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Effects of dietary cholesterol and bile salt in the golden hamster
EFFECTS
W.
OF
MARX,
DIETARY
PH.D.,
CHOLESTEROL AND GOLDEN HAMSTER L.
MARX,
Los
PH.D., ANGELES,
AND
H. J.
BILE
DEUEL,
SALT
IN
THE
JR., PH.D.
CALIF.
WAS established by a great number of investigators that a diet high in I Tcholesterol causes the development of atheromas in rabbits and chickens, but that in most other species so far investigated, such as the dog, cat, rat, and mouse, no atheromatosis is produced by cholesterol feeding.’ In recent years another small ro’dent has become available for experimental work, i.e., the golden hamster, and the present work was undertaken in order to investigate how this species would respond to a high cholesterol diet. After the experimental work reported below had been completed, two publications appeared dealing also with the effects of cholesterol feeding in hamsters.2s3 Since the results of these two groups of investigators are somewhat at variance, the findings of the present authors will be reported briefly. METHODS
The hamsters were first bought from local dealers, but in later experiments a more vigorous strain was obtained by breeding a few animals generous1.ysupplied by Dr. H. A. Mattill of the State University of Iowa. The age of the experimental animals varied somewhat; usually young adult hamsters were used. The hamsters were fed ad libitum a diet consisti,ng of one part of this laboratory’s stock diet for rats,* one part of Sherman diet, and one part of rolled oats, supplemented by lettuce twice weekly. For the experimental groups, cholesterolt was added to the diet at a level of 1 per cent; the sterol was dissolyed in hot cottonseed oil, and the warm solution mixed thoroughly with the other components of the diet. For one of the experimental groups, bile salt$ was mixed with the food at a level of 0.25 per cent in order to increase cholesterol absorption.4 From the Department of Biochemistry and Nutrition, School of Medicine, and the Laboratorler of the Allan Hancock Foundation, University of Southern California, Los Angeles. This study was supported by a grant from the Life Insurance Medical Research Fund. The authors wish to express their appreciation for the technical assistance of Stella Alogdelis, Emily R. Meserve, and Frank Shimoda. Received for publication Feb. 9. 1951. *Stock diet for rats: 34 per cent whole-wheat flour, 34 per cent oats, 15 per cent skim milk powder, 4 per cent alfalfa meal, 2 per cent yeast (strain G). 8 per cent cottonseed oil. 2 per cent fortified oil containing 300 I.U. of vitamin A and 50 I.U. of vitamin D per Gm.. 0.6 per cent NaCl. 0.6 per cent CaC03. tA part of the cholesterol was supplied by F. Fenger, The Armour Laboratories, and N. F. Payton. Suburban Chemical Co., Chicago. Ill. :Drled extract Of ox bile (U.&P.). in part supplied by F. Fenger. The Armour Laboratories. Chlcage, Ill. 124
MARX
ET AL.:
CHOLESTEROL
AND
BILE
SALT
IN GOLDEN
HAMSTER
125
After completion of the feeding period, the animals were autopsied (Nembutal anesthesia). Blood was taken from the vena cava or by heart puncture, and liver, kidneys, adrenals, and, in some instances? spleen, lungs, and brain were excised for determination of cholesterol. The aorta was taken for histological examination. Total cholesterol was determined as described by Nieft and Deuel.s Plasma was prepared from heparinized blood and extracted at room temperature by adding 14 volumes of a 1:l mixture of alcohol/acetone. Tissues were extracted with the same mixture, either using a Goldfinch apparatus* for continuous extraction or, after grinding the tissue in a mortar to a pulp (no sand added), by direct extraction in a volumetric flask provided with a reflux condenser and heated to boiling for about ten minutes. The latter procedure, which yielded better recoveries and was simpler and faster, was used in all later experiments. For histological examination, the aorta was fixed in Formalin, and strips, especially of the ascending portion and the convexity of the arc, were embedded in gelatin and studied on frozen sections stained with Nile blue sulfate. Cholesterol was identified by its birefringence and solubility in chloroform. Interesting specimens were also examined by other methods.? RESULTS
