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Effect of nialamide on cholesterol-induced atheroma in the rabbit
JOURNAL OF ATHEROSCLEROSIS RESEARCH
493
SHORT COMMUNICATIONS Effect of nialamide on cholesterolinduced atheroma in the rabbit c.
W. M. ADAMS, O. B. BAYLISS
AND
M. Z. M. IBRAHIM
Department of Pathology, Guy's Hospital Medical School, London (England) (Received August 10th, 1962)
INTRODUCTION It has been claimed by SHIMAMOTO et al. 1 - 3 , that the monoamino-oxidase inhibitor nialamide, chemically N -isonicotinoyl-l N -(f3-N -benzylcarboxamidoethyl) hydrazine, has an anti-atherogenic action in short term experiments on the rabbit arterial intima. This effect was ascribed to the preservation by nialamide of the "silicone-like" surface of vascular endothelium, similar to that which was reported as lowering platelet adhesiveness and reducing the size of experimental thrombi 3 . In view of the possibility that nialamide might protect the arterial wall from lipid infiltration, we have investigated its action on cholesterol-induced atheroma in the rabbit over a period of 12 weeks on a high cholesterol diet.
METHODS Male albino white rabbits of weight 1.3-3.3 kg were fed either a stock or a cholesterolenriched diet. The latter diet was prepared by mixing 1 g of cholesterol and 93 g of stock diet oiled with 6 g of corn oi1 4 . A group of animals on each diet received nialamide,S mg/kg body weight, orally once a day. Animals were killed at weekly intervals between 2 and 12 weeks on these regimens. Their aortas were dissected out, stripped free of adipose tissue and samples were taken for cholesterol estimation 5 and histology. The histological sample was fixed in Lillie's calcium acetate-formol and stained by the osmium tetroxide-a-naphthylaminealcian blue method 6 for cholesterol esters and phospholipid. Some sections were also stained by the perchloric acid-naphthoquinone method 7 for cholesterol. RESULTS These are summarized in Tables I-III. From 2-6 weeks, animals on a high cholesterol diet receiving nialamide had less cholesterol in their aortic arch (t = 3.48, P < 0.01), had a better histological assessment of their aortic arch (t = 2.16, P === 0.05) and had a tendency to less cholesterol in their proximal descending aorta (t = 1.50, P < 0.20) ]. Atheroscler. Res., 2 (1962) 493-496
494
SHORT COMMUNICATIONS
than the corresponding group on the cholesterol diet without nialamide. From 8-12 weeks on the cholesterol-enriched diet there was no difference between the nialamide and untreated group in either the cholesterol content of the aortic arch (t = 0.33, P> 0.70) or the histological assessment of atheroma in the arch (t = 0.20, P > 0.80). In this latter period on diet, there was a tendency towards increased cholesterol in the proximal descending aorta in the nialamide group (t = 1.84, P < 0.20). The body weights of the animals in all four groups showed an approximately uniform increase during the experiment. Those on stock diet had a mean percentage increase in weight of 38; those on stock diet with nialamide 38; those on high cholesterol diet 34; those on high cholesterol with nialamide 37. TABLE I EFFECT OF NIALAMIDE ON THE CHOLESTEROL CONTENT OF THE AORTIC ARCH OF THE CHOLESTEROL-FED RABBIT
Cholesterol content mg/l00 mg wet weight Weeks on diet
2 3 4 5 6 7 8
Stock diet alone
Stock with nialamide
Cholesterol diet alone
Cholesterol with nialamide
0.3
0.5
0.0
0.0
0.3 0.8 0.3 2.0 1.5
0.0 0.3 0.1 0.9 0.6 2.0 5.6 6.7 2.3 4.6 4.3
0.0 0.0 0.0 0.0
9
10 11 12
0.0 0.3 0.3
5.2 4.7 1.2 3.1 6.1
TABLE II EFFECT OF NIALAMIDE ON THE CHOLESTEROL CONTENT OF THE PROXIMAL DESCENDING AORTA* OF THE CHOLESTEROL-FED RABBIT
Cholesterol content mg/l00 mg wet weight Weeks on diet
2 3 4 5 6 7 8 9 10 11 12
*
Stock diet alone
Stock with nialamide
Cholesterol diet alone
Cholesterol with nialamide
0.0
0.0
0.2
0.0
0.0 0.3 0.3 0.1 0.9
0.0 0.2 0.0 0.2 0.2 0.3 0.5 1.6 2.5 2.6 4.0
0.2 0.2 0.1 0.1 0.1 0.2 0.4
0.6 1.8 1.5 2.5 3.0
The first 2 em of aorta below the origin of the left subclavian artery. ]. A theroscler. Res., 2 (1962) 493-496
SHORT COMMUNICAnONS
495
