280 500 700 25q aortic scqment
900 1100
PQ iotol cholesterol per
FIGURE2. Influence of extent of atherosclerosis on cholesterol esterification by White Carneau pigeon aorta in organ culture. Aortic segments wereobtained from pigeons fed cholesterol for up to I year and from control birds fed a cholesterol-free grain diet. Arterial segments were maintained in organ culture for 4 days with [ 1,2-3H]cholesterolas the substrate. The severity of atherosclerosis is expressed as the total cholesterol content of the arterial segment.
St. Clair: Cholesteryl Ester Metabolism 7000
233
.
viable
r . 0 . 8 3 P
Associated with the development of atherosclerosis are several alterations in the metabolism of the arterial wall.' Although all of these metabolic changes contribute to the ultimate expression of the disease, their specific role in the pathogenesis of the atherosclerotic lesion is unclear. One of the hallmarks of atherosclerosis is the accumulation of lipids within the arterial wall. This phenomenon is particularly true for cholesterol and cholesteryl esters, which increase to a greater extent in the atherosclerotic artery than does any other single component. In the severely diseased artery, it is not unusual to see as much as a 10-fold increase in the content of unesterified cholesterol and more than a 50-fold increase in the cholesteryl ester content.' Due to this major increase in the cholesteryl ester content of the arterial wall, considerable emphasis has been placed on understanding the mechanism of its accumulation. In this paper, I will review the evidence from our laboratory, and others, that relates to the significance of stimulation of cholesterol esterification in the arterial wall to the pathogenesis of atherosclerosis.
METHODS In the results to be described, four types of experimental approaches to the study of cholesterol esterification have been used. Details of these procedures are included in the references cited; the approaches include, arterial p e r f u s i ~ n , ~cell-free .~ hom~genate,'.~ organ c ~ l t u r e ,and ~ . ~arterial smooth muscle cell in c u l t ~ r e . ~ * ~ Each of these methods has particular advantages and disadvantages, some of which will be pointed out in the following results. Nevertheless, the conclusions drawn from these various approaches are remarkably consistent in suggesting an important role of cholesterol esterification in the pathogenesis of atherosclerosis.
c HOLESTEROL
ESTERIFICATION I N NORMAL AND ATHEROSCLEROTIC ARTERIAL TISSUE
Studies with arterial tissue either perfused or incubated with radiolabeled acetate indicated that arterial tissue could synthesize considerable quantities of fatty acids, with their syntheses directly proportional to the severity of atherosclerosis.z This increase in fatty acid synthesis is located in the area of the atherosclerotic plaque rather than in adjacent normal tissue. More than 95% of these newly synthesized fatty acids are found esterified to phospholipids, triglycerides, and cholesteryl esters. In normal arterial tissue, as shown in TABLE1, typically less than 5-1075 of the newly synthesized fatty acids are
228
St. Clair: Cholesteryl Ester Metabolism
229
esterified to cholesterol, with more than 90% found incorporated into phospholipids and triglycerides. In the atherosclerotic artery, however, there is a major shift in esterification of fatty acids toward a greater esterification to cholesterol, as was first described by Lofland et a1.2.10.11 In severely diseased aortas, for example, as much as 60% of the newly synthesized fatty acids may be found esterified to cholesterol. Such a stimulation in cholesterol esterification occurs in the experimentally induced lesions of several experimental animals (TABLEl), and in the "naturally occurring" atherosclerotic lesions of rnan.I2 Although an increase in the synthesis of phospholipids and triglycerides is often seen in atherosclerotic tissue, the relative extent of this increase is usually much less than for stimulation of cholesterol esterification. In addition, a stimulation in the synthesis of phospholipids and triglycerides is not a prerequisite for increased cholesterol esterification. Increased cholesterol esterification has been shown to occur in TABLE1 ATHEROSCLEROSIS O N INCORPORATION OF [l-"C]ACETATE INTO LIPIDS OF PERFUSED AORTICSEGMENTS
INFLUENCE OF
Rabbit* Control Cholesterol fed
N
Atherosclerosis Index ('70)
12 10
82
White Carneau Pigeons Control 60 Cholesterol fed 42 Squirrel Monkeysq Control Cholesterol fed
4 13
0
0
28 18 35
Cholesteryl Phospholipids 8225 45,300t (5.91
Triglycerides 826 1122
(1.4)
Esters
2750 9700t (3.5)
52,000 124,000t (2.4)
35,000 73,000t (2.1)
3500 37,000t (10.6)
84,288 141,770 (1.7)
84,598 63,649 (0.9)
2827 14,9371. (5.3)
*dpm/mg of DNA after a 4-hr perfusion with [ I-14C]acetate. t p < 0.05 versus control. $Valuesin parentheses represent the relative change from controls. §dpm/mg of wet weight after a 4-hr perfusion with [ l-'*C]acetate. (Results include abdominal and thoracic aorta.
