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Aortic cholesterol esterase in rabbits
Atherosclerosis, 19 (1974) 459-462 9 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
459
A O R T I C C H O L E S T E R O L ESTERASE I N RABBITS EFFECT OF DURATIONOF CHOLESTEROLFEEDING
DAVID KRITCHEVSKY, SHIRLEY A. TEPPER, JANET C. GENZANO ANO HIMANSHU V. KOTHARI Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pa. 19104 and Harrison Department of Surgical Research, University of Pennsylvania, Philadelphia, Pa. 19174 (U.S.A.)
(Received July 23rd, 1973)
SUMMARY The cholesteryl ester synthesizing (S) and hydrolysing (H) activities o f rabbit aorta have been assayed in rabbits fed an atherogenic regimen (2 ~ cholesterol in 6 corn oil). Both synthesis and hydrolysis o f cholesteryl esters increase over control levels, but the increase in synthetase activity is much greater than that o f hydrolase activity. The S/H ratio is sharply increased after 5-7 days on the atherogenic diet, a time when serum and liver cholesterol levels have risen but no atheromata are grossly visible.
Key words: Aortic cholesterol esterase - Experimental atherosclerosis
INTRODUCTION The level of aortic cholesteryl ester increases as the severity of atherosclerosis increases. This phenomenon has been observed in man 1 and in rabbits fed cholesterolcontaining 2,3 or cholesterol-free 4 atherogenic diets. Lofland et al. ~ have demonstrated accelerated cholesterol esterification in atherosclerotic pigeon arteries when compared with normal arteries and a similar effect has been demonstrated in rabbit aorta n,7. Recently St. Clair et al. 8 have reported that the cholesterol-esterifying activity of pigeon aorta becomes evident shortly after institution of cholesterol feeding. This Supported in part by Public Health Service Research grants HL-03299, HL-13722 and Research Career award HL-00734 from the National Heart and Lung Institute, RR-05540 from the Division of Research Resources; the Commonwealth of Pennsylvania, and the John A. Hartford Foundation, Inc.
460
D: K R I T C H E V S K Y , S. A. TEPPER, J. C. G E N Z A N O , H. V. K O T H A R I
effect is reported powders mes as a
seen prior to the appearance of atherosclerotic lesions. We have recently on the assay of cholesteryl ester synthetase and hydrolase activity in acetone o f rat and rabbit aortas 9 and this report describes the activity of these enzyfunction o f duration o f cholesterol feeding.
MATERIALS A N D M E T H O D S
Male rabbits o f the Dutch Belted strain were maintained on a diet of chow augmented with 2 ~ cholesterol and 6 ~ corn oil. At specified intervals, animals were bled by cardiac puncture, killed by cervical dislocation and aortas and livers excised. Serum was analyzed for total cholesterol ~0 and aliquots of liver were dissolved in 15 alc. K O H , the non-saponifiable material extracted with petroleum ether and, after drying over anh. Na2SO4, the extract was analyzed for total cholesterol 10. The aortas were graded for atherosclerotic lesions on a 0--4 scale 11 and frozen in dry ice-acetone. Acetone dry powder of individual aortas was prepared according to our previously published method 9. The enzyme was solubilized by suspending 1 g o f powder in 20 ml of 0.05 M phosphate buffer, p H 6.2, containing 0.05 M NH4CI. The suspension was stirred for 30 rain, then centrifuged at 10,000 • g for 10 rain. The clear supernatant contained 0.4-2.0 mg protein per mllL Synthetic activity was assayed using an emulsion containing 15.5 /~moles o f [4-14C]cholesterol, 46.5 #moles oleic acid, 31.0/zmoles of sodium taurocholate and 100/~moles of NH4C1 in 1.5 ml o f 0.154 M phosphate buffer, p H 6.2. Incubation mixtures contained 0.2 ml o f substrate in 1 ml o f solution. F o r measurement o f hydrolytic activity, a miceIlar substrate containing 1.54 /zmoles o f [4-14C]cholesteryl oleate, 3.75 #moles o f sodium taurocholate and 3.8 #moles o f lecithin in 1 ml o f 0.154 M phosphate buffer, p H 6.6 was prepared. The final incubation mixture (1.0 ml) contained 0.4 ml of substrate preparation. TABLE 1 SERUM AND LIVER LIPIDS, ATHEROSCLEROSISAND AORTIC CHOLESTEROL ESTERASE ACTIVITY IN RABBITS FED 2 ~ CHOLESTEROL IN 6 ~oo CORN OILa
Day
Cholesterol serum (mgldl)
0 5 12 20 30
Atheromata liver (gllO0 g)
49 ~ 6 b 0.52 • 0.05 173 dz 29 1.01 ~z 0.12 495 • 230 2.04 • 0.96 1874 • 316 4.16 • 0.77 925 4= 107 3.73 ~- 0.47
Hydrolase (HJ (nm/mg proteinlhr)
S/H
arch
Synthetase (SJ thoracic (nm/mg proteinlhr)
0 0 0.5 1.0 1.1
0 0 0 1.0 0.5
2.67 4- 0.33 3.33 z~ 0.61 5.15 • 0.75 4.83 + 1.69 4.65 ~ 0.90
1.17 1.68 1.81 1.62 1.73
Four rabbits taken at each time period. b Standard error.