DISCUSSION These results indicate that during the early stages of the high cholesterol diet, nialamide has a partial preventive effect on the infiltration of the rabbit arterial wall by cholesterol. This effect is not inconsistent with the protective action of nialamide on vascular endothelium reported by SHIMAMOTO et al. 1 ,2. However, although nialamide caused no loss of body weight, it is possible that this anti-atherogenic effect in the early stages of the dietary experiment was mediated through altered absorption, metabolism or excretion of cholesterol and other lipids. TABLE III EFFECT OF NIALAMIDE ON THE HISTOLOGICAL ASSESSMENT OF ATHEROMA IN THE AORTIC ARCH OF THE CHOLESTEROL-FED RABBIT
Histological grade* Weeks on diet
2 3 4 5 6 7 8 9 10 11 12
Stock diet alone
Stock with nialamide
Cholesterol diet alone
Cholesterol with nialamide
0
0
0
0
1 2 1 1 3
0 0 1 1 2 3 3 3 3 4 4
0 0 0 0 0 0 0
4 4 2 4 6
* Sum of degree of intimal thickening (scale 0-3) plus amount of stained cholesterol (scale 0-3). The degree of intimal thickening was graded: none, 0; less than width of media, 1; equal to width of media, 2; more than width of media, 3. The amount of stained cholesterol was graded: none, 0; few to moderate droplets, 1; abundant droplets and some lipid masses, 2; dense lipid masses, 3. In the later stages of the high cholesterol diet - the stage associated with the development of florid atheroma - nialamide had no protective effect on the arterial wall and the extent of cholesterol infiltration tended to be slightly more than in the untreated group. If cholesterol-induced atheroma in the rabbit can be compared to the factor of lipid infiltration in human atherogenesis, these results indicate that nialamide would be ineffective in the prevention or treatment of the lipid deposits of established human atherosclerosis. ACKNOWLEDGMENTS The dietary aspects of this study were carried out by Messrs. PFIZER LTD., Sandwich, England under the supervision of Dr. R. F. DAVIES. This study was otherwise supported by U.S.P.H.S. Grants Nos. H-6483 and HE-06483-02. ]. Atheroscler. Res., 2 (1962) 493-496
496
SHORT COMMUNICATIONS REFERENCES
1 2
3 4
5 6
7
T. SHIMAMOTO, Asian Med. ]., 3 (1960) 479. T. SHIMAMOTO, H. YAMAZAKI, M. INOUE, T. FUJITA, N. SAGAWA, T. SUNAGA AND T. ISHIOKA, Proc. lap. Acad., 36 (1960) 240. T. SHIMAMOTO AND T. FUJITA, Proc. lap. Acad., 37 (1961) 105. G. C. McMILLAN AND G. L. DUFF, A.M.A. Arch. Pathol., 59 (1955) 285. A. N. DAVISON, ]. DOBBING, R. S. MORGAN AND G. PAYLING WRIGHT, ]. Neurochem., 3 (19581959) 89. C. W. M. ADAMS, ]. Pathol. Bacteriol., 77 (1959) 648. C. W. M. ADAMS, Nature, 192 (1961) 331.
l. Atheroscler. Res., 2 (1962) 493-496
Sexual differences in the esterase activity of the aorta in rats ]. L. LACUARA, L. GERSCHENSON, H. C. MOGUILEVSKY AND M. R. MALINOW
Institute of Physiology, Buenos Aires Medical School, Buenos Aires (Argentina) (Received September 4th, 1962)
Previous work from this laboratory showed that in the aorta of rats the aerobic 1 ,2 and anaerobic 3 metabolisms as well as the concentration of nucleic acids 4 are at least partially regulated through gonadal mechanisms. The present report describes the esterase activity that extracts from the aorta of male and female rats exhibit against a-naphtyli:mtyrate. MATERIAL AND METHODS Adult male and female albino rats of our institute weighing 150-250 g and reared on the standard laboratory diet were used throughout. The following groups of already described animals 4 were studied: (1) intact males; (2) castrated males; (3) castrated males receiving pellets of testosterone; (4) intact females; (5) gonadectomized females; (6) gonadectomized females receiving pellets of estradiol. Two consecutive series were studied; further details concerning number of rats, time of follow-up, etc., have been reported. 4 Extracts of aorta and of liver were prepared as described 4 and the esterase activity toward a-naphtylbutyrate was determined as indicated by GOMORI5 and by KATZ et al. 6 RESULTS AND DISCUSSION As shown in Table I, the aorta has a esterolytic activity against a-naphtylbutyrate which is approximately 1 % from that of the liver. Males have higher activity than females in the aorta. Gonadectomy decreases this esterase activity in males, and increases it in females. Estradiol or testosterone does not alter this estcrolytic activity in the aorta of gonadectomized animals. Results in liver are similar to those iIi aorta, although spaying produced no changes. ]. Atheroscler. Res., 2 (1962) 496-498
493
SHORT COMMUNICATIONS Effect of nialamide on cholesterolinduced atheroma in the rabbit c.