The general condition of the hamsters was variable. As a consequence of the feeding of cholesterol, the livers were very much increased in size and had the pale yellowish color typical of extreme fatty degeneration. Supplementation with both cholesterol and bile salt enhanced this effect, but did not seem to cause any other serious symptoms. The results are shown in Table I. The plasma cholesterol was significantly increased in the group fed cholesterol. Addition of bile salt to the high cholesterol diet produced a further rise in the sterol content of the plasma. In the liver, extremely high concentrations of cholesterol were found, in particular in the group fed both cholesterol and bile salt, individual animals showing values up to 12 per cent cholesterol. As the mean total liver weight was approximately doubled in these animals, the extent of the accumulation of cholesterol in the liver was quite remarkable. Even in the group fed cholesterol without bile salt, the liver cholesterol was increased approximately twenty times as compared with the control group. A slight sex difference was observed in the liver cholesterol values, the male hamsters showing a somewhat greater accumulation of cholesterol. The gross appearance of the aortas was normal in both control and experimental animals. On histological examination, no fat drops were seen in untreated hamsters after staining with Nile blue sulfate. The most striking effect of the experimental diet was a fatty imbibition which was pronounced in four of the animals. The endothelium was loaded with fat and included foam cells. *Laboratory Construction Co.. Kansas City, MO. tThe authors are much obliged to Dr. R. B. Barden and the Department of Medicine. University of Southern California. for preparing and interpreting tions and for the loan of two microtomes.
of Anatomy of the School some of the Initial sec-
126
AMERICAN
HEART
JOURNAL
The foam cells in the hamster were peculiar in that they often carried a few minute, birefringent particles between the fat droplets. The demarcation of the particles was so definite that they were visible without polaroid lenses. The inner surface of the aorta and several rows of elastic lamellas in the medial coat had a fatty lining consisting of a thin film of extracellular fat with a high affinity for the red form of the dye. Only traces of cholesterol occurred in the endothelium and media. Structural changes were limited to the inward bulging of endothelial cells. Two animals only had small foci of hyperplastic endothelium,* while in four others, a slight uniform thickening was observed in the proximal portion of the intimal coat. A similar slight thickening was noted in the proximal intima of two of nine controls.
TABLE
I.
EFFECTS
OF
DIETARY
CHOLESTEROL AriD
AND CHICKEN
BILE
SALT
IN
THE
MEAN SPECIES
FEEDING PERIOD (WEEKS)
DIET*
Hamster
C+B
NO. ANIMALS
PI.ASMA PER 100
Cf N
B
Rabbit
*C
c N
f
I3 = C = N =
(MG. ML.)
LIVER (%)
+ 34 + 14 + 5
8.68 5.11 0.22
+ 0.88 f 0.17 zk 0.04
26-38
1.13 60
_t 12 + 3
2.03 0.21
2 It
0.11 0.02
152 1
45.5 35
rt- 36 + 2
1.33 0.17
+ f
0.10 0.02
10
685 100
-t 164 + 3
1.67 0.41
+ 0.21 + 0.07
+B
diet supplemerlted diet supplemented normal controls
CONTENT
282 160 57
E Chirken
CHOLESTEROL
RABBIT,
16-25 25
G Rat
RAT,
HAMSTER,
with with
cholesterol cholesterol
plus
bile
salt
For purposes of comparison, some results obtained under similar conditions in rats, rabbits, and chickens are included in Table I. Since the responses of these species to cholesterol feeding are well known ,l they are not dealt with in the text. DISCUSSION
The average plasma cholesterol level of the hamsters fed cholesterol without bile salt was similar to that reported by Goldman,’ but the hepatic accumulation Addition of bile salt to the diet of the sterol was found to be somewhat higher. further increased the cholesterol concentration in both plasma and liver. *In a preliminary progress report. only a first group of seven cholesterol-fed Fund. Annual Report for 1947).
this focal hamsters
hyperplasia had been
was mentioned processed (Life
as “lesions”; at Insurance Medical
the
time,
Research
MARX
ET
AL.
