- Email: [email protected]
Effect of dietary protein and cholesterol on atherosclerosis in swine
EXPERIMENTAL
AND
MOLECULAR
Effect P. P. GUPTA, Department
PATHOLOGY
22,
305-316
of Dietary Protein on Atherosclerosis H. D. TANDON,
of Pathology, All-India
Institute
(1975)
and Cholesterol in Swine
AND V. RAMALINGASWAMI
of Medical
Sciences, New Delhi 110016, India
Received June 3, 1974 Two groups of pigs, each consisting of six animals, were fed on isocaloric amounts of experimental diet with a high cholesterol content but no added fat and with varying levels of protein (5% vs 25% by weight of the diet) for 16 mo. Animals of the low-protein group were confirmed to have developed a proteindeficiency state by the characteristic microscopic changes in the viscera and hypoproteinemia due to reduced albumin fraction. They had a larger surface area of the aorta involved with atherosclerosis, and the lesions had a higher lipid and cholesterol and lower phospholipid content. The serum cholesterol was significantly higher, and the serum cholesterol esters contained larger proportion of oleic acid at the expense of linoleic acid than the animals of the high protein group. The results indicate that very low levels of dietary protein have a promotive effect on the induction of hypercholesterolemia and atherosclerotic lesions in the presence of cholesterol alone and in the absence of additional fats. The precise mechanism of this variation is not understood.
In a previous communication, (Gupta et al., 1974a), we have reported the effect of varying levels of dietary protein on the development and evolution of experimentally induced atherosclerosis in swine. The results have suggested that extremely low levels of dietary proteins have a promotive effect on the induction of atherosclerotic lesions whereas adequate levels have a protective effect. The atherogenic diet used contained high levels of saturated fats in addition to cholesterol. These studies have been extended to observe the long-term effects of feeding small dosesof cholesterol without the addition of standard fats on the atherosclerotic lesions in the two groups of pigs fed on diets containing high and low levels of dietary proteins, respectively. The results of this study are presented here. MATERIALS
AND METHODS
Twelve male Indian pigs (Sus Scrofa domestica) approximately 6-S mo of age and weighing 34-54 kg initially were used. The animals were placed in individual concrete floor pens and kept under observation for 1 mo on normal stock diet. They were then divided into two groups of six animals each and put on high and low-protein diets as shown in Table I, which also gives their composition. The ingredients of diets for the two groups of animals were essentially similar, differing only with respect to their protein content. The two diets were isocaloric. The daily food intakes in the two groups were equalized 305 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
GUPTA,
306
TANDON
AND TABLE
RAMALINGASWAMI I
COMPOSITION OF DIETS Ingredient
Sag0 Yellow maize Peanut cake Wheat bran Salt mixture0 Vitamin mixtured Cholesterol
Nutrient content (percent dry weight) Proteins
Carbohydrates
0.2 11.1 40.9 l-5.9
87.1 66.2 38.8 66.2
135 mg/kg Proteins Percent calories (wprox)
6.8
Percent
Fats
89.1
Group I (low protein)8
Group II (high protein)b
70 10
20 10 3.5 25 2 2
0.2 3.6 7.4 4.2
body wt/animal/day CarboFats hydrates 4.1
composition
1: 2 2
Proteins
28.3
As in Group Carbohydrates 61.2
I Fats
10.5
* Protein content 5%. b Protein content 25%. c Salt mixture: Manufactured by M/s. Shaw Wallace & Co. Ltd., P.O.B. No. 14, Madras-l, under the trade name “Starmin-Hog Special.” d All the water-soluble vitamins were mixed with finely ground sugar in such a proportion that each gram of the resultant mixture contained the following quantities: Thiamine hydrochloride 0.5 mg, riboflavin 0.4 mg, niacin 4.0 mg, calcium pentothenate 3.0 mg., pyridoxin hydrochloride 0.5 mg, para-amino benzoic acid 50.0 mg, inosito125 mg, pteroylglutamic acid 0.5 mg, biotin 0.05 mg, vitamin B-12 1.00 mg, ascorbic acid 10.0 mg, vitamin K 10.0 mg, and choline chloride 100 mg.
throughout the experiment by offering to the high-protein group of animals (Group II) on an average, the same quantities that were being consumed by the low-protein animals (Group I). A measured quantity of cholesterol ( 135 mg/kg body wt/day) made in the form of a cake with little sugar and starch were administered separately and individually to each animal in the morning before offering the bulk of the diet. The following investigations were carried out in all the animals before starting the experiment and at monthly intervals thereafter: total body weight, hemoglobin, hematocrit, total serum proteins, differential serum proteins by paper electrophoresis, total cholesterol, phospholipids, and triglycerides in the serum. The cholesterol esters of the terminal serum samples were also analyzed for their fatty acid composition. The methods of chemical estimation of proteins and lipids in the serum, the gross and microscopic grading of the atherosclerotic lesions, the extraction and purification of lipids in the tissues, their chemical determination, and fatty acids analysis by gas-liquid chromatography, were as described in our previous communications (Gupta, Tandon, and Ramalingaswami, 1969, 1970, and 1971; Gupta et al., 1974a, b). The animals in both groups were fed on their respective diets for 16 mo after which they were sacrificed. After autopsy, the aortic and coronary atherosclerosis was graded for severity both grossly and microscopically. In addition,
DIETARY
PROTEIN
AND
CHOLESTEROL
IN
SWINE
ATHEROSCLEROSIS
307
all the visceral organs were weighed and studied for any pathologic changes. Stripped portions of aortic intima involved with atherosclerosis from each animal were subjected to chemical estimation of lipids and their characterization. Means of values of each fraction obtained from all animals of a group were calculated and compared with those of the others. RESULTS Body weight. The .pigs fed the high-protein diet of the experiment, after which there was a slight Animals of the low-protein group gained weight of the experiment and thereafter showed a gradual,
90
-
HIGH
o---a
LOW PROTEIN
PROTEIN
gained steadily up to 13 mo decline in their body weight. only during the first 7 mo continuous decline (Fig. 1).
+CHOLESTEROL + CHOLESTEROL
es 1
iENGTI4
FIG. 1. Body weights and the serum of the two groups.
levels
OF
TIM
of total
OF
EXPERIMENT
cholesterol,
(MONTHS)
phospholipids,
and
triglycerides
in
GUFTA,
308
0
I
TANDON
AND
RAMALINGASWAMI
a.-.-.
HIGH
PROTEIN
+CHOLESTEROL
o--O
LOW
PROTEIN
+ CHOLESTEROL
2
3 LENGTH
4
5
6 OF
7 TIME
8 OF
9
IO
II
EXPERIMENT
I2
I3
I4
IS
I6
17
18
FIG. 2. Graph showing changes in the total serum proteins in the two groups The inset shows the electrophoretogram of serum proteins of a pig before starting ment (left) and after 16 mo of feeding the low-protein diet (right). Note the in the albumin fraction induced by protein deficiency.