microsomal preparations of arterial tissue in which no change in phospholipid synthesis was seen.13 We have also demonstrated increased cholesterol esterification in both homogenates4 and organ cultures of arterial tissue' in which there was either no change or a decrease in phospholipid and triglyceride syntheses. Further evidence for the independence of stimulation of cholesterol esterification compared with esterification to other lipids is the fact that the former is increased a t a time in the early progression of atherosclerosis when there is no change in the syntheses of phospholipids and triglycerides.14 In the pigeon, for example, as shown in FIGURE1, a significant increase in cholesterol esterification occurs after only 2 weeks of cholesterol feeding, a time at which no grossly visible atherosclerotic lesions are seen.4 In severely atherosclerotic aorta, cholesterol esterification was stimulated up to 50 times that seen in normal controls.
A n n a l s N e w York Academy of Sciences
230
*.
ll*
- 16 -I4
-12
-10
P
I
r3
3 t
L
:
-8
.u)
-6
pu
-4
d
P
e
3
-2
-5
1
2
3
4
5
6
7
8
Duration of Cholesterol Feeding (mo.)
F I C ~ ~ 1.R EInfluence of duration of cholesterol feeding on degree of atherosclerosis and rate of cholesterol esterification in cell-free preparation of White Carneau pigeon aorta. Results are
expressed as the change in incorporation of oleic acid into cholesteryl ester, phospholipid, and triglyceride relative to the rate observed in normal control aortas. For experimentaldetails, see Reference 4. Changes in the rate of cholesterol esterification are the result of the enzyme fatty acyl-CoA-cholesterol acyl transferase (ACAT). Although there was also a stimulation in incorporation of oleic acid into phospholipids and triglycerides, it occurred later and to only a fraction of the extent of that seen for cholesterol esterification. Similar results have been reported for rabbit aorta after only 3 days of cholesterol feeding.I5 The site of synthesis of cholesteryl esters within the arterial wall appears to be the smooth muscle cells or foam cells of the atherosclerotic lesion.16-1s The cholesteryl ester composition of foam cells is considerably different from that of the plasma cholesteryl esters, in that the foam cells a r e enriched with cholesteryl oleate a t the expense of cholesteryl lino1eate.19-2’ Presumably, a considerable proportion of the cholesteryl oleate of the foam cell is derived from local synthesis, because cholesteryl oleate synthesis is stimulated to the greatest extent in the atherosclerotic artery.3 Further evidence that a substantial proportion of the cholesteryl esters of the foam cells are derived from synthesis is found in the observation that foam cells nearly disappear from experimentally produced atherosclerotic lesions after removal of the atherogenic diet and the subsequent return to normal plasma cholesterol concentrations. When this procedure is performed, the cholesteryl ester concentration of the atherosclerotic artery decreases, with the decline occurring principally in foam cell cholesteryl esters. Apparently, there is little removal of extracellular cholesterol.22 Upon regression of atherosclerosis, there is a reduction in the rate of cholesterol esterification that parallels the loss of cholesteryl esters from the lesions and the disappearance of foam c e k 5
St. Clair: Cholesteryl Ester Metabolism MECHANISMS OF CHOLESTERYL ESTERSYNTHESIS
IN
23 1
ARTERIAL TISSUE
Several lines of evidence strongly suggest that the sterol portion of the cholesteryl esters that accumulates in the atherosclerotic artery comes from the blood and is not synthesized to any significant extent in the arterial wall. Thus, the synthesis of cholesteryl esters in the arterial wall is the end result of esterification of cholesterol originally derived from the plasma with fatty acids derived either from the plasma or from local synthesis.' The following three mechanisms of cholesterol esterification have been described for arterial tissue: Lecithin-Cholesterol-Fatty Acyl Transferase ( L C AT ) LCAT catalyzes the transfer of 1 mol of fatty acid from the 2 position of lecithin to cholesterol to form cholesteryl ester plus lysolecithin. This enzyme has been reported to be present in arterial tissue from humans and rabbits, with an approximate 50% increase