:
CHOLESTEROL
AND
BILE
SALT
IN
GOLDEN
HAMSTER
127
The aorta of the experimental hamsters showed only negligible morphological changes, though the wall was deeply infiltrated with stainable fat. Goldman likewise found only insignificant structural changes in the aorta of hamsters fed cholesterol.2 Altschul, on the other hand, observed large atheromas.3 These differences in the results obtained by the various authors are probably a consequence of differences in dietary conditions. In the experiments reported above, a solution of cholesterol in warm cottonseed oil was mixed thoroughly with the diet, and bile salt was added to the latter in order to increase absorption of the sterol from the gut. Goldman administered powdered cholesterol in gelatin capsules.2 Altschul fed dried egg yolk powder with milk and “yolk cake.“a As a consequence of these dietary variations, the following factors were probably different in the experiments of the mentioned groups: (1) the amount of cholesterol absorbed from the gut and (2) the presence and nature of certain other components in the diet which might have had an effect of their own on the aorta. The latter point was recently studied by Altschul.6 When the response to cholesterol feeding of the hamsters is compared with those of the other species studied, it is evident that the accumulation of cholesterol in the liver did not follow the pattern of its blood level. In the hamsters the plasma cholesterol rose to only intermediate levels, but the hepatic sterol content reached extremely high values. In chickens, the plasma cholesterol was more than five times as high as that in rats, but the liver cholesterol levels were similar in these two species. Regarding a relationship between the plasma cholesterol concentration and its effects on the aorta, it was found that macroscopic intimal cushions developed only in the chicken and the rabbit, the two species in which the plasma concentration rose to the highest levels. Among the other species, hamsters exhibited excessive fatty infiltration, while the rats, although showing the lowest of the experimental plasma sterol levels, developed, in a few instances, minor morphological changes. SUMMARY
The effects of a diet containing 1 per cent cholesterol and 0.25 per cent bile salt were studied in hamsters. After feeding periods of sixteen to twenty-five weeks, the plasma cholesterol was significantly increased; in the liver an extremely high accumulation of the sterol and a severe fatty degeneration were observed. Sections of the aorta revealed an almost normal structure associated with considerable fatty infiltration. The effects observed in hamsters fed 1 per cent cholesterol without supplementation by bile salt were qualitatively similar, but somewhat smaller in extent, These results obtained with hamsters were compared with the effects of cholesterol feeding in three other species, i.e., the rat. rabbit, and chicken.
128
AMERICAN
HEART
JOURNAL
REFERENCES
1.
2. 3. 4. 5. 6.
Reviewed by (a) Duff, G. L.: Experimental Cholesterol Arteriosclerosis and Its Relationship to Human Arteriosclerosis, Arch. Path. 20:81 and 259, 1935; (b) Hueper, W. C.: Arteriosclerosis, Arch. Path. 39:122 and 187, 1945; (c) Page, I. W.: Arteriosclerosis and Lipid Metabolism, Biol. Symp. 11:43, 1945; (d) Katz, L. N., and Dauber, D. V.: The Pathogenesis of Atherosclerosis, J. Mt. Sinai Hosp. 12:382, 1945-1946; (e) Gubner, R., and Ungerleider, H. E.: Arteriosclerosis, Am. J. Med. 6:60, 1949. Goldman. I.: Effect of Cholesterol Feeding in Hamsters. Arch. Path. 49:169. 1950. Altschul, ‘R.: Experimental Cholesterol Arteriosclerosis. ’ II. Changes Produced in Golden Hamsters and in Guinea Pigs, AM. HEART J. 40:401, 1950. Schoenheimer, R.: Ueber die Resorptionsbeschleunigung des Cholesterins bei Anwesenheit von Desoxvcholsaure. B&hem. Ztschr. 147:2X 1924. Nieft, M. L., and Deuel, H.‘J., Jr.: Studies on Cholesterol Esterases. I. Enzyme Systems in Rat Tissues, J. Biol. Chem. 177:143, 1949. Altschul, R.: Selected Studies on Arteriosclerosis, Springfield, Ill., 1950, Charles C Thomas, Publisher.