of animals. the experimarked fall
At the end of the study, these animals were weak, stunted, and had developed moderate edema in the dependent parts of the body. Hematological observations. Both hemoglobin and packed-cell volume (PCV) decreased markedly and were significantly (P less than 0.01) lower in the lowprotein group than in the high-protein group in which the levels gradually rose and remained elevated for most of the experiment. Serum proteins. There was a linear decline in the levels of serum total proteins of the low-protein group throughout the experimental period (Fig. 2); those in the high-protein group remained significantly unaltered. The difference between the two groups was highly significant (P < 0.01) at 16 mo. The decline in the serum total proteins of the low-protein animals seemed to have been contributed by a marked fall in the albumin fraction (Fig. 2). Serum lipids. The value for total cholesterol, phospholipids and triglycerides in the serum are given in Fig. 1. In both the groups, there was a slow and gradual rise in the levels of serum cholesterol, reaching a maximum after 8 mo. Although there was no significant difference between the two groups, the levels tended to be higher in the low-protein group during this period. After 8 mo, the serum cholesterol values in the high-protein group started declining, and reached basal levels towards the end of the experiment. On the other hand, in the lowprotein group, these values maintained a plateau after a slight initial decline and remained elevated for the rest of the experimental period. This dichotomization of cholesterol levels coincided with the break in weight gain in the low-protein group (Fig. 1). There was highly significant (P < 0.01) difference between serum cholesterol levels of the two groups at the end of the experiment. The serum phospholipids and triglycerides levels tended to be fluctuant in both the groups. There was an initial rise in these values in the first few months followed by a decline. Toward the end of the experiment, the triglyceride levels
DIETARY
PROTEIN
AND CHOLESTEROL
IN SWINE
TABLE FATTY
ACID
COMPOSITION
OF THE SERUM
ATHEROSCLEROSIS
309
II
CHOLESTEROL
ESTERS
IN THE
Two
GROUPS
Fatty acid (Percent of the total)
Group 12:0 14:0 15:0
16:0
16:l
16:2 18:0 18:ls
18:2’
18:3 20:0 20:3 20:4
I Low protein +cholesterol
0.5
0.3
1.8
13.4
3.8
3.1
1.2
52.4
17.5
0.4
1.7
1.6
2.3
High protein +cholesterol
0.8
0.6
1.4
12.8
1.8
2.2
0.7
31.1
40.4
1.7
1.7
1.7
3.1
II
(P
8 The levels of oleic (18: 1) and liioleic 0.01) different.
(18:2) acids between the two groups are significantly
in both groups were close to the basal levels while the phospholipid 1eveIs were much lower than the basal levels. There was no significant difference in these values between the two groups. Fatty acid composition of the serum cholesterol esters. The results of fatty acid analysis of the cholesterol esters of the terminal serum samples of the two groups are given in Table II. In both the groups, oleic ( 18 : 1) and linoleic ( 18:2) acids were the major fatty acids constituting about 70% of the total. The oleic acid level was significantly higher and the linoleic acid level significantly lower in the low-protein group than in the high-protein group, There was no significant difference in the proportion of the other fatty acids between the two groups. Atherosclerotic lesions. The percentage of intimal surface area involved with atherosclerosis in the two groups of animals is given in Table III. In animals in the low-protein group I, there was a significantly larger intimal surface area TABLE PERCENTAQE
OF INTIMAL
SURFACE INVOLVED IN THE Two
Pig no.
Aorta
Low protein +cholesterol
1 2 3 4 5 6
60 48 70 10 40 60
High protein +cholesterol
7 8 9 10 11 12
15 28 52 33 10 15
Group
III WITH ATKEROBCLEROTIC GROUPS
Mean of the values and their standard errors
LESIONS
Coronary arteries
I 48.0 f 8.7
II 25.5 f 6.4
No lesion -
-
310
GUPTA,
TANDON
AND RAMALINGASWAMI
DIETARY
PROTEIN
AND
CHOLESTEROL GROUP1 GROUPII-
-LOW HIGH
IN PROTElN PROTEIN
SWINE
ATHEROSCLEROSIS
311
+ CHOLESTEROL tCHOLESTEROL
251
n 0
q
TOML LIPID ESTERIFIEO CHOLESTEROL FREE CHOLESTEROL
N PHOSPHOLIPIDS q TRIGLYCERIDES
GROUP1
FIG. of the
4. Values two groups
GROUP
of total lipid and its various at the end of the experiment.
II
fractions
in
the
aortic
atherosclerotic
lesions
involved with atherosclerosis in the aorta (48.0 2 8.7% ) than that in the high-protein group II (25.5 + 6.4% ) as shown in Fig. 3. Coronary arteries were not significantly involved in either of the two groups except for small fatty streaks at the emergence of the main branches. Grossly and microscopically, the aortic atherosclerotic lesions were fatty streaks, and there was no difference in the severity of the lesions between the two groups. The distribution, the macroscopic and microscopic features of the lesions were similar to those already described in our previous communications (Gupta et al., 1969, 1974). Lipids in aortic atherosclerotic lesions. The level of total lipids and the various lipid fractions in the aortic lesions of the two groups of animals are given in Fig. 4. The values of total lipids and of each lipid fraction shown in the figure represent means of values obtained from all six animals in each group. In each animal, the total lipid and cholesterol levels were significantly higher and the phospholipid levels significantly lower in the aortic lesions of the lowprotein than those in the high-protein group. The cholesterol:phospholipid ratio was several times higher in the former ( 3 : 1) than that in the latter ( 1: 2). There was no significant difference in the triglyceride content of the lesions between the two groups. Changes in other organs. No significant change was seen in any of the visceral and musculoskeletal organs in the animals fed high-protein diet. Animals fed the low-protein diet showed the usual morphologic changes of protein deficiency state in various organs such as periportal fatty change in the liver, pancreatic acinar atrophy with loss of zymogen granules, marked atrophy of red pulp and lymphoid tissue in the spleen, brown atrophy of the myocardium, atrophy of mucosa of stomach and intestine, and marked reduction in endochondral bone formation.
312
GUFTA,
TANDON
AND RAMALINGASWAMI
DISCUSSION Our previous studies (Gupta et al., 1974a) have demonstrated that an adequate level of protein in the diet protects the pig from the atherogenic effects of a diet containing 25% saturated fats in addition to cholesterol whereas a very low level of protein has the opposite effect of aggravating the lesions. The present experiment was designed to study to what extent is cholesterol alone responsible for inducing and enhancing the lesions in the presence of a proteindeficiency state. The alterations in body weight, general appearance, serum proteins, hematological values, and the presence of characteristic morphological changes in the various organs observed in animals receiving a protein-deficient diet indicate that a state of severe protein deficiency was produced in these animals, with all the essential features of protein depletion described in man (Trowell, Davies, and Dean, 1954), monkeys (Deo, Sood, and Ramalingaswami, 1965) and pigs (Pond et al., 1965; Tumbleson et al., 1969). Th ese 1 changes are also similar to those described by us in pigs in our previous studies (Gupta et nl., 1974a, b). Protein deficiency is known to cause anemia (Sood et al., 1965) as observed in our protein-deficient animals. On the other hand, the hemoglobin and hematocrit showed a gradual rise in animals receiving a protein-adequate diet. This phenomenon is known to occur in growing pigs (Miller et al., 1961; McClellan et al., 1965). The animals were aged 6 mo at the time the experiment was started. While in the previous study ( Gupta et al., 1974a), atherogenesis was induced by the combined use of