Prevention of Plasma Cholesterol Elevation

Prevention of Plasma Cholesterol Elevation and Atheromatosis in the Cholesterol-Fed Bird by the Administration of Dihydrocholesterol By M. D. SIPERSTEIN, M.D., C. W. NICHOLS, JR., M.A., AND I. L. CHAIKOFF, M.D. The effect of the addition of dihydrocholesterol to a high cholesterol diet has been studied in the It is shown that in this species the elevated plasma cholesterol and resulting atherosclerosis can be reduced to normal levels by such a procedure. ACCORDING to present concepts, the level of plasma cholesterol or some component thereof, is a critical factor in the development of arteriosclerosis. It is not surprising, therefore, that, as a means of preventing arteriosclerosis, attention has been focused upon methods for lowering the level of circulating cholesterol. Until recently the feeding of a diet low in lipids has been the only method proposed for achieving this aim. But it is apparent that the control of diet is difficult, if not impractical, for the population as a whole. In this laboratory we have approached this problem from a different point of view, namely, we have sought methods that will maintain a normal cholesterol level in the plasma even in the presence of a high cholesterol intake. Our first attempt to prevent the rise in plasma cholesterol in cholesterol-fed chickens was by the feeding of ferric chloride.' Although the administration of this salt kept the level of plasma cholesterol from rising excessively, the prolonged feeding of large amounts of iron, as was to be expected, proved noxious.* This experiment did, however, show the feasability of such an approach to the control of the level of plasma cholesterol. From the Division of Physiology of the University of California School of Medicine, Berkeley, Calif. Aided by grants from the U. S. Public Health Service and the Life Insurance Medical Research Fund. * In these initial experiments we used a diet containing 3 per cent ferric chloride. Experiments with smaller doses, presumably nontoxic, are underway. 37 The recent demonstration by Peterson, that mixed soybean sterols prevented the rise of plasma cholesterol' and reduced the incidence of atherosclerosis3 in birds fed cholesterol, then led us to investigate the possible blocking action of animal sterols closely related to cholesterol. Dihydrocholesterolt was first chosen for study because of its close structural similarity to cholesterol-it differs only in the saturation of the double bond between carbons 5 and 6. It is shown here that the administration of dihydrocholesterol to birds fed a diet containing 1 per cent cholesterol almost completely prevented both the rise in plasma cholesterol and the ensuing atherosclerosis. EXPERIMENTAL Twenty-two White Leghorn cockerels, obtained at the age of 3 months from the Department of Poultry Husbandry, were fed Purina Broiler Chow (starter ration) for two weeks. At the end of that time a blood sample was taken for determination of the basal plasma lipids. The birds were then divided into three groups and fed the diets shown in table 1. These diets were fed ad libitum for 11 weeks. The average daily food consumption for each group is likewise shown in table 1, and their growth is shown in figure 1. During these 11 weeks, five blood samples were drawn from each Total plasma cholesterol was determined by a modification of the method of t We are indebted to Dr. R. E. Waterman of the Schering Corporation for generously supplying the dihydrocholesterol used in this study. Circulation, Volume VII, January, 1953

38 3PREVVENTIONN OFI'PLASMA CHOLESTEROL ELElVATION TABLE 1. Composition of Diets and Food Consumption Group number I 2 3 Composition of Diets All diets consisted of Purina Broiler Chow (starter ration) to which was added one or more of the following constituents: Wesson Oil Cholesterol Dihydrocholesterol 1% 1% 2% Group symbol in Figures 1 to A Food Consumption Each value represents the average amount (Gm.) consumed per bird per day. 1st week 2nd week 3rd week th week 5th week 6th week 7th week 8th week 9th week 1th week 11th week Gpn. Gmnt. 7 81 7 9 72 82 76 79 7 88 8 83 i 88 73 88 81 82 15 81 92 76 Gm. 73 76 79 89 8 89 9 85 92 91 8 Average 85 78 85 12--- 111,; 13-/- 1- W91 81?O. 5 I 2 3 5 6 7 8 9 1 FIG. 1. Growth curves for the three experimental groups. O Birds fed basal diet. A-Birds fed basal diet to which was added 1 per cent cholesterol. E Birds fed basal diet to which were added 1 per cent cholesterol and 2 per cent dihydrocholesterol. i 1-9- W 8 a. & 7- -J 6-/ cr/ W 5-- U- W -/ ( 3--/ L2- = _ too COESTEROL 2 2 / CHOLESTEROL + DIHYDROCHOLESTEROL ~~~~CONTROL,' ~~~~~~~~~~I I 2 3 5 6 7 8 9 1 11 FIG. 2. Influence of dihydrocholesterol feeding upon plasma cholesterol levels of the cholesterol-fed o Average plasma cholesterol levels of eight birds A-Average plasma cholesterol levels of eight birds O Average plasma cholesterol levels of six birds fed a basal diet plus 1 per cent cholesterol plus 2 per cent dihydrocholesterol.