3.13 • 0.43 5.58 • 0.52 9.31 ~ 0.29 7.83 _+ 1.35 8.03 J_ 1.27
461
AORTIC CHOLESTEROL ESTERASE IN RABBITS TABLE 2
SERUM CHOLESTEROL~ AORTIC ATHEROSCLEROSIS AND AORTIC CHOLESTEROL ESTERASEACTIVITYOF RABBITS FED 2 ~ CHOLESTEROL IN 6 ~ CORN OILa
Day
Serumcholesterol Atheromata Synthetase (S) (mg/dl) (nm/mg arch thoracic protein/hr)
Hydrolase(H) (nm/mg protein/hr)
S/H
0 7 11
84 • 8 `0 685 4- 147 506 4- 140
4.98 • 1.27 7.85 • 2.93 3.23 • 0.92
0.72 1.74 2.74
0 0 0
0 0 0
3.60 ~ 0.74 13.65 • 6.75 8.85 -k 2.33
F o u r rabbits t a k e n at each time period. b S t a n d a r d Error.
Incubations were carried out by shaking for 4 hours at 37~ in a Dubnoff metabolic shaker. The reaction was stopped by mixing 50 #1 o f incubation mixture with 50 #1 o f acetone-ethanol (1:1) and warming the mixture. The reaction mixture was spotted on Silica Gel G plates and subjected to Chromatographic development in hexane-ethyl ether-acetic acid, 83:16:1. The areas o f the plate containing free and esterified cholesterol were visualized in iodine vapor and, after sublimation of the iodine, the areas were scraped into scintillation vials and assayed by liquid scintillation spectrometry. Synthesis and hydrolysis of cholesteryl ester is expressed as nmoles converted per mg protein per hour. The labeled substrates were purchased from the New England Nuclear Corp., Boston, Mass. RESULTS AND DISCUSSION i
In the first experiment (Table 1), four rabbits were killed on days 0, 5, 12, 20 and 30 after institution of the atherogenic regimen. In this experiment both aortic arch and thoracic aorta were graded for atherosclerosis but only the thoracic aorta was used for cholesterol esterase assay. A sharp rise in serum and liver cholesterol levels shortly after dietary intake of cholesterol is seen. The rise in serum and liver cholesterol levels by day 5 was 253 ~ and 94 ~ , respectively. The rise in aortic cholesteryl ester synthetase by day 5 is also evident. The level of both hydrolytic and synthetic activity peaked at day 12 and remained close to that level thereafter. Although an aortic cholesteryl ester synthesizing enzyme has not been identified, for the sake o f brevity, we refer to this activity as synthetase. The synthetase/hydrolase ratio (S/H) was 1.17 in the control group. This value corresponds well to the value o f 1:1 observed in our earlier experiments 9. The S/H ratio rose with cholesterol feeding primarily because the rise in synthetase activity was 78 ~ on day 5 and 197 ~ , 150 ~ and 157 ~ at the other three periods. In contrast, hydrolase activity rose by 25 ~ on day 5, 93 ~ on day 12, 81 ~ on day 20 and 74 ~ on day 30. It is noteworthy that cholesteryl ester synthetase activity