W. M. ADAMS, O. B. BAYLISS
AND
M. Z. M. IBRAHIM
Department of Pathology, Guy's Hospital Medical School, London (England) (Received August 10th, 1962)
INTRODUCTION It has been claimed by SHIMAMOTO et al. 1 - 3 , that the monoamino-oxidase inhibitor nialamide, chemically N -isonicotinoyl-l N -(f3-N -benzylcarboxamidoethyl) hydrazine, has an anti-atherogenic action in short term experiments on the rabbit arterial intima. This effect was ascribed to the preservation by nialamide of the "silicone-like" surface of vascular endothelium, similar to that which was reported as lowering platelet adhesiveness and reducing the size of experimental thrombi 3 . In view of the possibility that nialamide might protect the arterial wall from lipid infiltration, we have investigated its action on cholesterol-induced atheroma in the rabbit over a period of 12 weeks on a high cholesterol diet.
METHODS Male albino white rabbits of weight 1.3-3.3 kg were fed either a stock or a cholesterolenriched diet. The latter diet was prepared by mixing 1 g of cholesterol and 93 g of stock diet oiled with 6 g of corn oi1 4 . A group of animals on each diet received nialamide,S mg/kg body weight, orally once a day. Animals were killed at weekly intervals between 2 and 12 weeks on these regimens. Their aortas were dissected out, stripped free of adipose tissue and samples were taken for cholesterol estimation 5 and histology. The histological sample was fixed in Lillie's calcium acetate-formol and stained by the osmium tetroxide-a-naphthylaminealcian blue method 6 for cholesterol esters and phospholipid. Some sections were also stained by the perchloric acid-naphthoquinone method 7 for cholesterol. RESULTS These are summarized in Tables I-III. From 2-6 weeks, animals on a high cholesterol diet receiving nialamide had less cholesterol in their aortic arch (t = 3.48, P < 0.01), had a better histological assessment of their aortic arch (t = 2.16, P === 0.05) and had a tendency to less cholesterol in their proximal descending aorta (t = 1.50, P < 0.20) ]. Atheroscler. Res., 2 (1962) 493-496
494
SHORT COMMUNICATIONS
than the corresponding group on the cholesterol diet without nialamide. From 8-12 weeks on the cholesterol-enriched diet there was no difference between the nialamide and untreated group in either the cholesterol content of the aortic arch (t = 0.33, P> 0.70) or the histological assessment of atheroma in the arch (t = 0.20, P > 0.80). In this latter period on diet, there was a tendency towards increased cholesterol in the proximal descending aorta in the nialamide group (t = 1.84, P < 0.20). The body weights of the animals in all four groups showed an approximately uniform increase during the experiment. Those on stock diet had a mean percentage increase in weight of 38; those on stock diet with nialamide 38; those on high cholesterol diet 34; those on high cholesterol with nialamide 37. TABLE I EFFECT OF NIALAMIDE ON THE CHOLESTEROL CONTENT OF THE AORTIC ARCH OF THE CHOLESTEROL-FED RABBIT
Cholesterol content mg/l00 mg wet weight Weeks on diet
2 3 4 5 6 7 8
Stock diet alone
Stock with nialamide
Cholesterol diet alone
Cholesterol with nialamide
0.3
0.5
0.0
0.0
0.3 0.8 0.3 2.0 1.5
0.0 0.3 0.1 0.9 0.6 2.0 5.6 6.7 2.3 4.6 4.3
0.0 0.0 0.0 0.0
9
10 11 12
0.0 0.3 0.3
5.2 4.7 1.2 3.1 6.1
TABLE II EFFECT OF NIALAMIDE ON THE CHOLESTEROL CONTENT OF THE PROXIMAL DESCENDING AORTA* OF THE CHOLESTEROL-FED RABBIT
Cholesterol content mg/l00 mg wet weight Weeks on diet
2 3 4 5 6 7 8 9 10 11 12
*
Stock diet alone
Stock with nialamide
Cholesterol diet alone
Cholesterol with nialamide
0.0
0.0
0.2
0.0
0.0 0.3 0.3 0.1 0.9
0.0 0.2 0.0 0.2 0.2 0.3 0.5 1.6 2.5 2.6 4.0
0.2 0.2 0.1 0.1 0.1 0.2 0.4
0.6 1.8 1.5 2.5 3.0
The first 2 em of aorta below the origin of the left subclavian artery. ]. A theroscler. Res., 2 (1962) 493-496
SHORT COMMUNICAnONS
495