M. D. SIPERSTEIN, C. W. NICHOLS, JR. AND I. L. CHAIKOFF 39 Sackett, phospholipids by the method of King" and fatty acids by the method of Bauer and Hirsch.6- The plasma levels of cholesterol and phospholipids remained relatively constant in the control birds (figs. 2 and 3), while the fatty acid values in this group (fig. ) decreased during the course of the study. The chickens receiving 1 per cent cholesterol in their diet manifested the usual pronounced rise in plasma phospholipids and fatty acids failed to show any significant elevation. It should be noted that the effect of dihydrocholesterol is not due to a lower consumption of cholesterol and fat (table 1). Indeed, the total food intake of the dihydrocholesterol-fed birds (group 3) was actually slightly greater than that of birds fed cholesterol alone (group 2). Furthermore, the gain in weight (fig. 1) shown by the birds of o 5- o W --/ CHOLESTEROL 35-- J 3--/ CHOLESTEROL + ~~~~DIHY DR OC HOLE STE ROL 2W5 2 3 5 6 7 8 9 1 11 FIG. 3. Influence of dihydrocholesterol feeding upon plasma phospholipid levels of the cholesterolfed o Average plasma phospholipid levels of eight birds A-Average plasma phospholipid levels of eight birds El-Average plasma phospholipid levels of six birds fed a basal diet plus 1 per cent cholesterol plus 2 per cent dihydrocholesterol. cholesterol, phospholipids, and fatty acids (figs. 2, 3, and ). Thus by the end of the second week, the average plasma cholesterol value was 665 mg. per 1 ml., by the end of the sixth week, 855 mg. per 1 ml., and 98 mg. per 1 ml. by the end of the tenth week. The addition of dihydrocholesterol to the cholesterol diet completely prevented the hyperlipemic effects of the cholesterol feeding. Not only did the plasma cholesterol levels in the birds of group 3 remain in the same range as those in the control birds, but, likewise, FIG.. Influence of dihydrocholesterol feeding upon plasma fatty acid levels of the cholesterol-fed -Average plasma fatty acid levels of eight birds A Average plasma fatty acid levels of eight birds El-Average plasma fatty acid levels of six birds fed a basal diet plus 1 per cent cholesterol plus 2 per cent dihydrocholesterol. group 3 was of the same order as that observed in the controls (group 1). At the end of the 11 weeks, autopsies were performed on all birds. The aorta of each chicken was removed and examined for gross atheromata. Each lesion was carefully measured, and an estimate made of its area. The total areas of the atheromatous plaques of the thoracic and of the abdominal aortas were then calculated. The effect of the dihydrocholesterol feeding on the development of atheromata in thoracic