462
D. KRITCHEVSKY, S. A. TEPPER, J. C. GENZANO, H. V. KOTHAR!
was a t its p e a k before a n y a t h e r o m a t o u s lesions were seen in the t h o r a c i c a o r t a . I n the second experiment, a n i m a l s were killed at days 0, 7 a n d 11 a n d the entire a o r t a , t h o r a c i c a n d arch, was t a k e n for assay. Results are s h o w n in T a b l e 2. T h e striking rise in serum cholesterol after 7 days o f cholesterol diet is similar to t h a t seen in the first experiment. T h e synthetase activity is increased b y 295 ~o on d a y 7 a n d the h y d r o l a s e activity is increased b y o n l y 58 ~ . A s in the first e x p e r i m e n t , a s h a r p increase in S / H ratio is seen after 7 days o n the atherogenic r e g i m e n (142 ~ ) a n d the increase over s t a r t i n g levels is 281 ~ on the eleventh day. N o lesions were grossly visible in a n y o f the a o r t a s o n either day. O u r findings in r a b b i t s c o m p l e m e n t those o f St. Clair et al. s in pigeons, in t h a t cholesteryl ester synthetase is elevated after a s h o r t p e r i o d o f cholesterol feeding a n d before a n y atherosclerotic lesions are visible. W e have further s h o w n t h a t b o t h aortic cholesteryl ester synthetase (S) a n d h y d r o l a s e (H) increase u p o n cholesterol feeding b u t the S / H r a t i o rises because the rise in synthetic activity is several times higher t h a n t h a t o f h y d r o l y t i c activity.
REFERENCES 1 SMITH, E. B., The influence of age and atherosclerosis on the chemistry of aortic intima, Part 1
(The lipids), J. Atheroscler. Res., 5 (1965) 224. 2 SWELt,,L., LAW, M. D. ANDTREADWELL,C. R., Tissue cholesterol ester and triglyceride fatty acid composition of rabbits fed cholesterol diets high and low in linoleic acid, J. Nutr., 76 (1962) 429. 3 NEWMAN,H. A. I. AND ZILVERSMIT,D. B., Accumulation of lipid and nonlipid constituents in rabbit atheromata, J. Atheroscler. Res., 4 (1964) 261. 4 KRITCHEVSKY,D. AND TEPPER, S. A., Experimental atherosclerosis in rabbits fed cholesterol-free diets: Influence of chow components, J. Atheroscler. Res., 8 (1968) 357. 5 LOFLAND, JR., H. g., MOURY, D. M., HOFFMAN, C. W. AND CLARKSON, T. B., Lipid metabolism in
pigeon aorta during atherogenesis, J. Lipid Res., 6 (1965) 112. 6 HASHIMOTO,S., DAYTON,S. ANDALFIN-SLATER,R. B., Esterification of cholesterol by homogenates of atherosclerotic and normal aortas, Life Sci., 12 (1973) Part 2: 12. 7 KRITCHEVSKY,D., TEPPER, S. A. AND KITAGAWA,M., Experimental atherosclerosis in rabbits fed cholesterol-free diets, Part 3 (Comparison of fructose and lactose with otber carbohydrates), Natr. Reports Int., 7 (1973) 193. 8 ST. CLAIR, R. W., LOFt,AND,H. B. AND CLARKSON,T. B., Influence of duration of cholesterol feeding on esterification of fatty acids by cell-free preparations of pigeon aorta, Circulation Res., 27 (1970) 213. 9 KOTHARI,H. V., MILLER,B. F. AND KRITCHEVSKY,D., Aortic cholesterol esterase: Characteristics of normal rat and rabbit enzyme, Biochim. Biophys. Acts, 296 (1973) 446. 10 PEARSON,S., STERN,S. AND McGAvACK, T. H., A rapid accurate method for the determination of total cholesterol in serum, Analyt. Chem., 25 (1953) 813. 11 DUFF, G. L. AND MCMILLAN,G. C., The effect of alloxan diabetes on experimental cholesterol atherosclerosis in the rabbit, J. Exptl. Med., 89 (1949) 611. 12 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265.