saturated fat to the extent of 25% by weight in the diet and cholesterol. In the present study only cholesterol was used, there was no added fat, and the bulk of the calories came from carbohydrates (Table I). This study extends and confirms our previous observations that a diet containing a low level of protein to the extent of 5% in the diet promotes the development of atherosclerosis. The lesions observed were confined to the aorta. They were more extensive though histologically not more severe, in the low-protein than in the high-protein group. The surface area involved in the low-protein group in the present experiment was less (48.0 * 8.7% ) than that observed in the last experiment (67 * 3.80% ). The difference is understandable, since the atherogenie stimulus was milder comprising cholesterol alone, even though the amount of cholesterol fed in the present experiment was proportionately higher than that in the previous experiment. What seems interesting, however, is that protein deprivation makes the animal prone to develop atherosclerosis, in the presence of small doses of cholesterol even in the absence of additional saturated fats. The surface area involved in the high-protein group (25.5 * 6.4% ) was not too different from that in the same experimental group in our last study (30.5 A 10.88%) or its control (31.5 + 13.44%). The striking difference in the degree of atherosclerosis as observed in the two groups could be ascribed to two factors-low levels of protein and/or excessive quantities of carbohydrates in the diet of animals of Group I. There is extensive evidence that dietary protein levels play a significant role in this connection. Several authors have reported in studies using chickens that atherosclerosis and serum cholesterol levels were more dependent on the levels of dietary protein than those of dietary fats. Nishida, Takenaka, and Kummerow, 1958; Leivelle, Feigenbaum, and Fisher, 1960; March and Bielly, 1959; Kokatnur et al., 1956
DIETARY
PROTEIN
AND
CHOLESTEROL
IN SWINE
ATHEROSCLEROSIS
313
have recorded similar observations in chickens. It has been well established by studies that decreased protein intake aggravates the development of atherosclerosis in birds (Stamler, Pick, and Katz, 1958a, b; Pick, Stamler and Katz, 1959; Pick et al., 1965; Kokatnur et al., 1956, 1958); rats (Fillios et al., 1958); rabbits (Polack et aI., 1965); monkeys (Mann et al., 1953), dogs (Li and Freeman, 1946) ; and pigs ( Greer et al., 1966), As was observed in our previous study (Gupta et al., 1974a), in this experiment also the low-protein group of animals maintained higher serum cholesterol levels for most of the experimental period. This seems to be the determining factor for greater severity of atherosclerosis in these animals. Several investigators have reported that restricted protein intake increases serum cholesterol concentrations in many species of animals (Rose and Balloun, 1969; Johnson, Leveille, and Fisher, 1958; Fisher et al., 1959; Chaikoff et al., 1961; Greer et al., 1966; Tumbleson et al., 1969). On the contrary, Barnes et al. (1959) and Moreland ( 1965) did not observe any effect on the levels of dietary protein in swine. This may be because in these studies biochemically demonstrable hypoproteinemia was not produced. Moreland (1965) used 8% protein in the low-protein diet and this level of protein was perhaps too high to produce hypoproteinemia and lesions associated with protein deficiency. Similarly, Barnes et al. (1959) also did not observe any change in the serum cholesterol values as long as their low-protein animals continued to be normoproteinemic, but after they had removed all protein from the diet and the animals developed hypoproteinemia, they observed some elevation of serum cholesterol. In this study also we observed hypercholesterolemia in low-protein animals only after they had developed clinical and biochemical signs of protein deficiency. The mechanism of hypercholesterolemia in experimental protein deficiency has been studied in several species and has been reviewed in our earlier communication (Gupta et al., 1974). Apart from the higher serum cholesterol values in the protein-deficient animals, their fatty acid composition in the serum cholesterol esters also showed some striking differences from those of the high-protein groups. The serum cholesterol esters of the low-protein group contained a larger proportion of oleic acid ( 18: 1) at the expense of linoleic acid ( 18:2). Several studies (Bottcher and Woodford, 1961; Schrade et al., 1961) have provided evidence that subjects with atherosclerosis tend to have a lower percentage of linoleic acid ( 18:2) and a higher percentage of oleic acid ( 18: 1) in their serum cholesterol esters as compared with normal subjects. Similar data have been reported in experimental atherosclerosis in rabbits (Zilversmit et cd., 1961; Swell et al., 1961, 1962). Thus, an increase in the oleic acid content at the expense of linoleic acid in the serum cholesterol esters of the low-protein animals could be a factor accounting for the proneness of these animals to develop more extensive lesions in our animals. The lesional lipids in the low-protein group had a higher cholesterol content and cholesterol:phospholipid ratio than in the high-protein group. The higher cholesterol content in the lesions may be related to the higher serum cholesterol values in these animals. Several experimental studies (Newman and Zilversmit, 1962; Newman et al., 1968; Day and Wilkinson, 1967) indicate that in aortic atheroscIerotic lesions, choIestero1 and its esters are derived primarily from the circulating blood and the rate of their accumulation is directly related to the severity
314
GUPTA,
TANDON
AND
RAMALINGASWAMI
of hypercholesterolemia. Our observation is similar to that of Fisher et al. (1959) who studied the effect of different fats and varying levels of protein and cholesterol on the biochemical changes in the aorta of chicken. They observed a higher cholesterol content in the aorta of the animals fed a low-protein diet as compared with those on a high-protein diet. The decrease in the phospholipid content in the atherosclerotic lesions of lowprotein animals in this study may be due to its retarded local synthesis. Studies with labeled phosphate (Shore et al., 1955; Zilversmit et al., 1954) and acetate (Newmann et al., 1961) have shown that the source of phospholipids in the atherosclerotic lesions is primarily from its synthesis in situ. It has been reported that in chronic protein deficiency, the synthesis of phospholipids in the body tissues is greatly retarded (MacDonald, 1960; Chatterjee and Mukherjee, 1968). The role played by the larger carbohydrate component of the protein-deficient diet cannot be dismissed lightly. The carbohydrate content of the proteindeficient diet was larger in this as compared with that of the same group in our previous experiment. It is known that dietary carbohydrates do influence the concentration and composition of serum, liver, and depot lipids (MacDonald, 1962; 1967). Among different types of carbohydrates, however, starch, which was the main carbohydrate constituent in our experimental diet, has been known to be the least atherogenic, if at all (MacDonald, 1962; Adams et al., 1959; Pick and Katz, 1965). ACKNOWLEDGMENT The
study
was supported
by a financial
grant
from
the
Indian
Council
of Medical
Research.