PREVENTION OF PLASMA CHOLESTEROL ELEVATION and abdominal aortas in cholesterol-fed birds is shown in table 2. The prevention of the characteristic lesion, observed in both portions of the aortas, is quite striking. Thus the average area of grossly visible lesions in the thoracic portion of the aortas of the birds fed only and.5 mm.2 for thoracic and abdominal aortas respectively. The significance of the experimental approach presented here to the control of plasma cholesterol, and possibly atherogeresis, in man is now under investigation. TABLE 2.-Effect of Dihydrocholesterol upon the Degree of Atherosclerosis in the Bird no. 1 2 3 5 6 7 8 Average... Diet 9 1% cholesterol (group 2) 1 1% cholesterol (group 2) 11 1% cholesterol (group 2) 12 1% cholesterol (group 2) 13 1% cholesterol (group 2) 1 1% cholesterol (group 2) 15 1% cholesterol (group 2) 16 1% cholesterol (group 2) Average... 17 1% cholesterol + 2% dihydrocholesterol (group 3) 18 1% cholesterol + 2% dihydrocholesterol (group 3) 19 1% cholesterol + 2% dihydrocholesterol (group 3) 2 1% cholesterol + 2% dihydrocholesterol (group 3) 21 1% cholesterol + 2% dihydrocholesterol (group 3) 22 1% cholesterol + 2% dihydrocholesterol (group 3) Average........ cholesterol was 13 mm.', while five of the six birds that received dihydrocholesterol along with cholesterol showed no gross lesions whatsoever in this portion of the aorta. The average atheromatous lesion in the abdominal aorta of the birds fed cholesterol measured 5 mm.', whereas that in the birds fed dihydrocholesterol plus cholesterol was 9.5 mm.2 It should be noted that two of the birds fed the control diet showed atheromatous lesions. The average degree of severity of this group was.5 mm.2 Qholesterol-Fed Bird Gross atheromata found in: Thoraicic aorta Abdominal aorta 1nmM.2 MM.2 o o ~~6~~ o.5 mm.2.5 mm.' ) ~~~17 12 ~~~51 23 ~~~57 1 25 51 ~~~87 t 1 2C )2 ~~~32 11 27 ~omm.2 8 9 8 5 MM.'2 1.3 mm.' 9.5 mm.' SUMMARY 1. Birds fed a diet containing 1 per cent cholesterol developed characteristic lipemia with average plasma cholesterol values ranging from 665 to 98 mg. per 1 ml. during the 11 week period. 2. Simultaneous feeding of dihydrocholesterol with this 1 per cent cholesterol diet resulted in complete suppression of the lipemia. During the period of observation the average plasma cholesterol values varied from 12 to 179 mg.

M. D. SIPERSTEIN, C. W. NICHOLS, JR. AND I. L. CHAIKOFF 1 per 1 ml. In control birds fed a stock diet containing no cholesterol, average values for plasma cholesterol ranged from 11 to 17 mg. per 1 ml. 3. The development of atherosclerosis in the cholesterol-fed birds was greatly inhibited by the simultaneous administration of dihydrocholesterol. Indeed no measurable difference in the extent of atherosclerosis was noted between birds fed the control diet and those fed cholesterol plus dihydrocholesterol. ACKNOWLEDGMENT The technical assistance of Mr. Jacob V. Sherokoff is gratefully acknowledged. SUMARIO ESPAfROL El efecto de la adici6n de dihidrocolesterol a una dieta alta en colesterol se ha estudiado en pollos. Se ha mostrado que en esta especie el colesterol elevado en el plasma y la ateroesclerosis resultante se puede reducir a niveles normales con este procedimiento. REFERENCES 1SIPERSTEIN, M. D., NICHOLS, C. W., JR., AND CHAIKOFF, I. L.: Effects of ferric chloride and bile on plasma cholesterol and atherosclerosis in the cholesterol-fed bird. Science. In press. 2 PETERSON, D. W.: Effect of soybean sterols in the diet on plasma and liver cholesterol in chicks. Proc. Soc. Exper. Biol. & Med. 78: 13, 1951. 3-, NiCHOLS, C. W., JR., AND SHNEOUR, E. A.: Some relationships among dietary sterols, plasma and liver cholesterol levels, and atherosclerosis in chicks. J. Nutrition 7: 57, 1952. SACKETT, G. E.: Modification of Bloor's method for the determination of cholesterol in whole blood or blood serum. J. Biol. Chem. 6: 23, 1925. 5KING, E. J.: The calorimetric determination of phosphorous. Biochem. J. 26: 292, 1932. 6BAUER, F. C., AND HIRSCH, E. F.: A new method for the calorimetric determination of the total esterified fatty acids in human sera. Arch. Biochem. 2: 22, 199.