459
A O R T I C C H O L E S T E R O L ESTERASE I N RABBITS EFFECT OF DURATIONOF CHOLESTEROLFEEDING
DAVID KRITCHEVSKY, SHIRLEY A. TEPPER, JANET C. GENZANO ANO HIMANSHU V. KOTHARI Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pa. 19104 and Harrison Department of Surgical Research, University of Pennsylvania, Philadelphia, Pa. 19174 (U.S.A.)
(Received July 23rd, 1973)
SUMMARY The cholesteryl ester synthesizing (S) and hydrolysing (H) activities o f rabbit aorta have been assayed in rabbits fed an atherogenic regimen (2 ~ cholesterol in 6 corn oil). Both synthesis and hydrolysis o f cholesteryl esters increase over control levels, but the increase in synthetase activity is much greater than that o f hydrolase activity. The S/H ratio is sharply increased after 5-7 days on the atherogenic diet, a time when serum and liver cholesterol levels have risen but no atheromata are grossly visible.
Key words: Aortic cholesterol esterase - Experimental atherosclerosis
INTRODUCTION The level of aortic cholesteryl ester increases as the severity of atherosclerosis increases. This phenomenon has been observed in man 1 and in rabbits fed cholesterolcontaining 2,3 or cholesterol-free 4 atherogenic diets. Lofland et al. ~ have demonstrated accelerated cholesterol esterification in atherosclerotic pigeon arteries when compared with normal arteries and a similar effect has been demonstrated in rabbit aorta n,7. Recently St. Clair et al. 8 have reported that the cholesterol-esterifying activity of pigeon aorta becomes evident shortly after institution of cholesterol feeding. This Supported in part by Public Health Service Research grants HL-03299, HL-13722 and Research Career award HL-00734 from the National Heart and Lung Institute, RR-05540 from the Division of Research Resources; the Commonwealth of Pennsylvania, and the John A. Hartford Foundation, Inc.
460
D: K R I T C H E V S K Y , S. A. TEPPER, J. C. G E N Z A N O , H. V. K O T H A R I
effect is reported powders mes as a
seen prior to the appearance of atherosclerotic lesions. We have recently on the assay of cholesteryl ester synthetase and hydrolase activity in acetone o f rat and rabbit aortas 9 and this report describes the activity of these enzyfunction o f duration o f cholesterol feeding.
MATERIALS A N D M E T H O D S
Male rabbits o f the Dutch Belted strain were maintained on a diet of chow augmented with 2 ~ cholesterol and 6 ~ corn oil. At specified intervals, animals were bled by cardiac puncture, killed by cervical dislocation and aortas and livers excised. Serum was analyzed for total cholesterol ~0 and aliquots of liver were dissolved in 15 alc. K O H , the non-saponifiable material extracted with petroleum ether and, after drying over anh. Na2SO4, the extract was analyzed for total cholesterol 10. The aortas were graded for atherosclerotic lesions on a 0--4 scale 11 and frozen in dry ice-acetone. Acetone dry powder of individual aortas was prepared according to our previously published method 9. The enzyme was solubilized by suspending 1 g o f powder in 20 ml of 0.05 M phosphate buffer, p H 6.2, containing 0.05 M NH4CI. The suspension was stirred for 30 rain, then centrifuged at 10,000 • g for 10 rain. The clear supernatant contained 0.4-2.0 mg protein per mllL Synthetic activity was assayed using an emulsion containing 15.5 /~moles o f [4-14C]cholesterol, 46.5 #moles oleic acid, 31.0/zmoles of sodium taurocholate and 100/~moles of NH4C1 in 1.5 ml o f 0.154 M phosphate buffer, p H 6.2. Incubation mixtures contained 0.2 ml o f substrate in 1 ml o f solution. F o r measurement o f hydrolytic activity, a miceIlar substrate containing 1.54 /zmoles o f [4-14C]cholesteryl oleate, 3.75 #moles o f sodium taurocholate and 3.8 #moles o f lecithin in 1 ml o f 0.154 M phosphate buffer, p H 6.6 was prepared. The final incubation mixture (1.0 ml) contained 0.4 ml of substrate preparation. TABLE 1 SERUM AND LIVER LIPIDS, ATHEROSCLEROSISAND AORTIC CHOLESTEROL ESTERASE ACTIVITY IN RABBITS FED 2 ~ CHOLESTEROL IN 6 ~oo CORN OILa
Day
Cholesterol serum (mgldl)
0 5 12 20 30
Atheromata liver (gllO0 g)
49 ~ 6 b 0.52 • 0.05 173 dz 29 1.01 ~z 0.12 495 • 230 2.04 • 0.96 1874 • 316 4.16 • 0.77 925 4= 107 3.73 ~- 0.47
Hydrolase (HJ (nm/mg proteinlhr)
S/H
arch
Synthetase (SJ thoracic (nm/mg proteinlhr)
0 0 0.5 1.0 1.1
0 0 0 1.0 0.5
2.67 4- 0.33 3.33 z~ 0.61 5.15 • 0.75 4.83 + 1.69 4.65 ~ 0.90
1.17 1.68 1.81 1.62 1.73
Four rabbits taken at each time period. b Standard error.