REFERENCES FISHER, M., and KOVAL, C. J. ( 1969). The influence of dietary carbohydrates on and liver damage and serum cholesterol in the rat. Fed. Proc. 18, 178. BARNES, R. H., KWONG, E., FIALA, G,, Recheigl, M., Lutz, P. N., and LOOSLI, J. K. (1959). Dietary fat and protein and serum cholesterol. I. Adult swine. J. Nutr. 69, 261. BOTTCHER, C. J. F., and WOODFORD, F. P. ( 1961). Lipid and fatty acid composition of plasma lipoproteins in cases of aortic atherosclerosis. J. Atheroscler. Res. 1, 434. CHAIKOFF, I. L., NXXOLS, C. W., JR., GAFFEY, W., and LINUSAY, S. (1961). The effect of dietary protein level on the development of naturally occurring aortic atherosclerosis in the chicken. J. Atheroscler. Res. 1, 461. CHATTERJEE, K. K., and MUKHERJEE, K. L. (1968). Phospholipids of the liver in children suffering from protein-calorie malnutrition. Brit. J. Nutr. 22, 145. DAY, A. J., and WILKINSON, G. K. (1967). Incorporation of Cl4 labelled acetate into lipid by isolated foam cells and by atherosclerotic arterial intima. Circ. Res. 21, 593. DEO, M. G., SOOD, S. K., and RAMALINGASWALII, V. ( 1965). Experimental protein deficiencyPathological features in the rhesus monkey. Arch. Puthol., 80, 14. FILLIOS, L. C., NAITO, C., ADAMS, S. B., PORTMAN, 0. W., and MARTIN, R. S. (1958). Variations in cardiovascular sudanophilia with changes in the dietary level of protein. Amer. J. Physiol., 194, 275. FISHER, M., FEIGENBAUM, A., LEVEILLE, G. A., WEISS, H. S., and GRIhfhfINGER, P. (1959). Biochemical observations on aortas of chickens: Effect of different fats and varying levels, of protein, fat and cholesterol. J. Nutr. 69, 163. GREER, S. A. N., HAYS, V. W., SPEER, V. C., and h~cCALL, J. T. (1966). Effect of dietary fat, protein and cholesterol on atherosclerosis in swine. J. Nut?. 90, 183. GUPTA, P. P., TANDON, H. D., and RAhKALINGASWAhfI, V. (1969). Spontaneous vascular lesions in Indian pigs. J. Pathol. 99, 19. ADAMS,
kidney
M.,
DIETARY
PROTEIN
AND
CHOLESTEROL
IN
SWINE
ATHEROSCLEROSIS
315
GUPTA, P. P., TANWN, H. D., and RAMALINGASWAMI, V. ( 1970). Experimental atherosclerosis in rabbits with special reference to reversal. J. Pathol. 101, 309. GUPTA, P. P., TANDON, H. D., and RAMALINGASWAMI, V. (1971). Further observations on the reversibility of experimentally induced atherosclerosis in rabbits. J. Pathol. 105, 229. GUPTA, P. P., TANWN, H. D., and RAMALINGASWAMI, V. (1974a). Experimental atherosclerosis in swine: effect of dietary protein and high fat. Erp. Mol. PathoE. 20, 115. GUPTA, P. P., TANDON, H. D., and RAMALINGASWAMI, V. ( 1974b). Liver changes in pigs fed a diet low in protein and high in fat and cholesterol. Ind. J. Med. Res., in press. JOHNSON, D., LE~E~LLE, G. A., and FISHER, H. (1958). Iniluence of amino acid deficiencies and protein level on the plasma cholesterol of the chick. J. Nutr. 66, 367. KOKATNUR, M., RAND, N. T., KUMMEROW, F. A., and SCOTT, H. M. (1956). Dietary proteina factor which may reduce serum cholesterol levels. Circulation 14, 1962. KOKATNUR, M., RAND, N. T., KUMMEROW, F. A., and Sccrrr, H. M. (1958). Effects of dietary protein and fat on changes of serum cholesterol in mature birds. J. Nutr. 64, 177. LEVEILLE, G. A., FEIGENBAUM, A. S., and FISHER, H. ( 1960). The effect of dietary protein, fat and cholesterol on serum protein components of the growing chick. Arch. Biochem. Biophys. 86, 67. Lr, T. W., and FREEMAN, S. ( 1946). Experimental lipaemia and hypercholesterolemia for protein depletion and by cholesterol feeding in dogs. Amer. J. Physiol. 145, 660. MACDONALD, I. (1960). Hepatic lipid of malnourished children. Metab. Clin. Exp. 9, 838. MACDONALD, I. ( 1962). Some-influence of dietary carbohydrates on liver and depot lipids. J. Physiol. 162, 334. MACDONALD, I. ( 1967). Inter-relationship between the influences of dietary carbohydrates and fats on fasting serum lipids. Amer. J. Clin. N&r. 20, 345. MANN, G. V., ANDRUS, S. B., MCNALLY, A., and STARE, F. J. (1953). Experimental atherosclerosis in cebus monkeys. J. Exp. Med. 98, 195. MARCH, B. E., and BIELLY, J. ( 1959). Dietary modification of serum cholesterol in the chick. J. Nutr. 69, 105. MCCLELLAN, R. O., VOGT, G. S., and RAGAN, H. A. ( 1965). Age related changes in haematological and serum biochemical parameters in immature swine. In “Swine in Biomedical Research.” Proc. Int. Symp., Richland, Washington, July 19-21, p. 597. MILLER, E. R., ULLRE, D. E., ACKERMAN, I., SCHMIDT, D. A., LUECKE, R. W., and HOEFER, J. A. ( 1961). Swine haematology from birth to maturity. J. Animal. SC., 20, 890. MORELAND, F. ( 1965). Experimental atherosclerosis of swine, In “Comparative Atherosclerosis” (J. C. Roberts, Jr. and R. Straus, els.) p. 21. Harper & Row, New York. NEUTMAN, H. A. I., MCCANDESS, E. L., and ZILVERSM~, D. B. ( 1961). The synthesis of Cl’-lipids in rabbit atheromatous lesions. J. Biol. Chem. 236, 1264. NEUTMAN, H. A. I., and ZILVERSMIT, D. B. (1968). Q uantitative aspects of cholesterol flux in aortas of cholesterol-fed rabbits. J. Atheroscler. Res., 8, 745. NEWMAN, H. A. I., and ZILVERSMIT, D. B. ( 1962). Quantitative aspects of cholesterol flux in rabbit atherosclerotic lesions. J. Biol. Chem. 237, 2078. NIS~DA, T., TAKENAKA, F. T., and KUMMEROW, F. A. ( 1958). Effect on dietary protein and heated fat on serum cholesterol and beta-lipoprotein levels and on the incidence of experimental atherosclerosis in chicks. Circ. Res. 6, 194. PICK, R., STAMLER, J., and KATZ, L. N. (1959). Effects of high protein intakes on cholesterolemia and atherosclerosis in growing and mature chickens fed high fat, high cholesterol diets. Circ. Res. 7, 866. PICK, R., and KATZ, L. N. ( 1965). Multiplicity of factors influencing atherosclerosis. Actu Cardiol. Suppl. 11, 169. PICK, R., JAIN, S., KA’IZ, L. N., and JOHNSON, P. (1965). Effect of dietary protein level on regression of cholesterol-induced hypercholesterolemia and atherosclerosis of cockerels. J. Atheroscler. Res. 5, 16. POLCAK, J., MELICHAR, F., SEVELOVA, D., DVORAK, I., and SKALOVA, M. (1965). The effect of a meat enriched diet on the development of experimental atherosclerosis in rabbits. J. Atheroscler. Res. 5, 174. POND, W. G., BARNES, R. H., REID, I., CROOK, L., KWONG, E., and MOORE, A. U. (1965). Protein deficiency in baby pigs, In “Swine in Biomedical Research.” p. 213. Proc. Int. Symp. Richland, Washington, 19-21 July.
316
GUPTA,
TANDON
AND
RAMALINGASWAMI
ROSE, R. J., and BALLOUN, S. L. ( 1969). Effect of restricted energy and protein intake on atherosclerosis and associated physiological factors in cockerels. J. Nutr. 98, 335 SCHFLADE, W., BI~GLER, R., and BOHLE, E. ( 1961). Fatty acid distribution in the lipid fractions of healthy persons of different age, patients with atherosclerosis and patients with idiopathic hyperlipidaemia. J. Atheroscler. Res. 1, 47. SHORE, M. L., ZILVERSMIT, D. B., and ACKERMAN, R. F. ( 1955). Plasma phospholipid deposition and aortic phospholipid synthesis in experimental atherosclerosis. Amer. 1. Physiol. 181, 527. SOOD, S. K., DEO, M. G., and RAMALINGASWAIII, V. (1965). A nemia in experimental protein deficiency in Rhesus monkey with special reference to iron metabolism. Blood 26, 421. STAMLER, J., PICK, R., and KATZ, L. N. ( 1958a). Effects of dietary proteins, methionine and vitamins on plasma lipids and atherosclerosis in cholesterol fed cockerels. Circ. Res. 6, 442. STAMLER, J., PICK, R., and KATZ, L. N. (1958b). Effects of dietary protein and carbohydrate level on cholesterolemia and atherogenesis in cockerels on high-fat, high-cholesterol mash.
Circ. Res. 6, 447. SWELL, L., Low, M. D., SCHOOLS, P. E., JR., and TREADWELL, C. R. (1961). Tissue lipid fatty acid composition in pyridoxin-deficient rats. J. Nzctr. 74, 148. SWELL, L., Low, M. D., and TREADWELL, C. R. (1962). Sterol ester formation in rat liver homogenates. PTOC. Sot. Erp. Biol. Med. 109, 176. TROWELL, H. C., DAVIES, J. N. P., and DEAN, R. F. A. (1954). “Kwashiorkor,” ed. 1. Arnold, London. TUMBLESON, M. E., TINSLEY, 0. W., CORWIN, L. A., JR., FLATT, R. E., and FLYNN, M. A. ( 1969). Undernutrition in young miniature swine. J. Nutr. 99, 505. ZILVERWIT, D. B., SHORE, M. L., and ACKERSIAN, R. F. ( 1954). The origin of aortic phospholipid in rabbit atheromatosis. Circulation 9, 581. ZILVERSMIT, D. B. MCCANDLESS, E. L., JORDAN, P. H., JR., HENLY, W. S., and ACKERMAN, R. F. ( 1961). The synthesis of phospholipids in human atheromatous lesions. Circdution
23, 370.