W.
OF
MARX,
DIETARY
PH.D.,
CHOLESTEROL AND GOLDEN HAMSTER L.
MARX,
Los
PH.D., ANGELES,
AND
H. J.
BILE
DEUEL,
SALT
IN
THE
JR., PH.D.
CALIF.
WAS established by a great number of investigators that a diet high in I Tcholesterol causes the development of atheromas in rabbits and chickens, but that in most other species so far investigated, such as the dog, cat, rat, and mouse, no atheromatosis is produced by cholesterol feeding.’ In recent years another small ro’dent has become available for experimental work, i.e., the golden hamster, and the present work was undertaken in order to investigate how this species would respond to a high cholesterol diet. After the experimental work reported below had been completed, two publications appeared dealing also with the effects of cholesterol feeding in hamsters.2s3 Since the results of these two groups of investigators are somewhat at variance, the findings of the present authors will be reported briefly. METHODS
The hamsters were first bought from local dealers, but in later experiments a more vigorous strain was obtained by breeding a few animals generous1.ysupplied by Dr. H. A. Mattill of the State University of Iowa. The age of the experimental animals varied somewhat; usually young adult hamsters were used. The hamsters were fed ad libitum a diet consisti,ng of one part of this laboratory’s stock diet for rats,* one part of Sherman diet, and one part of rolled oats, supplemented by lettuce twice weekly. For the experimental groups, cholesterolt was added to the diet at a level of 1 per cent; the sterol was dissolyed in hot cottonseed oil, and the warm solution mixed thoroughly with the other components of the diet. For one of the experimental groups, bile salt$ was mixed with the food at a level of 0.25 per cent in order to increase cholesterol absorption.4 From the Department of Biochemistry and Nutrition, School of Medicine, and the Laboratorler of the Allan Hancock Foundation, University of Southern California, Los Angeles. This study was supported by a grant from the Life Insurance Medical Research Fund. The authors wish to express their appreciation for the technical assistance of Stella Alogdelis, Emily R. Meserve, and Frank Shimoda. Received for publication Feb. 9. 1951. *Stock diet for rats: 34 per cent whole-wheat flour, 34 per cent oats, 15 per cent skim milk powder, 4 per cent alfalfa meal, 2 per cent yeast (strain G). 8 per cent cottonseed oil. 2 per cent fortified oil containing 300 I.U. of vitamin A and 50 I.U. of vitamin D per Gm.. 0.6 per cent NaCl. 0.6 per cent CaC03. tA part of the cholesterol was supplied by F. Fenger, The Armour Laboratories, and N. F. Payton. Suburban Chemical Co., Chicago. Ill. :Drled extract Of ox bile (U.&P.). in part supplied by F. Fenger. The Armour Laboratories. Chlcage, Ill. 124
MARX
ET AL.:
CHOLESTEROL
AND
BILE
SALT
IN GOLDEN
HAMSTER
125
After completion of the feeding period, the animals were autopsied (Nembutal anesthesia). Blood was taken from the vena cava or by heart puncture, and liver, kidneys, adrenals, and, in some instances? spleen, lungs, and brain were excised for determination of cholesterol. The aorta was taken for histological examination. Total cholesterol was determined as described by Nieft and Deuel.s Plasma was prepared from heparinized blood and extracted at room temperature by adding 14 volumes of a 1:l mixture of alcohol/acetone. Tissues were extracted with the same mixture, either using a Goldfinch apparatus* for continuous extraction or, after grinding the tissue in a mortar to a pulp (no sand added), by direct extraction in a volumetric flask provided with a reflux condenser and heated to boiling for about ten minutes. The latter procedure, which yielded better recoveries and was simpler and faster, was used in all later experiments. For histological examination, the aorta was fixed in Formalin, and strips, especially of the ascending portion and the convexity of the arc, were embedded in gelatin and studied on frozen sections stained with Nile blue sulfate. Cholesterol was identified by its birefringence and solubility in chloroform. Interesting specimens were also examined by other methods.? RESULTS