DISCUSSION These results indicate that during the early stages of the high cholesterol diet, nialamide has a partial preventive effect on the infiltration of the rabbit arterial wall by cholesterol. This effect is not inconsistent with the protective action of nialamide on vascular endothelium reported by SHIMAMOTO et al. 1 ,2. However, although nialamide caused no loss of body weight, it is possible that this anti-atherogenic effect in the early stages of the dietary experiment was mediated through altered absorption, metabolism or excretion of cholesterol and other lipids. TABLE III EFFECT OF NIALAMIDE ON THE HISTOLOGICAL ASSESSMENT OF ATHEROMA IN THE AORTIC ARCH OF THE CHOLESTEROL-FED RABBIT
Histological grade* Weeks on diet
2 3 4 5 6 7 8 9 10 11 12
Stock diet alone
Stock with nialamide
Cholesterol diet alone
Cholesterol with nialamide
0
0
0
0
1 2 1 1 3
0 0 1 1 2 3 3 3 3 4 4
0 0 0 0 0 0 0
4 4 2 4 6
* Sum of degree of intimal thickening (scale 0-3) plus amount of stained cholesterol (scale 0-3). The degree of intimal thickening was graded: none, 0; less than width of media, 1; equal to width of media, 2; more than width of media, 3. The amount of stained cholesterol was graded: none, 0; few to moderate droplets, 1; abundant droplets and some lipid masses, 2; dense lipid masses, 3. In the later stages of the high cholesterol diet - the stage associated with the development of florid atheroma - nialamide had no protective effect on the arterial wall and the extent of cholesterol infiltration tended to be slightly more than in the untreated group. If cholesterol-induced atheroma in the rabbit can be compared to the factor of lipid infiltration in human atherogenesis, these results indicate that nialamide would be ineffective in the prevention or treatment of the lipid deposits of established human atherosclerosis. ACKNOWLEDGMENTS The dietary aspects of this study were carried out by Messrs. PFIZER LTD., Sandwich, England under the supervision of Dr. R. F. DAVIES. This study was otherwise supported by U.S.P.H.S. Grants Nos. H-6483 and HE-06483-02. ]. Atheroscler. Res., 2 (1962) 493-496
496
SHORT COMMUNICATIONS REFERENCES
1 2
3 4
5 6
7
T. SHIMAMOTO, Asian Med. ]., 3 (1960) 479. T. SHIMAMOTO, H. YAMAZAKI, M. INOUE, T. FUJITA, N. SAGAWA, T. SUNAGA AND T. ISHIOKA, Proc. lap. Acad., 36 (1960) 240. T. SHIMAMOTO AND T. FUJITA, Proc. lap. Acad., 37 (1961) 105. G. C. McMILLAN AND G. L. DUFF, A.M.A. Arch. Pathol., 59 (1955) 285. A. N. DAVISON, ]. DOBBING, R. S. MORGAN AND G. PAYLING WRIGHT, ]. Neurochem., 3 (19581959) 89. C. W. M. ADAMS, ]. Pathol. Bacteriol., 77 (1959) 648. C. W. M. ADAMS, Nature, 192 (1961) 331.
l. Atheroscler. Res., 2 (1962) 493-496
Sexual differences in the esterase activity of the aorta in rats ]. L. LACUARA, L. GERSCHENSON, H. C. MOGUILEVSKY AND M. R. MALINOW
Institute of Physiology, Buenos Aires Medical School, Buenos Aires (Argentina) (Received September 4th, 1962)
Previous work from this laboratory showed that in the aorta of rats the aerobic 1 ,2 and anaerobic 3 metabolisms as well as the concentration of nucleic acids 4 are at least partially regulated through gonadal mechanisms. The present report describes the esterase activity that extracts from the aorta of male and female rats exhibit against a-naphtyli:mtyrate. MATERIAL AND METHODS Adult male and female albino rats of our institute weighing 150-250 g and reared on the standard laboratory diet were used throughout. The following groups of already described animals 4 were studied: (1) intact males; (2) castrated males; (3) castrated males receiving pellets of testosterone; (4) intact females; (5) gonadectomized females; (6) gonadectomized females receiving pellets of estradiol. Two consecutive series were studied; further details concerning number of rats, time of follow-up, etc., have been reported. 4 Extracts of aorta and of liver were prepared as described 4 and the esterase activity toward a-naphtylbutyrate was determined as indicated by GOMORI5 and by KATZ et al. 6 RESULTS AND DISCUSSION As shown in Table I, the aorta has a esterolytic activity against a-naphtylbutyrate which is approximately 1 % from that of the liver. Males have higher activity than females in the aorta. Gonadectomy decreases this esterase activity in males, and increases it in females. Estradiol or testosterone does not alter this estcrolytic activity in the aorta of gonadectomized animals. Results in liver are similar to those iIi aorta, although spaying produced no changes. ]. Atheroscler. Res., 2 (1962) 496-498