AND
MOLECULAR
Effect P. P. GUPTA, Department
PATHOLOGY
22,
305-316
of Dietary Protein on Atherosclerosis H. D. TANDON,
of Pathology, All-India
Institute
(1975)
and Cholesterol in Swine
AND V. RAMALINGASWAMI
of Medical
Sciences, New Delhi 110016, India
Received June 3, 1974 Two groups of pigs, each consisting of six animals, were fed on isocaloric amounts of experimental diet with a high cholesterol content but no added fat and with varying levels of protein (5% vs 25% by weight of the diet) for 16 mo. Animals of the low-protein group were confirmed to have developed a proteindeficiency state by the characteristic microscopic changes in the viscera and hypoproteinemia due to reduced albumin fraction. They had a larger surface area of the aorta involved with atherosclerosis, and the lesions had a higher lipid and cholesterol and lower phospholipid content. The serum cholesterol was significantly higher, and the serum cholesterol esters contained larger proportion of oleic acid at the expense of linoleic acid than the animals of the high protein group. The results indicate that very low levels of dietary protein have a promotive effect on the induction of hypercholesterolemia and atherosclerotic lesions in the presence of cholesterol alone and in the absence of additional fats. The precise mechanism of this variation is not understood.
In a previous communication, (Gupta et al., 1974a), we have reported the effect of varying levels of dietary protein on the development and evolution of experimentally induced atherosclerosis in swine. The results have suggested that extremely low levels of dietary proteins have a promotive effect on the induction of atherosclerotic lesions whereas adequate levels have a protective effect. The atherogenic diet used contained high levels of saturated fats in addition to cholesterol. These studies have been extended to observe the long-term effects of feeding small dosesof cholesterol without the addition of standard fats on the atherosclerotic lesions in the two groups of pigs fed on diets containing high and low levels of dietary proteins, respectively. The results of this study are presented here. MATERIALS
AND METHODS
Twelve male Indian pigs (Sus Scrofa domestica) approximately 6-S mo of age and weighing 34-54 kg initially were used. The animals were placed in individual concrete floor pens and kept under observation for 1 mo on normal stock diet. They were then divided into two groups of six animals each and put on high and low-protein diets as shown in Table I, which also gives their composition. The ingredients of diets for the two groups of animals were essentially similar, differing only with respect to their protein content. The two diets were isocaloric. The daily food intakes in the two groups were equalized 305 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
GUPTA,
306
TANDON
AND TABLE
RAMALINGASWAMI I
COMPOSITION OF DIETS Ingredient
Sag0 Yellow maize Peanut cake Wheat bran Salt mixture0 Vitamin mixtured Cholesterol
Nutrient content (percent dry weight) Proteins
Carbohydrates
0.2 11.1 40.9 l-5.9
87.1 66.2 38.8 66.2
135 mg/kg Proteins Percent calories (wprox)
6.8
Percent
Fats
89.1
Group I (low protein)8
Group II (high protein)b
70 10
20 10 3.5 25 2 2
0.2 3.6 7.4 4.2
body wt/animal/day CarboFats hydrates 4.1
composition
1: 2 2
Proteins
28.3
As in Group Carbohydrates 61.2
I Fats
10.5
* Protein content 5%. b Protein content 25%. c Salt mixture: Manufactured by M/s. Shaw Wallace & Co. Ltd., P.O.B. No. 14, Madras-l, under the trade name “Starmin-Hog Special.” d All the water-soluble vitamins were mixed with finely ground sugar in such a proportion that each gram of the resultant mixture contained the following quantities: Thiamine hydrochloride 0.5 mg, riboflavin 0.4 mg, niacin 4.0 mg, calcium pentothenate 3.0 mg., pyridoxin hydrochloride 0.5 mg, para-amino benzoic acid 50.0 mg, inosito125 mg, pteroylglutamic acid 0.5 mg, biotin 0.05 mg, vitamin B-12 1.00 mg, ascorbic acid 10.0 mg, vitamin K 10.0 mg, and choline chloride 100 mg.
throughout the experiment by offering to the high-protein group of animals (Group II) on an average, the same quantities that were being consumed by the low-protein animals (Group I). A measured quantity of cholesterol ( 135 mg/kg body wt/day) made in the form of a cake with little sugar and starch were administered separately and individually to each animal in the morning before offering the bulk of the diet. The following investigations were carried out in all the animals before starting the experiment and at monthly intervals thereafter: total body weight, hemoglobin, hematocrit, total serum proteins, differential serum proteins by paper electrophoresis, total cholesterol, phospholipids, and triglycerides in the serum. The cholesterol esters of the terminal serum samples were also analyzed for their fatty acid composition. The methods of chemical estimation of proteins and lipids in the serum, the gross and microscopic grading of the atherosclerotic lesions, the extraction and purification of lipids in the tissues, their chemical determination, and fatty acids analysis by gas-liquid chromatography, were as described in our previous communications (Gupta, Tandon, and Ramalingaswami, 1969, 1970, and 1971; Gupta et al., 1974a, b). The animals in both groups were fed on their respective diets for 16 mo after which they were sacrificed. After autopsy, the aortic and coronary atherosclerosis was graded for severity both grossly and microscopically. In addition,
DIETARY
PROTEIN
AND
CHOLESTEROL
IN
SWINE
ATHEROSCLEROSIS
307
all the visceral organs were weighed and studied for any pathologic changes. Stripped portions of aortic intima involved with atherosclerosis from each animal were subjected to chemical estimation of lipids and their characterization. Means of values of each fraction obtained from all animals of a group were calculated and compared with those of the others. RESULTS Body weight. The .pigs fed the high-protein diet of the experiment, after which there was a slight Animals of the low-protein group gained weight of the experiment and thereafter showed a gradual,
90
-
HIGH
o---a
LOW PROTEIN
PROTEIN
gained steadily up to 13 mo decline in their body weight. only during the first 7 mo continuous decline (Fig. 1).
+CHOLESTEROL + CHOLESTEROL
es 1
iENGTI4
FIG. 1. Body weights and the serum of the two groups.
levels
OF
TIM
of total
OF
EXPERIMENT
cholesterol,
(MONTHS)
phospholipids,
and
triglycerides
in
GUFTA,
308
0
I
TANDON
AND
RAMALINGASWAMI
a.-.-.
HIGH
PROTEIN
+CHOLESTEROL
o--O
LOW
PROTEIN
+ CHOLESTEROL
2
3 LENGTH
4
5
6 OF
7 TIME
8 OF
9
IO
II
EXPERIMENT
I2
I3
I4
IS
I6
17
18
FIG. 2. Graph showing changes in the total serum proteins in the two groups The inset shows the electrophoretogram of serum proteins of a pig before starting ment (left) and after 16 mo of feeding the low-protein diet (right). Note the in the albumin fraction induced by protein deficiency.