The general condition of the hamsters was variable. As a consequence of the feeding of cholesterol, the livers were very much increased in size and had the pale yellowish color typical of extreme fatty degeneration. Supplementation with both cholesterol and bile salt enhanced this effect, but did not seem to cause any other serious symptoms. The results are shown in Table I. The plasma cholesterol was significantly increased in the group fed cholesterol. Addition of bile salt to the high cholesterol diet produced a further rise in the sterol content of the plasma. In the liver, extremely high concentrations of cholesterol were found, in particular in the group fed both cholesterol and bile salt, individual animals showing values up to 12 per cent cholesterol. As the mean total liver weight was approximately doubled in these animals, the extent of the accumulation of cholesterol in the liver was quite remarkable. Even in the group fed cholesterol without bile salt, the liver cholesterol was increased approximately twenty times as compared with the control group. A slight sex difference was observed in the liver cholesterol values, the male hamsters showing a somewhat greater accumulation of cholesterol. The gross appearance of the aortas was normal in both control and experimental animals. On histological examination, no fat drops were seen in untreated hamsters after staining with Nile blue sulfate. The most striking effect of the experimental diet was a fatty imbibition which was pronounced in four of the animals. The endothelium was loaded with fat and included foam cells. *Laboratory Construction Co.. Kansas City, MO. tThe authors are much obliged to Dr. R. B. Barden and the Department of Medicine. University of Southern California. for preparing and interpreting tions and for the loan of two microtomes.
of Anatomy of the School some of the Initial sec-
126
AMERICAN
HEART
JOURNAL
The foam cells in the hamster were peculiar in that they often carried a few minute, birefringent particles between the fat droplets. The demarcation of the particles was so definite that they were visible without polaroid lenses. The inner surface of the aorta and several rows of elastic lamellas in the medial coat had a fatty lining consisting of a thin film of extracellular fat with a high affinity for the red form of the dye. Only traces of cholesterol occurred in the endothelium and media. Structural changes were limited to the inward bulging of endothelial cells. Two animals only had small foci of hyperplastic endothelium,* while in four others, a slight uniform thickening was observed in the proximal portion of the intimal coat. A similar slight thickening was noted in the proximal intima of two of nine controls.
TABLE
I.
EFFECTS
OF
DIETARY
CHOLESTEROL AriD
AND CHICKEN
BILE
SALT
IN
THE
MEAN SPECIES
FEEDING PERIOD (WEEKS)
DIET*
Hamster
C+B
NO. ANIMALS
PI.ASMA PER 100
Cf N
B
Rabbit
*C
c N
f
I3 = C = N =
(MG. ML.)
LIVER (%)
+ 34 + 14 + 5
8.68 5.11 0.22
+ 0.88 f 0.17 zk 0.04
26-38
1.13 60
_t 12 + 3
2.03 0.21
2 It
0.11 0.02
152 1
45.5 35
rt- 36 + 2
1.33 0.17
+ f
0.10 0.02
10
685 100
-t 164 + 3
1.67 0.41
+ 0.21 + 0.07
+B
diet supplemerlted diet supplemented normal controls
CONTENT
282 160 57
E Chirken
CHOLESTEROL
RABBIT,
16-25 25
G Rat
RAT,
HAMSTER,
with with
cholesterol cholesterol
plus
bile
salt
For purposes of comparison, some results obtained under similar conditions in rats, rabbits, and chickens are included in Table I. Since the responses of these species to cholesterol feeding are well known ,l they are not dealt with in the text. DISCUSSION
The average plasma cholesterol level of the hamsters fed cholesterol without bile salt was similar to that reported by Goldman,’ but the hepatic accumulation Addition of bile salt to the diet of the sterol was found to be somewhat higher. further increased the cholesterol concentration in both plasma and liver. *In a preliminary progress report. only a first group of seven cholesterol-fed Fund. Annual Report for 1947).
this focal hamsters
hyperplasia had been
was mentioned processed (Life
as “lesions”; at Insurance Medical
the
time,
Research
MARX
ET
AL.