3.13 • 0.43 5.58 • 0.52 9.31 ~ 0.29 7.83 _+ 1.35 8.03 J_ 1.27
461
AORTIC CHOLESTEROL ESTERASE IN RABBITS TABLE 2
SERUM CHOLESTEROL~ AORTIC ATHEROSCLEROSIS AND AORTIC CHOLESTEROL ESTERASEACTIVITYOF RABBITS FED 2 ~ CHOLESTEROL IN 6 ~ CORN OILa
Day
Serumcholesterol Atheromata Synthetase (S) (mg/dl) (nm/mg arch thoracic protein/hr)
Hydrolase(H) (nm/mg protein/hr)
S/H
0 7 11
84 • 8 `0 685 4- 147 506 4- 140
4.98 • 1.27 7.85 • 2.93 3.23 • 0.92
0.72 1.74 2.74
0 0 0
0 0 0
3.60 ~ 0.74 13.65 • 6.75 8.85 -k 2.33
F o u r rabbits t a k e n at each time period. b S t a n d a r d Error.
Incubations were carried out by shaking for 4 hours at 37~ in a Dubnoff metabolic shaker. The reaction was stopped by mixing 50 #1 o f incubation mixture with 50 #1 o f acetone-ethanol (1:1) and warming the mixture. The reaction mixture was spotted on Silica Gel G plates and subjected to Chromatographic development in hexane-ethyl ether-acetic acid, 83:16:1. The areas o f the plate containing free and esterified cholesterol were visualized in iodine vapor and, after sublimation of the iodine, the areas were scraped into scintillation vials and assayed by liquid scintillation spectrometry. Synthesis and hydrolysis of cholesteryl ester is expressed as nmoles converted per mg protein per hour. The labeled substrates were purchased from the New England Nuclear Corp., Boston, Mass. RESULTS AND DISCUSSION i
In the first experiment (Table 1), four rabbits were killed on days 0, 5, 12, 20 and 30 after institution of the atherogenic regimen. In this experiment both aortic arch and thoracic aorta were graded for atherosclerosis but only the thoracic aorta was used for cholesterol esterase assay. A sharp rise in serum and liver cholesterol levels shortly after dietary intake of cholesterol is seen. The rise in serum and liver cholesterol levels by day 5 was 253 ~ and 94 ~ , respectively. The rise in aortic cholesteryl ester synthetase by day 5 is also evident. The level of both hydrolytic and synthetic activity peaked at day 12 and remained close to that level thereafter. Although an aortic cholesteryl ester synthesizing enzyme has not been identified, for the sake o f brevity, we refer to this activity as synthetase. The synthetase/hydrolase ratio (S/H) was 1.17 in the control group. This value corresponds well to the value o f 1:1 observed in our earlier experiments 9. The S/H ratio rose with cholesterol feeding primarily because the rise in synthetase activity was 78 ~ on day 5 and 197 ~ , 150 ~ and 157 ~ at the other three periods. In contrast, hydrolase activity rose by 25 ~ on day 5, 93 ~ on day 12, 81 ~ on day 20 and 74 ~ on day 30. It is noteworthy that cholesteryl ester synthetase activity