of animals. the experimarked fall
At the end of the study, these animals were weak, stunted, and had developed moderate edema in the dependent parts of the body. Hematological observations. Both hemoglobin and packed-cell volume (PCV) decreased markedly and were significantly (P less than 0.01) lower in the lowprotein group than in the high-protein group in which the levels gradually rose and remained elevated for most of the experiment. Serum proteins. There was a linear decline in the levels of serum total proteins of the low-protein group throughout the experimental period (Fig. 2); those in the high-protein group remained significantly unaltered. The difference between the two groups was highly significant (P < 0.01) at 16 mo. The decline in the serum total proteins of the low-protein animals seemed to have been contributed by a marked fall in the albumin fraction (Fig. 2). Serum lipids. The value for total cholesterol, phospholipids and triglycerides in the serum are given in Fig. 1. In both the groups, there was a slow and gradual rise in the levels of serum cholesterol, reaching a maximum after 8 mo. Although there was no significant difference between the two groups, the levels tended to be higher in the low-protein group during this period. After 8 mo, the serum cholesterol values in the high-protein group started declining, and reached basal levels towards the end of the experiment. On the other hand, in the lowprotein group, these values maintained a plateau after a slight initial decline and remained elevated for the rest of the experimental period. This dichotomization of cholesterol levels coincided with the break in weight gain in the low-protein group (Fig. 1). There was highly significant (P < 0.01) difference between serum cholesterol levels of the two groups at the end of the experiment. The serum phospholipids and triglycerides levels tended to be fluctuant in both the groups. There was an initial rise in these values in the first few months followed by a decline. Toward the end of the experiment, the triglyceride levels
DIETARY
PROTEIN
AND CHOLESTEROL
IN SWINE
TABLE FATTY
ACID
COMPOSITION
OF THE SERUM
ATHEROSCLEROSIS
309
II
CHOLESTEROL
ESTERS
IN THE
Two
GROUPS
Fatty acid (Percent of the total)
Group 12:0 14:0 15:0
16:0
16:l
16:2 18:0 18:ls
18:2’
18:3 20:0 20:3 20:4
I Low protein +cholesterol
0.5
0.3
1.8
13.4
3.8
3.1
1.2
52.4
17.5
0.4
1.7
1.6
2.3
High protein +cholesterol
0.8
0.6
1.4
12.8
1.8
2.2
0.7
31.1
40.4
1.7
1.7
1.7
3.1
II
(P
8 The levels of oleic (18: 1) and liioleic 0.01) different.
(18:2) acids between the two groups are significantly
in both groups were close to the basal levels while the phospholipid 1eveIs were much lower than the basal levels. There was no significant difference in these values between the two groups. Fatty acid composition of the serum cholesterol esters. The results of fatty acid analysis of the cholesterol esters of the terminal serum samples of the two groups are given in Table II. In both the groups, oleic ( 18 : 1) and linoleic ( 18:2) acids were the major fatty acids constituting about 70% of the total. The oleic acid level was significantly higher and the linoleic acid level significantly lower in the low-protein group than in the high-protein group, There was no significant difference in the proportion of the other fatty acids between the two groups. Atherosclerotic lesions. The percentage of intimal surface area involved with atherosclerosis in the two groups of animals is given in Table III. In animals in the low-protein group I, there was a significantly larger intimal surface area TABLE PERCENTAQE
OF INTIMAL
SURFACE INVOLVED IN THE Two
Pig no.
Aorta
Low protein +cholesterol
1 2 3 4 5 6
60 48 70 10 40 60
High protein +cholesterol
7 8 9 10 11 12
15 28 52 33 10 15
Group
III WITH ATKEROBCLEROTIC GROUPS
Mean of the values and their standard errors
LESIONS
Coronary arteries
I 48.0 f 8.7
II 25.5 f 6.4
No lesion -
-
310
GUPTA,
TANDON
AND RAMALINGASWAMI
DIETARY
PROTEIN
AND
CHOLESTEROL GROUP1 GROUPII-
-LOW HIGH
IN PROTElN PROTEIN
SWINE
ATHEROSCLEROSIS
311
+ CHOLESTEROL tCHOLESTEROL
251
n 0
q
TOML LIPID ESTERIFIEO CHOLESTEROL FREE CHOLESTEROL
N PHOSPHOLIPIDS q TRIGLYCERIDES
GROUP1
FIG. of the
4. Values two groups
GROUP
of total lipid and its various at the end of the experiment.
II
fractions
in
the
aortic
atherosclerotic
lesions
involved with atherosclerosis in the aorta (48.0 2 8.7% ) than that in the high-protein group II (25.5 + 6.4% ) as shown in Fig. 3. Coronary arteries were not significantly involved in either of the two groups except for small fatty streaks at the emergence of the main branches. Grossly and microscopically, the aortic atherosclerotic lesions were fatty streaks, and there was no difference in the severity of the lesions between the two groups. The distribution, the macroscopic and microscopic features of the lesions were similar to those already described in our previous communications (Gupta et al., 1969, 1974). Lipids in aortic atherosclerotic lesions. The level of total lipids and the various lipid fractions in the aortic lesions of the two groups of animals are given in Fig. 4. The values of total lipids and of each lipid fraction shown in the figure represent means of values obtained from all six animals in each group. In each animal, the total lipid and cholesterol levels were significantly higher and the phospholipid levels significantly lower in the aortic lesions of the lowprotein than those in the high-protein group. The cholesterol:phospholipid ratio was several times higher in the former ( 3 : 1) than that in the latter ( 1: 2). There was no significant difference in the triglyceride content of the lesions between the two groups. Changes in other organs. No significant change was seen in any of the visceral and musculoskeletal organs in the animals fed high-protein diet. Animals fed the low-protein diet showed the usual morphologic changes of protein deficiency state in various organs such as periportal fatty change in the liver, pancreatic acinar atrophy with loss of zymogen granules, marked atrophy of red pulp and lymphoid tissue in the spleen, brown atrophy of the myocardium, atrophy of mucosa of stomach and intestine, and marked reduction in endochondral bone formation.
312
GUFTA,
TANDON
AND RAMALINGASWAMI
DISCUSSION Our previous studies (Gupta et al., 1974a) have demonstrated that an adequate level of protein in the diet protects the pig from the atherogenic effects of a diet containing 25% saturated fats in addition to cholesterol whereas a very low level of protein has the opposite effect of aggravating the lesions. The present experiment was designed to study to what extent is cholesterol alone responsible for inducing and enhancing the lesions in the presence of a proteindeficiency state. The alterations in body weight, general appearance, serum proteins, hematological values, and the presence of characteristic morphological changes in the various organs observed in animals receiving a protein-deficient diet indicate that a state of severe protein deficiency was produced in these animals, with all the essential features of protein depletion described in man (Trowell, Davies, and Dean, 1954), monkeys (Deo, Sood, and Ramalingaswami, 1965) and pigs (Pond et al., 1965; Tumbleson et al., 1969). Th ese 1 changes are also similar to those described by us in pigs in our previous studies (Gupta et nl., 1974a, b). Protein deficiency is known to cause anemia (Sood et al., 1965) as observed in our protein-deficient animals. On the other hand, the hemoglobin and hematocrit showed a gradual rise in animals receiving a protein-adequate diet. This phenomenon is known to occur in growing pigs (Miller et al., 1961; McClellan et al., 1965). The animals were aged 6 mo at the time the experiment was started. While in the previous study ( Gupta et al., 1974a), atherogenesis was induced by the combined use of