:
CHOLESTEROL
AND
BILE
SALT
IN
GOLDEN
HAMSTER
127
The aorta of the experimental hamsters showed only negligible morphological changes, though the wall was deeply infiltrated with stainable fat. Goldman likewise found only insignificant structural changes in the aorta of hamsters fed cholesterol.2 Altschul, on the other hand, observed large atheromas.3 These differences in the results obtained by the various authors are probably a consequence of differences in dietary conditions. In the experiments reported above, a solution of cholesterol in warm cottonseed oil was mixed thoroughly with the diet, and bile salt was added to the latter in order to increase absorption of the sterol from the gut. Goldman administered powdered cholesterol in gelatin capsules.2 Altschul fed dried egg yolk powder with milk and “yolk cake.“a As a consequence of these dietary variations, the following factors were probably different in the experiments of the mentioned groups: (1) the amount of cholesterol absorbed from the gut and (2) the presence and nature of certain other components in the diet which might have had an effect of their own on the aorta. The latter point was recently studied by Altschul.6 When the response to cholesterol feeding of the hamsters is compared with those of the other species studied, it is evident that the accumulation of cholesterol in the liver did not follow the pattern of its blood level. In the hamsters the plasma cholesterol rose to only intermediate levels, but the hepatic sterol content reached extremely high values. In chickens, the plasma cholesterol was more than five times as high as that in rats, but the liver cholesterol levels were similar in these two species. Regarding a relationship between the plasma cholesterol concentration and its effects on the aorta, it was found that macroscopic intimal cushions developed only in the chicken and the rabbit, the two species in which the plasma concentration rose to the highest levels. Among the other species, hamsters exhibited excessive fatty infiltration, while the rats, although showing the lowest of the experimental plasma sterol levels, developed, in a few instances, minor morphological changes. SUMMARY
The effects of a diet containing 1 per cent cholesterol and 0.25 per cent bile salt were studied in hamsters. After feeding periods of sixteen to twenty-five weeks, the plasma cholesterol was significantly increased; in the liver an extremely high accumulation of the sterol and a severe fatty degeneration were observed. Sections of the aorta revealed an almost normal structure associated with considerable fatty infiltration. The effects observed in hamsters fed 1 per cent cholesterol without supplementation by bile salt were qualitatively similar, but somewhat smaller in extent, These results obtained with hamsters were compared with the effects of cholesterol feeding in three other species, i.e., the rat. rabbit, and chicken.
128
AMERICAN
HEART
JOURNAL
REFERENCES
1.
2. 3. 4. 5. 6.
Reviewed by (a) Duff, G. L.: Experimental Cholesterol Arteriosclerosis and Its Relationship to Human Arteriosclerosis, Arch. Path. 20:81 and 259, 1935; (b) Hueper, W. C.: Arteriosclerosis, Arch. Path. 39:122 and 187, 1945; (c) Page, I. W.: Arteriosclerosis and Lipid Metabolism, Biol. Symp. 11:43, 1945; (d) Katz, L. N., and Dauber, D. V.: The Pathogenesis of Atherosclerosis, J. Mt. Sinai Hosp. 12:382, 1945-1946; (e) Gubner, R., and Ungerleider, H. E.: Arteriosclerosis, Am. J. Med. 6:60, 1949. Goldman. I.: Effect of Cholesterol Feeding in Hamsters. Arch. Path. 49:169. 1950. Altschul, ‘R.: Experimental Cholesterol Arteriosclerosis. ’ II. Changes Produced in Golden Hamsters and in Guinea Pigs, AM. HEART J. 40:401, 1950. Schoenheimer, R.: Ueber die Resorptionsbeschleunigung des Cholesterins bei Anwesenheit von Desoxvcholsaure. B&hem. Ztschr. 147:2X 1924. Nieft, M. L., and Deuel, H.‘J., Jr.: Studies on Cholesterol Esterases. I. Enzyme Systems in Rat Tissues, J. Biol. Chem. 177:143, 1949. Altschul, R.: Selected Studies on Arteriosclerosis, Springfield, Ill., 1950, Charles C Thomas, Publisher.