in its activity in atherosclerotic arterial Others, however, have failed to confirm this observation.4~'8~24-26 If LCAT is responsible for the esterification of cholesterol in atherosclerotic arterial tissue, cholesteryl esters should be formed from lecithin labeled in the 2 position with [ I-'4C]oleic acid. It is apparent from the results shown in TABLE 2 that only minimal activity of LCAT was observed. This finding is consistent with the observations of Hashimoto et al.24It seems apparent that if LCAT is present in arterial tissue, its activity is low, as in most tissues,*' and that LCAT probably does not account for a significant proportion of the stimulation in cholesterol esterification that occurs in the atherosclerotic artery. Cholesteryl Ester Synth etase (cholesterol es t erase) Cholesterol esterification in arterial tissue has also been reported to occur at acid pH by an enzyme that catalyzes the esterification of fatty acids to cholesterol. This enzyme does not require cofactors and is thought to be soluble rather than TABLE 2 INCORPORATION OF [l-'4c]oLEIC ACID AND 2-[l-"C~~leO~/]LECITHIN INTO CHOLESTERYL ESTERS BY CELL-FREE PREPARATIONS OF AORTA
Substrate [ 1-"CIOleate [ l-14C]Oleate (1 mM EDTA and 10 pM glutathione)
2-1l-L4C-o/eoyl]Lecithin 2-1 I - 14C-o/eoyl]Lecithin (1 m M EDTA and 10 pM glutathione)
White Carneau Pieeon Rabbit Control Chol-fed Control Chol-fed (dpm/mg of protein/hr) 9859*
36,285
1329
12,59 1
6043 22
32,283 120
1346 2
1 1,348
0
0
7
81
17
*Results are averages of duplicate determinations. For experimental details, see Reference 4.
Annals New York Academy of Sciences
232
membrane bound. An increase in the activity of the acid pH cholesteryl ester synthetase has been reported to occur in atherosclerotic rabbit and rhesus monkey aorta.28Recently, an acid cholesteryl ester hydrolase has been described in arterial tissue and is thought to be of lysosomal origin. This finding, along with the observation that the lysosomal cholesteryl ester hydrolase may be reversiblez9 and under certain conditions in vitro may act as a synthetase rather than as a hydrolase, leaves some question as to whether the acid pH cholesteryl ester synthetase of the arterial wall actually functions as a synthetase in vivo.
Fatty A cy/-CoA-CholesterolAcyl Transferase ( A CA T )
ACAT is a microsomal enzyme that catalyzes the esterification of a coenzyme A-activated fatty acid to cholesterol. I t is a particulate enzyme with a pH optimum of about 7.4. Oleic acid is the fatty acid most readily esterified by ACAT. With fatty acid used as the substrate, esterification requires ATP and coenzyme A and is stimulated by Mg2+.' This preference for esterification ofoleic acid may be of considerable significance to the pathogenesis of atherosclerosis, because cholesteryl oleate is the individual cholesteryl ester that accumulates to the greatest extent in the early atherosclerotic lesion. As is shown in FIGUREI , ACAT may be stimulated nearly 50-fold in atherosclerotic aortas from cholesterol-fed pigeons. Thus, it appears that the bulk of the stimulation in cholesterol esterification that occurs in the atherosclerotic artery is due to ACAT. The mechanism of this stimulation is unclear, however. Hashimoto et a/.*' and Brecher and ChobanianZRhave suggested that the increased esterification of cholesterol is due to increased amounts of enzyme (enzyme induction). Because the microsomal preparations from the atherosclerotic arteries used in these studies contained from 2.5 to 5 times as much cholesterol as did normal microsomes. one
f
$66000 2
. 5
46000
e
8
r o
cholesterol- fed control
r = 0 9 3 Pc.001 dpmCE'vs uq cholesterol
26000
B
B -e
14000
P 10000 P 6000 B
%
40
80
I20
160
200 240 280 500 700 25q aortic scqment
900 1100
PQ iotol cholesterol per
FIGURE2. Influence of extent of atherosclerosis on cholesterol esterification by White Carneau pigeon aorta in organ culture. Aortic segments wereobtained from pigeons fed cholesterol for up to I year and from control birds fed a cholesterol-free grain diet. Arterial segments were maintained in organ culture for 4 days with [ 1,2-3H]cholesterolas the substrate. The severity of atherosclerosis is expressed as the total cholesterol content of the arterial segment.
St. Clair: Cholesteryl Ester Metabolism 7000
233
.
viable
r . 0 . 8 3 P