saturated fat to the extent of 25% by weight in the diet and cholesterol. In the present study only cholesterol was used, there was no added fat, and the bulk of the calories came from carbohydrates (Table I). This study extends and confirms our previous observations that a diet containing a low level of protein to the extent of 5% in the diet promotes the development of atherosclerosis. The lesions observed were confined to the aorta. They were more extensive though histologically not more severe, in the low-protein than in the high-protein group. The surface area involved in the low-protein group in the present experiment was less (48.0 * 8.7% ) than that observed in the last experiment (67 * 3.80% ). The difference is understandable, since the atherogenie stimulus was milder comprising cholesterol alone, even though the amount of cholesterol fed in the present experiment was proportionately higher than that in the previous experiment. What seems interesting, however, is that protein deprivation makes the animal prone to develop atherosclerosis, in the presence of small doses of cholesterol even in the absence of additional saturated fats. The surface area involved in the high-protein group (25.5 * 6.4% ) was not too different from that in the same experimental group in our last study (30.5 A 10.88%) or its control (31.5 + 13.44%). The striking difference in the degree of atherosclerosis as observed in the two groups could be ascribed to two factors-low levels of protein and/or excessive quantities of carbohydrates in the diet of animals of Group I. There is extensive evidence that dietary protein levels play a significant role in this connection. Several authors have reported in studies using chickens that atherosclerosis and serum cholesterol levels were more dependent on the levels of dietary protein than those of dietary fats. Nishida, Takenaka, and Kummerow, 1958; Leivelle, Feigenbaum, and Fisher, 1960; March and Bielly, 1959; Kokatnur et al., 1956
DIETARY
PROTEIN
AND
CHOLESTEROL
IN SWINE
ATHEROSCLEROSIS
313
have recorded similar observations in chickens. It has been well established by studies that decreased protein intake aggravates the development of atherosclerosis in birds (Stamler, Pick, and Katz, 1958a, b; Pick, Stamler and Katz, 1959; Pick et al., 1965; Kokatnur et al., 1956, 1958); rats (Fillios et al., 1958); rabbits (Polack et aI., 1965); monkeys (Mann et al., 1953), dogs (Li and Freeman, 1946) ; and pigs ( Greer et al., 1966), As was observed in our previous study (Gupta et al., 1974a), in this experiment also the low-protein group of animals maintained higher serum cholesterol levels for most of the experimental period. This seems to be the determining factor for greater severity of atherosclerosis in these animals. Several investigators have reported that restricted protein intake increases serum cholesterol concentrations in many species of animals (Rose and Balloun, 1969; Johnson, Leveille, and Fisher, 1958; Fisher et al., 1959; Chaikoff et al., 1961; Greer et al., 1966; Tumbleson et al., 1969). On the contrary, Barnes et al. (1959) and Moreland ( 1965) did not observe any effect on the levels of dietary protein in swine. This may be because in these studies biochemically demonstrable hypoproteinemia was not produced. Moreland (1965) used 8% protein in the low-protein diet and this level of protein was perhaps too high to produce hypoproteinemia and lesions associated with protein deficiency. Similarly, Barnes et al. (1959) also did not observe any change in the serum cholesterol values as long as their low-protein animals continued to be normoproteinemic, but after they had removed all protein from the diet and the animals developed hypoproteinemia, they observed some elevation of serum cholesterol. In this study also we observed hypercholesterolemia in low-protein animals only after they had developed clinical and biochemical signs of protein deficiency. The mechanism of hypercholesterolemia in experimental protein deficiency has been studied in several species and has been reviewed in our earlier communication (Gupta et al., 1974). Apart from the higher serum cholesterol values in the protein-deficient animals, their fatty acid composition in the serum cholesterol esters also showed some striking differences from those of the high-protein groups. The serum cholesterol esters of the low-protein group contained a larger proportion of oleic acid ( 18: 1) at the expense of linoleic acid ( 18:2). Several studies (Bottcher and Woodford, 1961; Schrade et al., 1961) have provided evidence that subjects with atherosclerosis tend to have a lower percentage of linoleic acid ( 18:2) and a higher percentage of oleic acid ( 18: 1) in their serum cholesterol esters as compared with normal subjects. Similar data have been reported in experimental atherosclerosis in rabbits (Zilversmit et cd., 1961; Swell et al., 1961, 1962). Thus, an increase in the oleic acid content at the expense of linoleic acid in the serum cholesterol esters of the low-protein animals could be a factor accounting for the proneness of these animals to develop more extensive lesions in our animals. The lesional lipids in the low-protein group had a higher cholesterol content and cholesterol:phospholipid ratio than in the high-protein group. The higher cholesterol content in the lesions may be related to the higher serum cholesterol values in these animals. Several experimental studies (Newman and Zilversmit, 1962; Newman et al., 1968; Day and Wilkinson, 1967) indicate that in aortic atheroscIerotic lesions, choIestero1 and its esters are derived primarily from the circulating blood and the rate of their accumulation is directly related to the severity
314
GUPTA,
TANDON
AND
RAMALINGASWAMI
of hypercholesterolemia. Our observation is similar to that of Fisher et al. (1959) who studied the effect of different fats and varying levels of protein and cholesterol on the biochemical changes in the aorta of chicken. They observed a higher cholesterol content in the aorta of the animals fed a low-protein diet as compared with those on a high-protein diet. The decrease in the phospholipid content in the atherosclerotic lesions of lowprotein animals in this study may be due to its retarded local synthesis. Studies with labeled phosphate (Shore et al., 1955; Zilversmit et al., 1954) and acetate (Newmann et al., 1961) have shown that the source of phospholipids in the atherosclerotic lesions is primarily from its synthesis in situ. It has been reported that in chronic protein deficiency, the synthesis of phospholipids in the body tissues is greatly retarded (MacDonald, 1960; Chatterjee and Mukherjee, 1968). The role played by the larger carbohydrate component of the protein-deficient diet cannot be dismissed lightly. The carbohydrate content of the proteindeficient diet was larger in this as compared with that of the same group in our previous experiment. It is known that dietary carbohydrates do influence the concentration and composition of serum, liver, and depot lipids (MacDonald, 1962; 1967). Among different types of carbohydrates, however, starch, which was the main carbohydrate constituent in our experimental diet, has been known to be the least atherogenic, if at all (MacDonald, 1962; Adams et al., 1959; Pick and Katz, 1965). ACKNOWLEDGMENT The
study
was supported
by a financial
grant
from
the
Indian
Council
of Medical
Research.