462
D. KRITCHEVSKY, S. A. TEPPER, J. C. GENZANO, H. V. KOTHAR!
was a t its p e a k before a n y a t h e r o m a t o u s lesions were seen in the t h o r a c i c a o r t a . I n the second experiment, a n i m a l s were killed at days 0, 7 a n d 11 a n d the entire a o r t a , t h o r a c i c a n d arch, was t a k e n for assay. Results are s h o w n in T a b l e 2. T h e striking rise in serum cholesterol after 7 days o f cholesterol diet is similar to t h a t seen in the first experiment. T h e synthetase activity is increased b y 295 ~o on d a y 7 a n d the h y d r o l a s e activity is increased b y o n l y 58 ~ . A s in the first e x p e r i m e n t , a s h a r p increase in S / H ratio is seen after 7 days o n the atherogenic r e g i m e n (142 ~ ) a n d the increase over s t a r t i n g levels is 281 ~ on the eleventh day. N o lesions were grossly visible in a n y o f the a o r t a s o n either day. O u r findings in r a b b i t s c o m p l e m e n t those o f St. Clair et al. s in pigeons, in t h a t cholesteryl ester synthetase is elevated after a s h o r t p e r i o d o f cholesterol feeding a n d before a n y atherosclerotic lesions are visible. W e have further s h o w n t h a t b o t h aortic cholesteryl ester synthetase (S) a n d h y d r o l a s e (H) increase u p o n cholesterol feeding b u t the S / H r a t i o rises because the rise in synthetic activity is several times higher t h a n t h a t o f h y d r o l y t i c activity.
REFERENCES 1 SMITH, E. B., The influence of age and atherosclerosis on the chemistry of aortic intima, Part 1
(The lipids), J. Atheroscler. Res., 5 (1965) 224. 2 SWELt,,L., LAW, M. D. ANDTREADWELL,C. R., Tissue cholesterol ester and triglyceride fatty acid composition of rabbits fed cholesterol diets high and low in linoleic acid, J. Nutr., 76 (1962) 429. 3 NEWMAN,H. A. I. AND ZILVERSMIT,D. B., Accumulation of lipid and nonlipid constituents in rabbit atheromata, J. Atheroscler. Res., 4 (1964) 261. 4 KRITCHEVSKY,D. AND TEPPER, S. A., Experimental atherosclerosis in rabbits fed cholesterol-free diets: Influence of chow components, J. Atheroscler. Res., 8 (1968) 357. 5 LOFLAND, JR., H. g., MOURY, D. M., HOFFMAN, C. W. AND CLARKSON, T. B., Lipid metabolism in
pigeon aorta during atherogenesis, J. Lipid Res., 6 (1965) 112. 6 HASHIMOTO,S., DAYTON,S. ANDALFIN-SLATER,R. B., Esterification of cholesterol by homogenates of atherosclerotic and normal aortas, Life Sci., 12 (1973) Part 2: 12. 7 KRITCHEVSKY,D., TEPPER, S. A. AND KITAGAWA,M., Experimental atherosclerosis in rabbits fed cholesterol-free diets, Part 3 (Comparison of fructose and lactose with otber carbohydrates), Natr. Reports Int., 7 (1973) 193. 8 ST. CLAIR, R. W., LOFt,AND,H. B. AND CLARKSON,T. B., Influence of duration of cholesterol feeding on esterification of fatty acids by cell-free preparations of pigeon aorta, Circulation Res., 27 (1970) 213. 9 KOTHARI,H. V., MILLER,B. F. AND KRITCHEVSKY,D., Aortic cholesterol esterase: Characteristics of normal rat and rabbit enzyme, Biochim. Biophys. Acts, 296 (1973) 446. 10 PEARSON,S., STERN,S. AND McGAvACK, T. H., A rapid accurate method for the determination of total cholesterol in serum, Analyt. Chem., 25 (1953) 813. 11 DUFF, G. L. AND MCMILLAN,G. C., The effect of alloxan diabetes on experimental cholesterol atherosclerosis in the rabbit, J. Exptl. Med., 89 (1949) 611. 12 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265.