REFERENCES FISHER, M., and KOVAL, C. J. ( 1969). The influence of dietary carbohydrates on and liver damage and serum cholesterol in the rat. Fed. Proc. 18, 178. BARNES, R. H., KWONG, E., FIALA, G,, Recheigl, M., Lutz, P. N., and LOOSLI, J. K. (1959). Dietary fat and protein and serum cholesterol. I. Adult swine. J. Nutr. 69, 261. BOTTCHER, C. J. F., and WOODFORD, F. P. ( 1961). Lipid and fatty acid composition of plasma lipoproteins in cases of aortic atherosclerosis. J. Atheroscler. Res. 1, 434. CHAIKOFF, I. L., NXXOLS, C. W., JR., GAFFEY, W., and LINUSAY, S. (1961). The effect of dietary protein level on the development of naturally occurring aortic atherosclerosis in the chicken. J. Atheroscler. Res. 1, 461. CHATTERJEE, K. K., and MUKHERJEE, K. L. (1968). Phospholipids of the liver in children suffering from protein-calorie malnutrition. Brit. J. Nutr. 22, 145. DAY, A. J., and WILKINSON, G. K. (1967). Incorporation of Cl4 labelled acetate into lipid by isolated foam cells and by atherosclerotic arterial intima. Circ. Res. 21, 593. DEO, M. G., SOOD, S. K., and RAMALINGASWALII, V. ( 1965). Experimental protein deficiencyPathological features in the rhesus monkey. Arch. Puthol., 80, 14. FILLIOS, L. C., NAITO, C., ADAMS, S. B., PORTMAN, 0. W., and MARTIN, R. S. (1958). Variations in cardiovascular sudanophilia with changes in the dietary level of protein. Amer. J. Physiol., 194, 275. FISHER, M., FEIGENBAUM, A., LEVEILLE, G. A., WEISS, H. S., and GRIhfhfINGER, P. (1959). Biochemical observations on aortas of chickens: Effect of different fats and varying levels, of protein, fat and cholesterol. J. Nutr. 69, 163. GREER, S. A. N., HAYS, V. W., SPEER, V. C., and h~cCALL, J. T. (1966). Effect of dietary fat, protein and cholesterol on atherosclerosis in swine. J. Nut?. 90, 183. GUPTA, P. P., TANDON, H. D., and RAhKALINGASWAhfI, V. (1969). Spontaneous vascular lesions in Indian pigs. J. Pathol. 99, 19. ADAMS,
kidney
M.,
DIETARY
PROTEIN
AND
CHOLESTEROL
IN
SWINE
ATHEROSCLEROSIS
315
GUPTA, P. P., TANWN, H. D., and RAMALINGASWAMI, V. ( 1970). Experimental atherosclerosis in rabbits with special reference to reversal. J. Pathol. 101, 309. GUPTA, P. P., TANDON, H. D., and RAMALINGASWAMI, V. (1971). Further observations on the reversibility of experimentally induced atherosclerosis in rabbits. J. Pathol. 105, 229. GUPTA, P. P., TANWN, H. D., and RAMALINGASWAMI, V. (1974a). Experimental atherosclerosis in swine: effect of dietary protein and high fat. Erp. Mol. PathoE. 20, 115. GUPTA, P. P., TANDON, H. D., and RAMALINGASWAMI, V. ( 1974b). Liver changes in pigs fed a diet low in protein and high in fat and cholesterol. Ind. J. Med. Res., in press. JOHNSON, D., LE~E~LLE, G. A., and FISHER, H. (1958). Iniluence of amino acid deficiencies and protein level on the plasma cholesterol of the chick. J. Nutr. 66, 367. KOKATNUR, M., RAND, N. T., KUMMEROW, F. A., and SCOTT, H. M. (1956). Dietary proteina factor which may reduce serum cholesterol levels. Circulation 14, 1962. KOKATNUR, M., RAND, N. T., KUMMEROW, F. A., and Sccrrr, H. M. (1958). Effects of dietary protein and fat on changes of serum cholesterol in mature birds. J. Nutr. 64, 177. LEVEILLE, G. A., FEIGENBAUM, A. S., and FISHER, H. ( 1960). The effect of dietary protein, fat and cholesterol on serum protein components of the growing chick. Arch. Biochem. Biophys. 86, 67. Lr, T. W., and FREEMAN, S. ( 1946). Experimental lipaemia and hypercholesterolemia for protein depletion and by cholesterol feeding in dogs. Amer. J. Physiol. 145, 660. MACDONALD, I. (1960). Hepatic lipid of malnourished children. Metab. Clin. Exp. 9, 838. MACDONALD, I. ( 1962). Some-influence of dietary carbohydrates on liver and depot lipids. J. Physiol. 162, 334. MACDONALD, I. ( 1967). Inter-relationship between the influences of dietary carbohydrates and fats on fasting serum lipids. Amer. J. Clin. N&r. 20, 345. MANN, G. V., ANDRUS, S. B., MCNALLY, A., and STARE, F. J. (1953). Experimental atherosclerosis in cebus monkeys. J. Exp. Med. 98, 195. MARCH, B. E., and BIELLY, J. ( 1959). Dietary modification of serum cholesterol in the chick. J. Nutr. 69, 105. MCCLELLAN, R. O., VOGT, G. S., and RAGAN, H. A. ( 1965). Age related changes in haematological and serum biochemical parameters in immature swine. In “Swine in Biomedical Research.” Proc. Int. Symp., Richland, Washington, July 19-21, p. 597. MILLER, E. R., ULLRE, D. E., ACKERMAN, I., SCHMIDT, D. A., LUECKE, R. W., and HOEFER, J. A. ( 1961). Swine haematology from birth to maturity. J. Animal. SC., 20, 890. MORELAND, F. ( 1965). Experimental atherosclerosis of swine, In “Comparative Atherosclerosis” (J. C. Roberts, Jr. and R. Straus, els.) p. 21. Harper & Row, New York. NEUTMAN, H. A. I., MCCANDESS, E. L., and ZILVERSM~, D. B. ( 1961). The synthesis of Cl’-lipids in rabbit atheromatous lesions. J. Biol. Chem. 236, 1264. NEUTMAN, H. A. I., and ZILVERSMIT, D. B. (1968). Q uantitative aspects of cholesterol flux in aortas of cholesterol-fed rabbits. J. Atheroscler. Res., 8, 745. NEWMAN, H. A. I., and ZILVERSMIT, D. B. ( 1962). Quantitative aspects of cholesterol flux in rabbit atherosclerotic lesions. J. Biol. Chem. 237, 2078. NIS~DA, T., TAKENAKA, F. T., and KUMMEROW, F. A. ( 1958). Effect on dietary protein and heated fat on serum cholesterol and beta-lipoprotein levels and on the incidence of experimental atherosclerosis in chicks. Circ. Res. 6, 194. PICK, R., STAMLER, J., and KATZ, L. N. (1959). Effects of high protein intakes on cholesterolemia and atherosclerosis in growing and mature chickens fed high fat, high cholesterol diets. Circ. Res. 7, 866. PICK, R., and KATZ, L. N. ( 1965). Multiplicity of factors influencing atherosclerosis. Actu Cardiol. Suppl. 11, 169. PICK, R., JAIN, S., KA’IZ, L. N., and JOHNSON, P. (1965). Effect of dietary protein level on regression of cholesterol-induced hypercholesterolemia and atherosclerosis of cockerels. J. Atheroscler. Res. 5, 16. POLCAK, J., MELICHAR, F., SEVELOVA, D., DVORAK, I., and SKALOVA, M. (1965). The effect of a meat enriched diet on the development of experimental atherosclerosis in rabbits. J. Atheroscler. Res. 5, 174. POND, W. G., BARNES, R. H., REID, I., CROOK, L., KWONG, E., and MOORE, A. U. (1965). Protein deficiency in baby pigs, In “Swine in Biomedical Research.” p. 213. Proc. Int. Symp. Richland, Washington, 19-21 July.
316
GUPTA,
TANDON
AND
RAMALINGASWAMI
ROSE, R. J., and BALLOUN, S. L. ( 1969). Effect of restricted energy and protein intake on atherosclerosis and associated physiological factors in cockerels. J. Nutr. 98, 335 SCHFLADE, W., BI~GLER, R., and BOHLE, E. ( 1961). Fatty acid distribution in the lipid fractions of healthy persons of different age, patients with atherosclerosis and patients with idiopathic hyperlipidaemia. J. Atheroscler. Res. 1, 47. SHORE, M. L., ZILVERSMIT, D. B., and ACKERMAN, R. F. ( 1955). Plasma phospholipid deposition and aortic phospholipid synthesis in experimental atherosclerosis. Amer. 1. Physiol. 181, 527. SOOD, S. K., DEO, M. G., and RAMALINGASWAIII, V. (1965). A nemia in experimental protein deficiency in Rhesus monkey with special reference to iron metabolism. Blood 26, 421. STAMLER, J., PICK, R., and KATZ, L. N. ( 1958a). Effects of dietary proteins, methionine and vitamins on plasma lipids and atherosclerosis in cholesterol fed cockerels. Circ. Res. 6, 442. STAMLER, J., PICK, R., and KATZ, L. N. (1958b). Effects of dietary protein and carbohydrate level on cholesterolemia and atherogenesis in cockerels on high-fat, high-cholesterol mash.
Circ. Res. 6, 447. SWELL, L., Low, M. D., SCHOOLS, P. E., JR., and TREADWELL, C. R. (1961). Tissue lipid fatty acid composition in pyridoxin-deficient rats. J. Nzctr. 74, 148. SWELL, L., Low, M. D., and TREADWELL, C. R. (1962). Sterol ester formation in rat liver homogenates. PTOC. Sot. Erp. Biol. Med. 109, 176. TROWELL, H. C., DAVIES, J. N. P., and DEAN, R. F. A. (1954). “Kwashiorkor,” ed. 1. Arnold, London. TUMBLESON, M. E., TINSLEY, 0. W., CORWIN, L. A., JR., FLATT, R. E., and FLYNN, M. A. ( 1969). Undernutrition in young miniature swine. J. Nutr. 99, 505. ZILVERWIT, D. B., SHORE, M. L., and ACKERSIAN, R. F. ( 1954). The origin of aortic phospholipid in rabbit atheromatosis. Circulation 9, 581. ZILVERSMIT, D. B. MCCANDLESS, E. L., JORDAN, P. H., JR., HENLY, W. S., and ACKERMAN, R. F. ( 1961). The synthesis of phospholipids in human atheromatous lesions. Circdution
23, 370.